Abstract:
Disclosed herein is a method of examining tissue growth and/or conditioning and/or adhesion properties of cells on a synthetic and/or natural scaffold ( 30 ) as well as a perfusion bioreactor ( 10 ) for use in this method. The method comprises the following steps: fixing a scaffold ( 30 ) to a holding means ( 28 ) disposed or disposable in a perfusion bioreactor ( 10 ), generating a flow of nutrition solution along the surface of said scaffold ( 30 ) held by said holding means ( 28 ), generating and/or maintaining physiological conditions in said nutrition solution flowing along said scaffold ( 30 ) using heat exchange means ( 20, 24, 48, 50 ) and/or gas exchange means ( 52, 54, 56, 58, 60, 62, 64 ) associated with said bioreactor ( 10 ), such as to allow growth and/or conditioning of cells on said scaffold ( 30 ), and optically inspecting, in particular by microscopy, the scaffold ( 30 ) held by said holding means ( 28 ) through a window ( 66, 68, 70, 72 ) provided in said perfusion bioreactor ( 10 ) at different times or continuously during tissue growth and/or conditioning of cells on said scaffold ( 30 ) while maintaining said physiological conditions in said perfusion bioreactor ( 10 ).

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to the field of tissue engineering. In particular, the invention relates to a method of examining tissue growth and/or conditioning and/or adhesion properties of cells on a scaffold, and a perfusion bioreactor to be employed in such method. Herein, the scaffold may be either one of a synthetic or a natural scaffold. 
       RELATED PRIOR ART 
       [0002]    Worldwide, every year approximately 300,000 heart valves are implanted to patients. Currently, there are three different types of implants available, namely mechanical heart valves, biological heart valves and homografts. While mechanical artificial heart valves have good lifetimes, unfortunately, they bear a severe risk of thrombosis, meaning that patients with artificial mechanical heart valves have to use anticoagulation medication throughout their lives. This is highly undesirable, because due to the decreased coagulation, any injury or surgery involves a significant risk or danger for the patient. 
         [0003]    While biological heart valves and homografts have good haemodynamic properties, unfortunately their lifetime is rather limited. In addition, there is a risk that the patient&#39;s body does not accept the biological homograft implant or that an inflammatoric reaction may occur. 
         [0004]    In order to overcome the abovementioned difficulties, it has been attempted to produce artificial heart valves via tissue engineering. Tissue engineering utilizes living cells as engineering materials. For example, if an artificial heart valve is to be produced by tissue engineering, an artificial heart valve is first seeded with smooth muscle cells, then with fibroblasts and finally with endothelium cells, corresponding to the structure of human vessels. It has been shown that with this three layer construction, the attachment of the endothelium cells has been improved as compared to a simple construction, but the attachment is unfortunately generally not strong enough to withstand shear forces that will occur during operation (see Fang Ning-tao, Xie Shang-zhe,  Construction of tissue - engineered heart valves by using decellularized scaffolds and endothelial progenitor cells , Chin Med. 120, 2007, Vol. 8, pp. 696-702). It has been observed that the formation of the extra cellular matrix can be improved if the artificial heart valve is subjected to mechanical strain or a constant fluid flow (see Ralf Sodian, Thees Lemke, Matthias Loebe, Simon P. Hoerstrup,  New Pulsatile Bioreactor for Fabrication of Tissue-Engineered Patches , Tissue Engineering, Mar. 9, 2001, pp. 401-405), thereby allowing for an increased stability of the cell layers. This effect has been employed in a certain class of bioreactors, in which the implant is subjected to fluid flow and mechanical strain. Under these conditions, the endothelium cells will align according to the flow direction of the fluid flow (see Helmut Gulbins, et al,  Development of an artificial vessel lined with human vascular cells , The Journal of Thoracic and Cardiovascular Surgery, September 2004, pp. 372-377). 
         [0005]    Bioreactors for use in tissue engineering are for example disclosed in DE 199 19 625 C2, DE 103 49 688 A1, DE 103 22 024 A1 and DE 100 53 014 A1. 
         [0006]    While remarkable success has been made employing such prior art bioreactors, tissue engineering is still a fairly new discipline which is based on the principle of trial and error. In particular, still much research has to be done to find out optimum combinations of scaffolds, cell types and growth factors. For example, it may very well be that certain cells grow well on certain scaffolds under statical conditions, but will not withstand the physiological flow occurring when the tissue is employed in the vascular system. 
         [0007]    Unfortunately, determining the best combinations of scaffold, cells and tissue growth conditions is a very time consuming task. For example, seeding an artificial heart valve takes about 2.000.000 cells/cm 2  that have to be cultivated in advance. Whether the tissue will eventually be capable of withstanding the fluid-dynamical stress in the physiological environment can typically only be determined from the confluently colonized valve scaffold. This means that it may well take one month to grow a tissue that then turns out to be entirely useless. These problems are not limited to tissue engineering for heart valves or cardiovascular applications in general, which have been referred to above merely by way of example, but to practically all types of tissue engineering. 
       SUMMARY OF THE INVENTION 
       [0008]    It is hence an object of the invention to provide a method and an apparatus for examining tissue growth and/or conditioning and/or adhesion properties of cells to determine promising combinations of scaffold and cells with less experimental effort. In the following, whenever reference to the examination of conditioning of cells is made, this is meant to also refer to the examination of adhesion properties of the cells or detachment of the cells, without further mention. 
         [0009]    This object is achieved by a method according to claim  1  and a perfusion bioreactor of claim  4 . Preferable embodiments are defined in the depending claims. 
         [0010]    The method of the invention comprises the following steps:
       fixing a scaffold to a holding means disposed or disposable in a perfusion bioreactor,   generating a flow of nutrition solution along the surface of said scaffold held by said holding means,   generating and/or maintaining physiological conditions in said nutrition solution flowing along said scaffold using heat exchange means and/or gas exchange means associated with said bioreactor, such as to allow tissue growth and/or conditioning of cells on said scaffold, and   optically inspecting, in particular by microscopy, the colonized scaffold held by said holding means through a window provided in said perfusion bioreactor at different times or continuously during tissue growth and/or conditioning of cells on said scaffold while maintaining said physiological conditions in said perfusion bioreactor.       
 
         [0015]    According to the invention, the success and/or stability of tissue growth or conditioning can be optically inspected during the tissue growth and/or conditioning process itself, i.e. the scientist does not have to wait with evaluations until the growth of tissue and/or conditioning is finished. This is enabled by a new type of bioreactor that allows to simultaneously optically inspect the tissue through a window and at the same time maintain physiological conditions inside the bioreactor, so that tissue growth and/or conditioning may continue during or after the inspection. Accordingly, less promising combinations of scaffolds and cells can be recognized early on, and the experiment can be terminated at an early stage, without spending too much effort in vain. Herein, the “scaffold” may be a sample piece of a “full” or “true” scaffold for actual use in tissue engineering, and in particular, a sample piece of a scaffold for cardiovascular applications, such as a (possibly comparatively small) sample piece of a heart valve or a cardiovascular patch scaffold. However, the method is not limited to this and may be applied for scaffolds for generally any type of tissue. 
         [0016]    Also, by generating a flow of nutrition solution along the surface of said colonized scaffold held by said holding means, the effect the flow of nutrition solution has on the tissue can be directly observed. For example, by varying the flow during the experiment, the effect of the flow on the tissue growth and/or the conditioning can be directly examined. Also, by adjusting the flow of nutrition solution to flow rates similar to the physiological environment of the intended use, it can be directly determined whether the tissue will withstand the fluid dynamical stress it will encounter after implantation. 
         [0017]    In summary, by allowing for optical inspection of the tissue at any time during tissue growth and/or conditioning, valuable additional information can be obtained, information can be obtained much earlier, and a better understanding of the tissue growth and/or conditioning process and material biocompatibility properties can be gained. Accordingly, more empirical information can be obtained with less experimental effort. 
         [0018]    In a preferred embodiment, the flow of nutrition solution along said scaffold is adjusted to a flow speed of 0.04 to 167 mm/s, preferably at least 33 mm/s. These are flow speeds which may realistically be expected in-vivo e.g. in the cardiovascular system. The method and apparatus of the invention allow to directly verify whether the tissue would withstand the shear forces generated by such flow. However, it is to be understood that the method is not limited to tissue engineering for cardiovascular applications. 
         [0019]    In a preferred embodiment, the scaffold used in the method is comparatively small and has a size of 400 mm 2  or less, preferably 200 mm 2  or less. For such small sample scaffolds, much less cells have to be cultivated as compared to the actual tissue to be grown. However, it turns out that such small samples are sufficient to get a very realistic and reliable impression of the behavior of the tissue, in particular in view of the possibility of direct optical inspection and control of the nutrition solution flow rate. Accordingly, a large number of combinations of scaffolds and cells can be examined in experiment with moderate experimental effort. 
         [0020]    The invention also relates to a perfusion bioreactor for use in the above method. The perfusion bioreactor comprises a housing, holding means disposed or disposable in said housing for holding a scaffold to be seeded with cells, means for directing a flow of nutrition solution along the surface of a scaffold when held in said holding means, and at least one window, preferably two opposite windows, said at least one window being transmissive for at least one of visible light, IR or UV radiation and arranged such as to allow for optical, particularly microscopy inspection of the scaffold when held in said holding means. 
         [0021]    Preferably, the bioreactor further comprises heat exchange means and/or gas exchange means for use in generating or maintaining physiological conditions in the nutrition solution flowing along said scaffold. 
         [0022]    Preferably, the housing has an opening for inserting the holding means to and removing said holding means from the housing, and a lid or cap for closing the opening. Accordingly, the scaffold patch can be easily fixed in the holding means while the holding means are outside the housing, and then the holding means can be inserted to the housing with the scaffold patch fixed therein or thereon. 
         [0023]    Preferably, the holding means comprises clamping means for conveniently fixing the scaffold by clamping. The clamping means may for example comprise an annular seat for receiving said scaffold and an annular clamping ring that can be clamped in a position suitable for pressing said scaffold against said seat. The clamping ring may further define the area of the scaffold to be colonized or seeded by cells. The clamping ring may in particular be dimensioned to restrict the scaffold area to be seeded to 400 mm 2  or less, preferably 200 mm 2  or less, and most preferably 100 mm 2  or less. 
         [0024]    In a preferred embodiment, the holding means is part of a preferably cylindrical substrate chamber through which said nutrition solution may be fed and that is closed by said at least one window. The holding means may further comprise a channel for directing nutrition solution to flow along the surface of the scaffold. In this embodiment, the holding means thus forms a separate chamber, in which the desired flow conditions of the nutrition solution can be reliably and reproducibly generated. By providing the channel in the holding means as a sole entry to the substrate chamber, the flow rate of the nutrition solution into the substrate chamber can be controlled. Since the geometry of the substrate chamber is known, the flow rate of nutrition solution through the channel into the substrate chamber translates directly into a flow velocity along the scaffold. Accordingly, a predetermined flow velocity of the nutrition solution along the scaffold can be generated by controlling the flow rate of nutrition solution through said channel. This in turn allows to conduct experiments with precisely defined and reproducible flow velocities along the scaffold. 
         [0025]    Preferably, the perfusion bioreactor further comprises inlet and outlet ports for nutrition solution, a first channel for guiding nutrition solution from said inlet port to the holding means and/or a second channel for guiding nutrition solution from said holding means to said outlet port. 
         [0026]    Further, the heat exchange means preferably comprise inlet and outlet ports for feeding and discharging a heating fluid to and from said bioreactor, respectively. Herein, the heating fluid inlet port is preferably located closer to the nutrition solution outlet port, and the heating fluid outlet port is located closer to the nutrition solution inlet port. This way, the flow directions of the nutrition solution and the heating fluid are generally opposite to each other, hence allowing for an increased efficiency of the heat exchange. 
         [0027]    Note that with such ports, the perfusion bioreactor can be easily combined with external supplies for nutrition solution and heating fluid, respectively. Accordingly, the experimental setup can be easily assembled, and the bioreactor can be quickly exchanged, thereby allowing scientists to carry out different experiments with different cells and scaffolds quickly one after another by simply exchanging the perfusion bioreactors. In fact, as will be shown below with regard to a specific embodiment, the perfusion bioreactor of the invention can be manufactured at very reasonable costs so that in practice it may be provided as a disposable article. By providing the ports at the perfusion bioreactor, such one-way product can be easily combined with stationary equipment for providing the nutrition solution and/or the heating fluid. 
         [0028]    In a preferred embodiment, the housing of the perfusion bioreactor comprises a cavity for receiving said heating fluid, and at least a part of said first and/or second channel(s) is (are) disposed in said cavity such as to be at least partially surrounded by said heating fluid. This way, the temperature of the nutrition solution can be easily and efficiently adjusted upon flow through the first and/or second channels within the bioreactor. 
         [0029]    Preferably, the heating fluid receiving cavity is partitioned into at least two compartments by at least one partitioning wall, wherein said partitioning wall has at least one opening allowing a flow of heating fluid from one compartment to another. By providing separate compartments with restricted flow from one compartment to another, a more uniform temperature distribution in the heating fluid inside the bioreactor is achieved. 
         [0030]    Preferably, the first and/or second nutrition solution channel(s) is (are) formed by a bore in said partitioning wall. This is a very cost-efficient way of providing the channel within the bioreactor and at the same time ensures an efficient heat exchange between the heating fluid and the nutrition solution guided in said first and/or second channel(s). 
         [0031]    In a preferred embodiment, the gas exchange means comprises one or more gas exchange interfaces for exchanging gas with said nutrition solution. Herein, the one or more gas exchange interface(s) is (are) preferably associated with the first and/or the second nutrition channels, thereby allowing gas exchange with the nutrition solution while guided through said first and/or second channels. 
         [0032]    The one or more gas exchange interface(s) preferably comprise(s) a gas-permeable membrane, and is (are) connected with a gas inlet or outlet port via a gas channel. Accordingly, the gas exchange can be easily integrated within the perfusion bioreactor and only needs to be connected with a gas supply via the gas inlet or outlet ports. 
         [0033]    Preferably, the gas channel is formed by a bore in a housing wall. Herein, the term “housing wall” may refer to any bottom, top or side wall or lid of the perfusion bioreactor housing. Again, using a bore in a housing wall for the gas channel is favorable from a manufacturing point of view, as it allows to reduce the number of parts, the manufacturing and the assembly costs. 
         [0034]    Preferably, the length and width of the perfusion bioreactor housing corresponds to the length and width of a standard microtiter plate, also referred to “microplate” in the art. Microtiter plates or microplates are flat plates with multiple wells used as small test tubes. Microplates have become a standard tool in analytical research and clinical diagnostic testing laboratories, and typically have 6, 12, 24, 96 or 384 sample wells arranged in a 2:3 rectangular matrix. Since the microplates have become a de facto standard, most microscopes will be equipped to handlethe same. For example, most microscopes have means for receiving and holding microplates during microscopy. By adapting the size of the bioreactor to this format, the bioreactor of the invention can be most easily examined with a conventional microscope that need not be further adapted in any particular way. 
         [0035]    Importantly, the bioreactor as a whole can be easily placed on a microscope table to inspect the cell growth and/or conditioning on the scaffold, without interrupting the tissue growth and/or conditioning, since the bioreactor comprises heat and/or gas exchange means that allow to maintain the physiological conditions even if the bioreactor is transferred to a microscope. Accordingly, the bioreactor of the invention allows to optically inspect the scaffold held in said holding means at different times or continuously during tissue growth and/or conditioning while maintaining physiological conditions in the perfusion bioreactor. Accordingly, valuable information can be obtained not only after the tissue growth and/or conditioning is finished, but at any time during tissue growth and/or conditioning. This way, additional process related information can be obtained, which will promote a better understanding the processes of tissue growth and/or conditioning and will help to adapt the environment, particularly the flow velocity of the nutrition solution such as to obtain an optimal tissue growth and/or conditioning. Last but not least, visual inspection during an early stage of tissue growth and/or conditioning will allow to terminate an experiment early, if it is apparent that the tissue growth and/or conditioning is not satisfactory, which allows to save time and resources. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0036]      FIG. 1  is a perspective, partly transparent view of a perfusion bioreactor according to an embodiment of the invention, 
           [0037]      FIG. 2  is a perspective exploded view of the components of the bioreactor of  FIG. 1 , seen at an angle from above, 
           [0038]      FIG. 3  is a perspective exploded view of the components of the bioreactor of  FIG. 1 , seen at an angle from below, 
           [0039]      FIG. 4  is a plan view onto a bottom portion of the bioreactor housing, and 
           [0040]      FIG. 5  is a perspective view of holding means for holding a scaffold. 
       
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENT 
       [0041]    For the purposes of promoting an understanding of the principles of the invention, reference will now be made to the preferred embodiment illustrated in the drawings, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated device and method and such further applications of the principles of the invention as illustrated therein being contemplated therein as would normally occur now or in the future to one skilled in the art to which the invention relates. 
         [0042]      FIG. 1  shows a perspective and partially transparent view of a bioreactor  10  in the assembled state according to an embodiment of the invention. The same bioreactor  10  is also shown in  FIG. 2  which is a perspective, exploded view of the bioreactor  10  as seen from an upper angle, and in  FIG. 3 , which is the same exploded view as  FIG. 2  but seen from a lower angle.  FIG. 4  is a plan view onto a bottom portion of the bioreactor  10  of  FIGS. 1 to 3 . 
         [0043]    As is best seen  FIGS. 2 and 3 , the bioreactor  10  comprises a bottom portion  12  and a top portion  14 . The bottom portion  12  forms a cavity  16  for receiving a heating fluid (not shown) that is divided into a first compartment  16   a  and a second compartment  16   b  by a partitioning wall  18 . A heating fluid inlet port  20  with a Luer lock mechanism is provided for connecting the bioreactor  10  to a heating fluid supply (not shown), such as a supply of water at 37° C. From the inlet port  20 , the heating fluid flows to the first compartment  16   a , from there through narrow openings  22  in the partitioning wall  18  to the second compartment  16   b  and is discharged from the second compartment  16   b  through a heating fluid outlet port  24 . 
         [0044]    In the center of the bottom portion, a cylindrical structure  26  is formed. The cylindrical structure  26  is for receiving the holding means  28  for holding a small scaffold patch  30  (s.  FIGS. 2 and 3 ), said scaffold patch  30  typically having a diameter of only little more than 8 mm. In  FIG. 5 , the holding means  28  is separately shown in a perspective view. Note that the orientation of the holding means as depicted in  FIG. 5  is upside down as compared to the orientation in  FIGS. 2 and 3 . 
         [0045]    With reference to  FIG. 5 , the holding means  28  comprise clamping means for clamping the scaffold  30  (s.  FIGS. 2 and 3 , not shown in  FIG. 5 ), which clamping means comprise an annular seat  32  in a cylindrical body  34  and a clamping ring  36 . For fixing the scaffold patch  30  in the holding means  28 , the scaffold patch  30  is placed on the annular seat  32  and the clamping ring  36  is pressed into an enlarged diameter portion  38  adjacent to the annular seat  32  and clamped in position, thereby pressing the scaffold patch  30  against the annular seat  32  and fixing it in place. The clamping ring  36  further defines the area of the scaffold  30  that may be seeded or colonized by cells to a desired value, such as 400 mm 2  or less, preferably 200 mm 2  or less, and most preferably 100 mm 2  or less. 
         [0046]    As is further seen in  FIG. 5 , the cylindrical body  34  comprises a channel  40  for guiding nutrition solution to the inside of the cylindrical body  34 , said channel  40  being positioned such as to direct a flow of nutrition solution along the surface of the scaffold patch  30  fixed in the holding means  28 . Note that the clamping ring  36  has corresponding recesses  42  such as not to occlude the channel  40  in the cylindrical body  34 . 
         [0047]    With reference again to  FIGS. 1 to 4 , the bioreactor  10  comprises inlet and outlet ports  44 ,  46  for nutrition solution. The inlet/outlet ports  44 ,  46  are in fluid communication with first and second channels  48 ,  50 , respectively, that are formed by bores in the partitioning wall  18  and shown by broken lines in  FIG. 2 . The first and second channels  48 ,  50  are also in fluid communication with the channel  40  in the cylindrical body  34  of the holding means  28 . The nutrition solution inlet and outlet ports  44 ,  46  both have a Luer lock mechanism for easy and standardized connection to an external nutrition solution supply. 
         [0048]    Associated with the first and second channels  48 ,  50 , are respective gas exchange interfaces  52 . The gas exchange interfaces  52  comprise a water-tight but gas-permeable membrane  54  that can for example be made from silicone. The gas-permeable membrane  54  separates the first and second nutrition solution channels  48 ,  50  from the first and second gas channels  56 ,  58  formed as bores in the top portion  14 . The first and second gas channels  56 ,  58  are shown in  FIG. 1  and also shown in broken lines in  FIG. 3  and  FIG. 4 . The outlets  60  of the first and second gas channels  56 ,  58  at the lower surface of the top portion  14  are seen in  FIG. 3 . The first and second gas channels  56 ,  58  are connected with respective gas inlet and gas outlet ports  62 ,  64  disposed at the top portion  14 . 
         [0049]    As is seen in  FIGS. 3 and 4 , an opening  66  is formed in the bottom portion  12  and located in the center of the cylindrical structure  26 . The opening  66  is hermetically sealed by a light transparent glass plate  68  and sealing means (not shown). The glass plate  68  forms a window allowing optical inspection of the scaffold  30  held in said holding means  28 . 
         [0050]    Likewise, an opening  70  is formed in the top portion  14  and is closed by a light transparent glass plate  72 , a sealing ring  74  and a lid  78  that has a threaded portion  80  to be screwed into a corresponding threaded portion  82  of the opening  70 . The opening  70  and the glass plate  72  form a second window for optical inspection. For example, the scaffold patch may be illuminated through the opening  70  in the top portion  14 , and observed using a microscope through the opening  66  in the bottom portion  12 . The opening  70  is dimensioned such that the holding means  28  can be inserted therethrough and placed in the cylindrical structure  26 , with the channels  40  communicating with the first and second channels  48 ,  50  provided in the partitioning wall  18 . In the present disclosure, the scaffold  30  may be either one of a natural or a synthetic scaffold or a combination thereof. Next, the glass plate  72  and the sealing ring  74  can be placed on top of the holding means  28  and the lid  78  may be screwed into the opening  70 , thereby sealing the bioreactor  10 . 
         [0051]    Next, a method of examining tissue growth and/or conditioning of seeded cells and/or adhesion properties on the scaffold  30  will be described. Positioning of the holding means  28  for alignment of its channels  40  with the first and second channels  48 ,  50  may be facilitated by a notch-and-feather mechanism or the like (not shown). First, the holding means  28  is taken out of the housing, and a scaffold  30  to be seeded with cells is placed on the annular seat  32  of the cylindrical body  34  of the holding means  28  and clamped in place by inserting the clamping ring  36  to the enlarged diameter portion  38 . The lid  78  is screwed from the top portion  14  and the holding means  28  is inserted through the opening  70  into the cylindrical structure  26  in a way such that the channels  40  of the holding means  28  are aligned with the first and second nutrition solution channels  48 ,  50 . The glass plate  72  and the sealing ring  74  are placed on top of the holding means  28  and the lid  78  is closed by screwing its threaded portion  80  into the threaded portion  82  of the opening  70 . The heating fluid ports  20  and  24  are connected to corresponding heating fluid supply lines (not shown) of a heating fluid circle. Typically, water at a temperature of about 37° C. will be used as heating fluid. 
         [0052]    Note that the water entering the heating fluid inlet port  20  will first enter the first compartment  16   a . In order to flow into the second compartment  16   b , the water must pass the narrow openings  22 . This will cause an improved mixing of water already present in the first compartment  16   a  with fresh water supplied through the inlet port  20 . 
         [0053]    Further, supply lines (not shown) for nutrition solution are connected with the nutrition solution inlet and outlet ports  44 ,  46 . Nutrition solution will flow through the first channel  48  formed inside the partitioning wall  18  and will hence exchange heat with the water in the first and second compartments  16   a ,  16   b  such as to ensure a physiological temperature of the nutrition solution. The nutrition solution leaving the first channel  48  passes through the channel  40  in the cylindrical body  34  of the holding means  28  and is guided by said channel  40  to flow along the surface of the scaffold  30  fixed in the holding means  28 . Since the flow of nutrition solution is confined to a volume defined by the cylindrical body  34  also referred to as “substrate chamber” or “sample chamber”, herein, a precisely controllable flow of nutrition solution over the surface of the scaffold patch  30  can be generated. The flow velocity can be easily adjusted by the flow rate of nutrition solution entering the inlet port  44 . The nutrition solution then exits the cylindrical body  34  of the holding means  28  through the channel  40  and is guided through the second channel  50  to the nutrition solution outlet port  46  where it is discharged. The nutrition solution can then e.g. be filtered from dead cells and/or circulating cells and recirculated back to the inlet port  44 . 
         [0054]    In addition, gas supply lines (not shown) are connected to the gas inlet and outlet ports  62 ,  64  such as to exchange gas with the nutrition solution flowing in the first and second. channels  48 ,  50  via the corresponding gas exchange interfaces  52 . By the gas exchange, the gas content of the nutrition solution, such as the CO 2  content can be maintained at physiological conditions that specific cells encounter in-vivo, e.g. a 5% content of CO 2 . 
         [0055]    At any time during cell seeding and/or conditioning on the scaffold patch  30 , the whole bioreactor  10 , while still connected to the respective supply lines (not shown) can be placed on a microscope table, and the current state of the tissue growing and/or conditioning on the scaffold  30  can be examined by ordinary microscopy or fluorescence microscopy. For this purpose, an image of the lower side of the scaffold  30  can be taken through the glass plate  68  and the opening  66  in the bottom portion  12 , while the scaffold  30  is illuminated through the opening  70  and the glass plate  72 . Since the gas and heat exchange means are provided within in the bioreactor  10 , the physiological conditions can be maintained, while the scaffold  30  is under optical examination, and the tissue growth and/or conditioning can be maintained during or picked up immediately after the inspection. This means that an optical inspection of the tissue growth and/or conditioning can be made at any time or continuously during the tissue growth and/or conditioning process. 
         [0056]    To further facilitate the optical inspection, the length and width of the bioreactor  10  are chosen to correspond to the length and width of a standard microtiter plate as routinely used in microscopy. In the present example, the length of the bioreactor  10  housing is 127.9 mm and the width is 85.6 mm. 
         [0057]    While in conventional tissue engineering, tissues are only examined after the growth process is finished, the method and bioreactor of the invention allow obtaining valuable insights in the tissue growth and/or conditioning process as such, which promotes a better understanding thereof. This way formerly inaccessible information can be gained that will help to identify favorable combinations of scaffold types and cell types in the nutrition solution and to optimize the processing parameters, including the behavior of the colonized cells to flow shear stresses under physiological conditions. Also, by inspecting the tissue already during early stages of tissue growth and/or conditioning, clearly non-working experiments can be immediately terminated, such as to save time and resources. 
         [0058]    Note that the bioreactor  10  of the preferred embodiment is devised for particularly small scaffold patches  30  having an area of less than 400 mm 2 , typically even less than 200 mm 2  and preferably less than 100 mm 2 . Accordingly, the number of cells needed to seed such scaffold patches is still moderate and can be cultivated in reasonable time. Still, the behavior of the tissue under physiological flow conditions can be directly discerned, since the flow of nutrition solution across the surface of the scaffold patch  30  can be easily, reliably and reproducibly controlled. 
         [0059]    Finally, the construction of the bioreactor  10  shown in  FIGS. 1 to 5  is rather simple and can be manufactured at low cost. One of the reasons for the low cost is that the first and second nutrition solution channels  48 ,  50  and the gas channels  56 ,  58  are formed by bores in wall portions and that hence no additional components or complicated assembly are needed. Accordingly, the bioreactor  10  can be manufactured as a disposable article. 
         [0060]    The embodiment described above and the accompanying figures merely serve to illustrate the method and bioreactor of the invention and should not be taken to indicate any limitation thereof. The scope of the patent is solely determined by the following claims. 
       LIST OF REFERENCE SIGNS 
       [0000]    
       
           10  bioreactor 
           12  bottom portion 
           14  top portion 
           16  cavity 
           16   a  first compartment 
           16   b  second compartment 
           18  partitioning wall 
           20  heating fluid inlet port 
           22  narrow opening 
           24  heating fluid outlet port 
           26  cylindrical structure 
           28  holding means 
           30  small scaffold patch 
           32  annular seat 
           34  cylindrical body 
           36  clamping ring 
           38  enlarged diameter portion 
           40  channel 
           42  recess 
           44  nutrition solution inlet port 
           46  nutrition solution outlet port 
           48  first channel 
           50  second channel 
           52  gas exchange interfaces 
           54  membrane 
           56  first gas channel 
           58  second gas channel 
           60  outlet 
           62  gas inlet port 
           64  gas outlet port 
           66  opening 
           68  light transparent glass plate 
           70  opening 
           72  light transparent glass plate 
           74  sealing ring 
           78  lid 
           80  threaded portion of the lid  78   
           82  threaded portion of the opening  70