Patent Publication Number: US-2021180001-A1

Title: Bioreactor with freeze-thaw capabilities to enhance product recovery and related methods

Description:
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 62/661,413, the disclosure of which is incorporated herein by reference. The disclosures of 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 are incorporated herein by reference. 
    
    
     TECHNICAL FIELD 
     This document relates generally to the cell culturing arts and, more particularly, to a bioreactor provided with freeze/thaw capabilities to enhance the recovery of products from cells and related methods. 
     BACKGROUND 
     Bioreactors are frequently used for culturing cells. In many cases, an issue that arises is the inability to recover substantially all of the desired final product produced by the cells. This is because a substantial portion of the product (e.g., 50%) oftentimes remains trapped inside the cells, and cannot be released or retrieved, at least without opening the bioreactor or otherwise causing it to lose integrity (which could render the product worthless). 
     Accordingly, a need is identified for an improved bioreactor and methods for processing cells that overcome one or more of the foregoing issues, and perhaps others that have yet to be discovered. The bioreactor and methods would provide for the enhanced recovery of products of interest, that would normally remain trapped within the cells when the bioprocessing operation is completed. 
     SUMMARY 
     According to one aspect of the disclosure, a bioreactor comprises a first chamber having cells and a second chamber providing temperature control of the cells in the first chamber. In some embodiments, the second chamber comprises a freezer connected to the bioreactor. 
     In some embodiments, the second chamber comprises a jacket surrounding the first chamber. The jacket may comprise an inlet and an outlet for receiving a chilled fluid, which may be sufficiently cold to cause any or all of the cells in the bioreactor to freeze. In some embodiments, the jacket comprises a pipe for circulating a chilled fluid within the jacket. The jacket may further comprise a thermally conductive fluid. The jacket may comprise a bladder including a chilled fluid for contacting a wall of the first chamber, which bladder may optionally have an inlet and an outlet. The jacket may comprise a removable sleeve. In some embodiments, the freezer comprises a chiller for chilling a fluid and a pump for circulating the fluid. 
     The bioreactor may further comprise a heater for heating the bioreactor. In some embodiments, the heater is adapted for at least partially receiving the bioreactor. In some embodiments, the heater comprises a conductive block having a heating element applied thereto. 
     In some embodiments, the heater is integrated into the bioreactor, such as by being located at least partially within a wall of the bioreactor. The heater may comprise a wire within the wall and connected to a power supply, and/or the heater may comprise a passage within the wall and connected to a source of heated fluid. 
     In some embodiments, the bioreactor comprises a modular bioreactor. In some embodiments, the bioreactor comprises a structured fixed bed. In some embodiments, the structured fixed bed comprises a spiral bed. 
     A further aspect of the disclosure pertains to a method of bioprocessing using a bioreactor including a chamber having a liquid including cells. The method comprises freezing the cells in the bioreactor. 
     In some embodiments, the freezing step comprises placing a chilled fluid in thermal communication with the chamber. The chilled fluid may comprise a gas. The placing step may comprise delivering the chilled fluid to a jacket of the bioreactor. The placing step may comprise delivering the chilled fluid to a pipe external to the chamber. The method may comprise the step of at least partially immersing the pipe in a thermally conductive liquid. 
     In some embodiments, the placing step comprises delivering the chilled fluid to a bag external to the chamber. The placing step may comprise delivering the chilled fluid to a pipe in contact with a wall of the bioreactor adjacent to the chamber. The placing step may comprise delivering the chilled fluid to a bag in contact with a wall of the bioreactor adjacent to the chamber. In some embodiments, the freezing step comprises delivering a cold gas to the chamber. 
     The method in some embodiments may further include the step of heating the cells after the freezing step. The heating step may comprise placing the bioreactor at least partially within a heater. The heating step may in some embodiments comprise heating a wall of the chamber. The heating of the wall may comprise supplying power to a wire on or within the wall, and/or supplying a heated fluid to a passage within the wall. The method may further optionally include monitoring the temperature of the chamber during the heating step, and/or monitoring the temperature of the chamber during the freezing step. 
     In some embodiments, the method further comprises generating a product within the cells prior to the freezing step. The method may then further comprise thawing the cells and recovering the product. The freezing step may comprise applying a pre-chilled sleeve to the bioreactor, such as adjacent to or around an external wall of the chamber. 
     According to a further aspect of the disclosure, a method of bioprocessing using a bioreactor including a chamber including cells is disclosed. The method comprises generating a product within the cells, freezing the cells, thawing the cells, and recovering the product. The disclosure is also directed to a product obtained using the aforesaid method. 
     This disclosure also pertains to an apparatus comprising a bioreactor including cells and means for freezing the cells. The apparatus may further include means for thawing the cells. 
     Still a further aspect of the disclosure pertains to an apparatus, comprising a bioreactor including a chamber having cells and a freezer connected to the bioreactor for freezing the cells in the chamber. 
     In some embodiments, the freezer comprises a jacket at least partially surrounding the chamber of the bioreactor. In some embodiments, the jacket comprises an inlet and an outlet for receiving a chilled fluid. The jacket may comprise a pipe for circulating a chilled fluid within the jacket. The jacket may include a thermally conductive fluid. 
     The jacket may comprise a bladder including a chilled fluid for contacting a wall of the chamber. The bladder may comprise an inlet and an outlet. The jacket may comprise a removable sleeve. 
     In some embodiments, the jacket comprises a removable sleeve. In some embodiments, the freezer comprises a chiller for chilling a fluid and a pump for circulating the fluid. The freezer may be adapted for delivering a gas to the chamber to freeze the cells. A sterile filter may be provided for sterilizing the gas prior to delivery to the chamber. A dehydrator may also be provided for drying the gas prior to delivery to the chamber. 
     In some embodiments, the apparatus further includes a heater for heating the bioreactor. The heater may be adapted for at least partially receiving the bioreactor. The heater may comprise a conductive block having a heating element applied thereto, or may be integrated into the bioreactor (such as by being located at least partially within a wall of the bioreactor, and may be in the form of a wire within the wall and connected to a power supply or a passage within the wall and connected to a source of heated fluid). 
     In some embodiments, the bioreactor comprises a modular bioreactor. In some embodiments, the bioreactor comprises a structured fixed bed, which may comprise a spiral bed. 
     A further aspect of the disclosure pertains to an apparatus comprising a cell culture bed including one or more frozen cells. The cell culture bed may comprise a fiber matrix. The cell culture bed may comprise a structured fixed bed. 
     In accordance with a further aspect of the disclosure, a modular bioreactor comprises a base, a first wall adapted for connecting with the base to form a first chamber for receiving a first fluid for culturing cells, and a second wall adapted for connecting with the base to form a second chamber for receiving a second fluid for regulating a temperature of the first chamber. 
     In some embodiments, the first wall comprises an inner wall and the second wall comprises an outer wall. A height of the second wall may correspond to a height of the first chamber. A freezer may also be provided for freezing the cells in the first chamber. The apparatus may further include a cooler for cooling the second fluid to an amount sufficient to cause one or more cells in the first chamber to freeze. A bladder may be provided with the second fluid for positioning in the second chamber. The first wall may include an inlet for receiving the second fluid and outlet for releasing the second fluid. The second chamber may include a coil for receiving the second fluid. 
     A further aspect of the disclosure pertains to a bioreactor comprising a first chamber for culturing cells, and a second chamber providing temperature control of the cells in the first chamber, the second chamber including a liquid including an anti-freezing agent. 
    
    
     
       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 perspective view of a first embodiment of a bioreactor according to the disclosure. 
         FIGS. 2A, 2B, and 2C  illustrate a possible environment of use of the bioreactor of  FIG. 1 . 
         FIG. 3  is a perspective view of the bioreactor of  FIG. 1 , including several enlarged views. 
         FIGS. 3A, 3B and 3C  illustrate a matrix material for use in forming a structured fixed bed for culturing cells in any of the disclosed bioreactors. 
         FIG. 4  is a cross-sectional view of a second embodiment of a bioreactor according to the disclosure. 
         FIG. 5  is a partially cutaway view of a portion of an alternate version of the bioreactor of  FIG. 4 . 
         FIGS. 6A and 6B  are cross-sectionals view a further embodiment of a bioreactor; 
         FIGS. 7, 8, 9, and 10  are cross-sectional views of different manners of providing a bioreactor with freeze/thaw capabilities; 
         FIG. 11  is a schematic diagram of another embodiment of a bioreactor with freeze/thaw capabilities; and 
         FIGS. 12, 13, 14, 15, and 16  illustrate various manners of facilitating the heating or thawing of a bioreactor, and the frozen cells therein in particular. 
     
    
    
     DETAILED DESCRIPTION 
     Reference is now made to  FIGS. 1-3 , which illustrate one embodiment of a bioreactor  100  for culturing cells, according to one aspect of the disclosure. In some embodiments, the bioreactor  100  includes an external casing or housing  112  forming an interior compartment and a removable cover  114  for covering the interior compartment, which may include various openings or ports P with removable covers or caps C for allowing for the selective introduction or removal of fluid, gas (including by way of a sparger), probes, sensors, samplers, or the like. As indicated in  FIGS. 2A, 2B, and 2C , in some embodiments, the bioreactor  100  may be used in connection with an external reservoir  102  and conduits  104  (e.g., forward and return) to form a loop for circulating fluid to the bioreactor  100 . 
     Within the interior compartment formed by the bioreactor housing  112 , several compartments or chambers may be provided for transmitting a flow of fluid or gasses throughout the bioreactor  100 . As indicated in  FIG. 3 , in some embodiments, the chambers may include a first chamber  116  at or near a base of the bioreactor  100 . In some embodiments, the first chamber  116  may include an agitator for causing fluid flow within the bioreactor  100 . In some embodiments, the agitator may be in the form of a “drop-in” rotatable, non-contact magnetic impeller  118  (which as outlined further below may be captured or contained within a container (not shown) including a plurality of openings for admitting and releasing fluid). In some embodiments, as a result of the agitation provided, fluid may then flow upwardly (as indicated by arrows A in  FIG. 2 ) into an annular chamber  120  along the outer or peripheral portion of the bioreactor  100 . In some embodiments, the bioreactor is adapted to receive a fixed bed, such as a structured spiral bed  122 , which in use may contain and retain cells being grown. However, the bioreactor of the present disclosure can be used with any type of cell culturing arrangement, including fixed beds, packed beds, fluidized beds, or the like. As indicated in  FIG. 3 , in some embodiments, the spiral bed  122  may be in the form of a cartridge that may be dropped or placed into the chamber  120  at the point of use. In some embodiments, the spiral bed  122  can be pre-installed in the chamber during manufacture at a facility prior to shipping. 
     In some embodiments, fluid exiting the chamber  120  is passed to a chamber  124  on one (upper) side of the bed  122 , where the fluid is exposed to a gas (such as oxygen or nitrogen). In some embodiments, fluid may then flow radially inwardly to a central return chamber  126 . In some embodiments, the central return chamber can be columnar in nature and may be formed by an imperforate conduit or tube  128  or rather formed by the central opening of the structured spiral bed. In some embodiments, the chamber  126  returns the fluid to the first chamber  116  (return arrow R) for recirculation through the bioreactor  100 , such that a continuous loop results (“bottom to top” in this version). In some embodiments, a sensor, for example a temperature probe or sensor T may also be provided for sensing the temperature of the fluid in the chamber  126 . In some embodiments, additional sensors (such as, for example, pH, oxygen, dissolved oxygen, temperature) may also be provided at a location before the fluid enters (or re-enters) the chamber  116 . 
       FIG. 3A  shows one embodiment of a matrix material for use as a structured fixed bed in the bioreactor of the present disclosure and, in particular, a spiral (or “wound”) bed  122 . In some embodiments, one or more cell immobilization layers  122   a  are provided adjacent to one or more spacer layers  122   b  made from a mesh structure. In some embodiments, the layering may optionally be repeated several times to achieve a stacked or layered configuration. In some embodiments, the mesh structure included in spacer layers  122   b  forms a tortuous path for cells for causing fluid flow radially outwardly into the immobilization layer(s)  122   a  (see cells L in  FIG. 3B  suspended or entrapped in the material of the immobilization layer  122   a ), and a cell culture may form part of any invention claimed herein). Homogeneity of the cells is maintained within the structured fixed bed as a result of this type of arrangement. In some embodiments, other spacer structures can be used which form such tortuous paths. In some embodiments, as shown in  FIG. 3A , the structured fixed bed can be subsequently spirally or concentrically rolled along an axis or core (e.g., conduit  128 , which may be provided in multiple component parts). In some embodiments, the layers of the structured fixed bed are firmly wound. In some embodiments, the diameter of the core, the length and/or amount of the layers will ultimately define the size of the assembly or matrix. In some embodiments, thickness of each of the layers  122   a ,  122   b  may be between 0.1 and 5 mm, 01 and 10 mm, or 0.001 and 15 mm. 
     A second embodiment of a bioreactor  200  is described with reference to  FIGS. 4, 5, 6A, and 6B . In some embodiments, the bioreactor  200  includes first through fifth chambers  216 ,  220 ,  224 ,  226 , and  228  for circulating fluid in the manner described above. In this embodiment, the housing  212  is optionally comprised of a plurality of modular parts. In some embodiments, the parts include a base part  230 , one or more intermediate parts  250 , and a cover part  270 . In some embodiments, the parts  230 ,  250 ,  270  can be adapted to interact in a fluid-tight manner so as to form the bioreactor  200  with the chambers  216 ,  220 ,  224 ,  226 , and  228 , as noted. 
     In some embodiments, and as perhaps best understood from  FIG. 4 , the base part  230  can include a peripheral connector, shown in the form of a groove  232 , for receiving and engaging a corresponding peripheral connector, such as a tongue  252 , projecting from one of the intermediate parts  250 . In some embodiments, interiorly, the base part  230  can include an upstanding wall  234 , which defines the first chamber  216  for receiving a fluid agitator (not shown). In some embodiments, the wall  234  can includes openings or passages to allow for fluid to flow radially into an outer portion of the base part  230 , which defines a further or second chamber  220 . In some embodiments, as the flow is redirected vertically as a result of the presence of the base part  230 , turbulence is created, which thus promotes mixing and homogeneity of the fluid throughout the bioreactor and thus enhances the cell culturing process. 
     Two intermediate parts  250   a ,  250   b  are shown as being stacked, with a peripheral connector (groove  254 ) of the first (lower) part  250   a  engaging a corresponding connector (tongue  252 ) of the second (upper) part  250   b . As can be appreciated from  FIG. 4 , in some embodiments, each intermediate part  250   a ,  250   b  can include an outer side wall  256  supporting the tongue  252  and groove  254 , respectively. In some embodiments, radially inwardly, an inner wall  258  carries inner and outer connectors, which may be in the form of upstanding ledges  260 ,  262 , can be provided for receiving the corresponding ends of a tube  236 , which thus forms periphery of the fifth or return chamber  228 . 
     In some embodiments, the first or lower intermediate part  250   a  may also include openings, such as elongated arcuate slots  264 , which at least partially receive connectors, of the base part  230 , such as upstanding projections  234   a  from the wall  234 . In some embodiments, an interior ledge  466  can form central openings  266   a  in the intermediate parts  250   a ,  250   b  for permitting fluid to flow in an inner column defined by the wall  234 , as well as to receive any temperature sensor, dip tube or the like (which would be positioned after the fluid exits the fixed bed). In some embodiments, the second intermediate part  250   b  may be similarly constructed to promote interchangeability, in which case the openings (slots  264 ) in the second or upper intermediate part  250   b  allow for the creation of the thin falling flow or film of fluid within the fifth or return chamber  228 , as previously noted. 
     In some embodiments, extending between the inner and outer walls  256 ,  258  are a plurality of supports  268 . In some embodiments, the supports  268  include radially extending supports  268   a  and at least one circumferentially extending support  268   b , which together can create a perforated or reticulated plate-like structure that allows fluid flow (which structure in this or any embodiment may comprise a screen, net, grid, or other skeletal structure, and may be rigid, semi-rigid, or flexible). In fact, the supports  268  may be designed to enhance fluid flow through the bed(s) by maximizing the amount of open space created by the openings for permitting fluid to pass. In some embodiments, for culturing cells, a fixed bed, such as the spiral bed (not shown) wound around wall  234  may be positioned in the chamber  224  formed between the parts  250   a ,  250   b . In some embodiments, fluid passing from the upper intermediate part  250   b  can enters the fourth chamber  226  defined partially by cover part  270  and may flow to the column forming the fifth chamber  228  before returning to the first chamber  216  for recirculation. 
     In some embodiments, the cover part  270  includes a connector, such as tongue  272 , for fitting into the corresponding connector (groove  254 ) of the second intermediate part  250   b . In some embodiments, the cover part  270  can also include a first or central receiver, such as upstanding wall  274  for receiving a removable cap or lid  276 , which may include various ports P for connecting with conduits for delivering fluids or other substances to the bioreactor  400  (and the fifth chamber  228 ). In some embodiments, the cap or lid  276  may also carry the temperature sensor or probe T, as shown, as well as other sensors, and may also be adapted for providing additions or removing substances from the bioreactor  200 , or for regulating a product manufacturing process. As can be appreciated, in some embodiments, the cap or lid  276  can be well positioned to allow for sensing or fluid sampling to occur in connection with the return flow via chamber  228 . In some embodiments, a second peripherally positioned receiver, such as upstanding wall  277 , may also be adapted for connecting with a second cap or lid  278  for receiving sensors or depositing or withdrawing substances (including culture samples) from the bioreactor and, in particular, a peripheral portion thereof including the third chamber  226  in which cell culturing is completed. In some embodiments, the caps or lids  276 ,  278  may have different types of ports P and may be different sizes/shapes, or they may be identical to promote interchangeability. 
     In some embodiments, adhesives or glue may be used at the connections to hold the structures together. In some embodiments, threaded or locking (e.g., bayonet style) connections may also be used, such that a fluid-tight seal is maintained to prevent leakage and help ensure that sterility is maintained. In some embodiments, the arrangement of modular parts  230 ,  250 ,  270  allows for the bioreactor  200  to be pre-assembled, assembled or constructed on site rapidly, and potentially disassembled with similar rapidity. As it is possible to easily add additional tube(s) to form a heightened wall  234  or intermediate parts  250 , the number of fixed beds or height of the bioreactor  200  may be adjusted to suit a particular need or process setting depending on the application. 
     In some embodiments, the flow from one fixed bed to the next-adjacent one in the chamber is direct or uninterrupted. In some embodiments, the outer chamber  224  for receiving the bed creates a continuous flow path through the multiple beds present therein, which may be structured fixed beds, unstructured fixed beds, or other beds. In some embodiments, the continuous and substantially unimpeded flow helps to promote homogeneity as if the modules are actually a single bed and thus improves the predictability and quality of the cell culturing process. Homogeneity means that the cell distribution throughout the bed is homogeneous or having a somewhat equal spread. 
       FIGS. 6A and 6B  also illustrate an alternate embodiment of a modular bioreactor  200  including fixed beds  296 . In some embodiments, the base part  230  and cover part  270  can be adapted for connecting with an outer casing  292 , which creates a gap or space with the periphery of the intermediate parts  250 . As can be understood, the height of the outer casing  292  may extend substantially the full height of the third chamber  226  for culturing cells, or it may be subdivided into individual chambers associated with each corresponding portion of the cell culture chamber. In some embodiments, the gap G or space may be used for providing a heating or cooling effect to control the temperature of the beds associated with the intermediate parts  250 . The gap G or space may also simply supply insulation of the walls of the intermediate area of the bioreactor which are close to growing cells within the bed and likely to be sensitive to temperature variations. This insulation acts to prevent heat which is applied to the bottom of the base part  230  of the bioreactor from extending up to the adhered cells in the bed(s)  296 . 
       FIG. 6A  also illustrates the possible use of sparging in the bioreactor, which may be provided in any disclosed embodiment. In the illustrated arrangement, the sparging is provided by a sparger  294  located in the fifth chamber  228 . The bubbles generated as a result may thus flow upwardly countercurrent to the return fluid flow. 
     These figures also show that the intermediate parts  250  may engage internal tubes  236 , which are fluid impervious to thus provide the chamber  228  for returning flow to the base part  230 , where it may be agitated and returned to enter the beds from below and flow upwardly therethrough (in any embodiment disclosed). These tubes  236  may be provided such that one tube corresponds to each fixed bed  296  present, as shown, and two intermediate parts  250  engage each tube  236  (e.g., one from below and one from above). However, in this or any other disclosed embodiment, it should be appreciated that the innermost surface of the fixed bed, such as the innermost spiral wrap of a spiral bed, may be made to perform a similar function by making it or otherwise conditioning it so as to be impervious to fluid. For instance, the surface may be coated with a fluid-impervious or hydrophobic material, such that it still retains the fluid in the bed(s) and maintains a distinct, return flow of fluid through the central column formed by chamber  228 . 
     According to a further aspect of the disclosure, a bioreactor (which could be any of the above-described bioreactors  100 ,  200 , or any other known form) for processing the cells to generate a product of interest (e.g., a virus or protein) may be adapted to freeze the cells in the bed to a temperature below the freezing point of the liquid in the cells (e.g. from zero degrees down to −20° C., and more preferably within the range of about −5 to about −20 degrees Celsius) and then revert to an unfrozen state (e.g., to room temperature) afterwards, by thawing (including possibly with assisted heating). This temperature cycle causes the breaking of the cell membranes and the resulting release of the final product within the cells, thereby enhancing recovery of the product of interest. This process of recovery avoids any compromising of the integrity of the bioreactor. The bioreactor may be substantially drained of liquid prior to being subjected to freezing according to the disclosure in order to avoid damage to the bioreactor that might otherwise occur upon expansion of the frozen liquid, if completely filled, but could also be subjected to freezing during the draining of the liquid). 
     An embodiment of a bioreactor  300  is adapted to perform the above-described “freeze-thaw” operation without being placed inside of a freezer (which would be difficult, if not impossible, for most bioreactors in view of size constraints. Specifically, the bioreactor  300  may be connected directly to a freezer, the implementation of which may take various forms, as shown in  FIGS. 7, 8, 9, 10, and 11 , as examples. With reference to  FIG. 7 , the bioreactor  300  includes a housing  312  comprised of a plurality of modular parts, such as a base part  330 , one or more intermediate parts  350 , and a cover part  370 . A jacket  390  is also provided for surrounding at least the intermediate part  350 , which includes the one or more chambers for growing cells. The jacket  390  may comprise a cylindrical or annular part defining a space, which together with a chilled fluid forms an associated freezer, which may be used to thermally regulate the chamber(s) within the bioreactor  300  and associated with the intermediate part  350 . 
     The jacket  390  may alternatively be in the form of a portable device or removable sleeve. This device or sleeve may slip over or snap around the bioreactor  300 , and thus may be used when freezing is desired and not used otherwise. The sleeve can be pre-chilled/frozen, and may be reusable or made to be disposable 
     Causing the freezing of the liquid within the cells of the bioreactor  300  may be achieved in a variety of ways. One example is by providing a fluid (such as cold air) to the space covered by the jacket  390  (note inlet  392  and outlet  394  in  FIG. 8 ), such that it is in direct contact with an outer surface of the bioreactor  300 . The fluid may be provided by a pump M in communication with the inlet and outlet for cycling the gas through the jacket  390  in a continuous fashion, and a cooler or chiller N may serve to cool the fluid during the cycling external to the jacket  390 . The jacket  390 , pump M, and cooler or chiller N thus together serve as the freezer in this example. 
     Another option, as shown in  FIG. 9 , is to provide a vessel for receiving a chilled fluid (e.g., an anti-freezing agent, such as liquid glycol, or water mixed with liquid glycol, and possibly with an anti-bacterial agent) in the jacket  390 . This may be done by providing one or more cooling conduits, such as flexible or rigid pipes  396 , in the space bounded by the jacket  390 . Each pipe  396  provided may also have an inlet and outlet  396   a ,  396   b  for circulating a fluid in a circuit with a freezer and pump (see  FIG. 8 ). Using more than one pipe (such as in a stacked configuration) is possible and may allow for a better degree of thermal control and avoid possible “hot” spots by staggering the location of the respective inlet and outlet. The pipe(s)  396  may also be immersed in a fluid within jacket  390  serving as a good thermal conductor (e.g, liquid glycol) to improve thermal conductivity and allow for an enhanced degree of thermal transfer. In the typical case where the bioreactor  300  is cylindrical, the pipes  396  would be annular in nature, and may traverse the bioreactor  300  in a helical fashion. As discussed below, the pipes  396  may also be integrated into one or more walls of the bioreactor  300 . 
     Instead of a pipe(s)  396 , the vessel may comprise a bladder, such as one or more flexible bags  398 , as shown in  FIG. 10 . The bag  398  may also have an inlet  398   a  and outlet  398   b  for circulating chilled fluid. In view of the large surface area and flexible surface of the bag  398 , a better degree of thermal transfer may result due to the increase area of surface contact with the outer surface of the intermediate part  350 . Again, for the typical cylindrical bioreactor  300 , the bag  398  provides a generally annular shape so that the respective surfaces are in intimate thermal engagement to maximize the transfer of cooling. Alternatively, the bladder or bag  398  may not have an inlet or outlet and may have its temperature regulated by integrating a heating or cooling system into adjacent structure, such as for example the intermediate part  350  or another part. In any case, the bladder or bag may comprise a multilayer flexible bag made of polymer material (HDPE or PVC, as examples) designed to withstand freezing or cyclic temperature changes, and can be designed to fit around or within a chamber of any bioreactor according to the needs of the particular arrangement. 
     In any of these cases or others where the liquid in or around the cells is frozen, it may be allowed to thaw naturally by simply stopping the provision of chilled fluid to the jacket  390 . Alternatively, a warming fluid may be supplied to the jacket  390  to assist with the thawing procedure. Still another alternative, and as discussed further below, is to use a heater to assist in the thawing operation for the frozen (or partially frozen) cells, which may involve adding a warm fluid or buffer to the jacket  390  or the bioreactor  300  itself. 
     Upon thawing, the cell membranes of the once-frozen cells are broken. Fluid such as buffer may be introduced to the bioreactor  300  to harvest the product or material of interest (e.g., a virus or protein). The bioreactor  300  may be at least partially drained of fluid before the freezing step, thereby creating room for additional fluid (i.e., the buffer, which again may be warmed to facilitate thawing). Once liquefied, the fluid may be pumped out of the bioreactor  300  to a holding tank and may be purified thereafter (either separate of combined with the thawed fluid). 
     According to another aspect of the disclosure, and referring to  FIG. 11 , cells can be frozen by providing a cold medium to a bioreactor  500 , such as cold air at, for example, from −5 degrees C. to about −20 degrees C. or otherwise at a temperature sufficient to cause any or all cells to freeze. As indicated schematically in  FIG. 11 , this can be achieved by using a freezer  502  to supply the cold medium directly to the bioreactor  500 , and a chamber  500   a  thereof in which cells are cultured in particular, via an inlet  504  or other port, and may optionally be done with a sterilization filter  506 , which may be upstream or downstream of the freezer  502 . This is contrasted with the above embodiment in which an outer chamber of the bioreactor is used to receive a fluid for causing freezing of the cells. A temperature sensor or probe  508  may also be provided to monitor the temperature of the bioreactor  500  and, in particular, any portion including cells to be frozen. 
     If air is used as the medium for causing the cells to freeze, it may optionally be dehydrated so as to avoid having moisture condense and freeze in any delivery conduit and the sterilization filter  506 , if present, and render both components unusable. Dehydrated air can be provided by drying ambient air using a dehydrator  510 , as indicated, which make take the form of molecular sieves, a heat exchanger or other heat source, and which may be upstream or downstream of the freezer  502 . Additionally, the air may be dried prior to delivery (before or after being chilled) by exposing it to coils or some other source at a temperature that is below the air target temperature of −20 degrees C., i.e. −30 degrees C. Instead of air, other gases could also be used. 
     As noted above, subsequent to freezing the cells, it may be desirable to subject the cells to thawing. This may be achieved passively or naturally by simply withdrawing any cold influence from an associated freezer or freezing means and waiting. However, it is also possible to subject the cells to active thawing, such as by warming or heating the cells or an associated portion or chamber of the bioreactor. 
     In some embodiments, active heating may be achieved using externally applied or transferred heat, and various examples of achieving such heating are shown in  FIGS. 12-16 . In  FIG. 12 , a heat source or heater  602  for a bioreactor  600  comprises a conductive block  604 . This block  604  may be formed of metal and is adapted for receiving at least a portion of the bioreactor  600  (such as a base thereof). A heating element  606  may be applied to the block  604 , which as shown in  FIG. 13  may comprise a flexible strip, such as one made of a polymer film  608  on or in which an electric wire  610  is attached (e.g., glued or encapsulated), and which wire may be connected to a power supply S. Alternatively, the block  604  may be provided with one or more passages (not shown) for receiving a flow of fluid. In any case, the bioreactor  600  may be associated with the heater  602  to achieve the desired heat transfer, and cause the more rapid thawing of the once-frozen cells or portions of the bioreactor  600 . 
     As can be appreciated from  FIGS. 12 and 12A , a gap P may exist between the bioreactor  600  and the recessed portion of the block  604  receiving it, which can result in suboptimal heat transfer. Thus, according to a further aspect of the disclosure, and with reference to  FIGS. 14, 15, and 16 , the heater  602  may be instead or additionally integrated directly into the bioreactor  600  (which may take any form disclosed herein or otherwise).  FIG. 14  illustrates that a resistive heating element, such as an electrically conductive wire  612 , may be integrated into the wall  600   a  of the bioreactor  600 , such as by injection molding (and overmolding in particular, so that intimate contact with the wire and the material (plastic) of the bioreactor wall  600   a  results). Alternatively or additionally, the wire  612  may be externally applied to the wall  600   a  as indicated in  FIG. 15 , and in a manner that maximizes the heating effect (such as by applying it in a serpentine pattern, as shown, but any pattern that maximizes the coverage area could be used). As can be appreciated, this allows for the heating to be selectively applied to portions of the bioreactor  600  in a more precise manner, depending on the cell culture process and/or where the heating may be desired to maximize efficiency (e.g., only on portions of the bioreactor corresponding to a chamber where cells are cultured). 
     Referring to  FIG. 16 , a further option for temperature regulation to achieve active thawing is to integrate a passage  600   b  in a wall  600   a  of the bioreactor  600 . A fluid (gas or liquid) may be passed through the passage  600   b  to achieve the desired the thermal exchange (heating or cooling). In particular, the fluid may be caused to flow from an inlet I to and outlet O, and possibly in a recirculating loop in communication with a heater  614 . A temperature probe or sensor  620  may also be provided to monitor the temperature of the bioreactor  600  in a more accurate manner and helps to ensure that overheating and damage does not result. 
     This disclosure may also be considered to relate to a cell culturing bed with frozen cells, whether part of a bioreactor or otherwise. 
     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 35 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 e.g. component 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 preferred embodiments of the present invention 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, while the bioreactor is shown in a vertical orientation, it could be used in any orientation. Also, while the bioreactor is shown throughout independent of any isolator or cabinet, it should be understood that it could be used in combination with such structures in order to maintain a sterile environment. It should also 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.