Abstract:
A shear stress inducing apparatus and a shear stress inducing system is provided. The invention makes use of a conical disk rotating at a controlled angular speed within a culturing vessel to induce a uniform and position-independent shear stress in a saline solution containing a cell culture.

Description:
BACKGROUND OF THE INVENTION  
       [0001]     1. Field of Invention  
         [0002]     The present invention relates to a laboratory apparatus. More particularly, the present invention relates to a shear stress inducing apparatus and an automatic shear stress inducing system for cultured biological cells.  
         [0003]     2. Description of Related Art  
         [0004]     In recent years, biotechnology has had a rapid development in many parts of the world. Some of the most important researches center around DNA, in particular, the sequencing of human DNA in the so-called ‘human genome project’. In the medical community, investigation on the genetic factors controlling the production of proteins is a hot topic of research. Another area of concern relates to the care of the aging population. Some degenerative diseases in the aging community such as rheumatism have caught the attention of many medical researchers.  
         [0005]     In the area of research for rheumatic diseases, investigations are mostly centered around genetic control, mechanical stress and drug reaction on the relationship between cartilaginous cell reaction and the appearance of rheumatism. However, the current investigation on the effect of mechanical stress on cartilaginous cells is hampered by a lack of highly functional and efficient mechanical stress inducing apparatus for controlling the experimental settings. Thus, an economical and efficient digital controlled mechanical stress inducing apparatus capable of carrying out research on cells and tissues is in constant demand by rheumatic disease investigators.  
         [0006]     In addition, following the rapid advance in biotechnology, another branch of medical investigation in the 21 st  century is tissue engineering using various types of self, foreign, mature or embryonic cells from different cell lines as constituent elements. A few types of cartilaginous tissues engineered by some biotechnology firms have already claimed preliminary success in curing patients with rheumatic disease and damages to cartilaginous joints. Nevertheless, the research community still lacks a set of formal inspection and monitoring apparatus for determining the quality standard of engineered tissues.  
         [0007]     At present, a simple mechanical shear stress inducing apparatus having an acrylic conical disk mounted on the axle of an electric motor held to a fixed supporting frame is used. One major defect for this type of apparatus is its bulkiness. Normally, only one set of experiment can be carried out inside a temperature-controlled chamber at a time. In addition, the conventional mechanical stress inducing apparatus provides very little capacity to adjust the gap between the circular disk and the culturing vessel, the rotational speed of the disk and the amount of vibration of the disk axis or perform an experiment with complicated shear stress pattern.  
       SUMMARY OF THE INVENTION  
       [0008]     Accordingly, one object of the present invention is to provide a shear stress inducing apparatus and an automatic shear stress inducing system having a digital controlled mechanical stress inducing mechanism capable of producing precise and adjustable stress for cell or tissue research.  
         [0009]     A second object of this invention is to provide a shear stress inducing apparatus and an automatic shear stress inducing system that can be used to investigate the etiology and the pathology of rheumatism and provide a means of inspecting and monitoring the quality of engineered cartilaginous tissues.  
         [0010]     A third object of this invention is to provide a shear stress inducing apparatus and an automatic shear stress inducing system having a design capable of removing some of the defects in a conventional mechanical stress inducing apparatus.  
         [0011]     To achieve these and other advantages and in accordance with the purpose of the invention, as embodied and broadly described herein, the invention provides a shear stress inducing apparatus. The shear stress inducing apparatus comprises a platform, a culturing vessel, a conical disk, a stepper motor, an upper cover panel, a bottom base plate and two side base plates. The platform is a height-adjustable platform. The culturing vessel is placed on the platform. The conical disk is horizontally set inside the culturing vessel. Furthermore, the axis of the conical disk is vertically positioned relative to the bottom surface of the culturing vessel and aligned close to the circular center of the culturing vessel. The axle of the stepper motor is connected to the central axis of the conical disk for rotating the disk. In other words, the conical disk can be driven by the stepper motor to rotate through its axle connection. In addition, the upper cover panel is set up between the stepper motor and the conical disk. The stepper motor is fastened to the upper cover panel. In this invention, the upper cover panel also has a cultured solution extraction hole to facilitate sampling or replacement of solution without interfering with an on-going experiment. The bottom base plate is set up underneath the platform on each side of the platform. The two side base plates are connected to the bottom base plate and the upper cover panel as well. Hence, the upper cover panel, the bottom base plate and the two side base plates together form a base stand while the culturing vessel and the conical disk are set up inside the base stand. Furthermore, the shear stress inducing apparatus may also include two transparent acrylic observer windows positioned on each side between the bottom base plate and the upper cover panel for monitoring the experimental status and conditions of the culturing solution inside the vessel.  
         [0012]     This invention also provides an automatic shear stress inducing system. The system includes a temperature-controlled incubator, a plurality of shear stress inducing apparatus, a stepper motor driver, a controller and a multi-channel square-wave generation interface. The temperature-controlled incubator provides an enclosed area sustained at a high temperature and moisture content. The shear stress inducing apparatus are placed inside the temperature-controlled incubator. Each shear stress inducing apparatus comprises a platform, a culturing vessel, a conical disk and a stepper motor. Since the construct of a shear stress inducing apparatus has already been described, detailed explanation of the shear stress inducing apparatus is not repeated here. The stepper motor driver is electrically connected to the stepper motor of each shear stress inducing apparatus. The multi-channel square-wave generation interface is electrically coupled to the stepper motor driver and the controller is electrically coupled to the multi-channel square-wave generation interface. In other words, the controller sends an instruction to the multi-channel square-wave generation interface so that the multi-channel square-wave generation device is triggered to transmit a square wave signal to the stepper motor driver for controlling the rotating speed of the conical disk inside each shear stress inducing apparatus. In the meantime, the controller also transmits non-inverted/inverted signal to the stepper motor driver to control the rotational direction (positive or negative rotation) of the stepper motor inside each shear stress inducing apparatus.  
         [0013]     The shear stress inducing apparatus and automatic shear stress inducing system according to this invention is capable of producing highly accurate shear stress values so that experimental errors is reduced to a minimum.  
         [0014]     The rotational speed of each conical disk inside the automatic shear stress inducing system can be programmed to simulate or accelerate the shear stress of the cells inside a human body to determine any physiological changes.  
         [0015]     The shear stress inducing apparatus according to this invention is designed to operate inside a temperature-controlled incubator at a high temperature and relative humidity to simulate the internal environment of a human body for an extended period of time.  
         [0016]     Because the shear stress inducing apparatus occupies a relatively small volume, operates with a high degree of stability and has a low cost of production, a multiple of apparatus can be squeeze inside a standard temperature-controlled incubator. Therefore, considerable amount of experimental data can be produced within a very short time.  
         [0017]     The shear stress inducing apparatus of this invention is specially designed with a culture solution extraction hole. Hence, the cultured cells within the vessel can be sampled from the solution periodically for further inspection and analysis.  
         [0018]     The shear stress inducing apparatus of this invention has a very simple construction and hence is virtually maintenance free. Furthermore, after an experiment, the conical disk can be easily removed from the apparatus and disinfected in preparation for a brand new experiment. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0019]     The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification. The drawings illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.  
         [0020]      FIG. 1  is a diagram showing the principle behind the production of a shear stress.  
         [0021]      FIG. 2  is a diagram showing the geometric relationship between a conical disk and a culturing vessel in a shear stress inducing apparatus.  
         [0022]      FIG. 3  is a graph showing a function f relating the shear stress with the radius ratio for a conical disk having different distance of separation between the conical disk and a culturing vessel.  
         [0023]      FIG. 4  is a diagram showing the basic structural elements of a shear stress inducing apparatus according to one preferred embodiment of this invention.  
         [0024]      FIG. 5  is a diagram of an actual shear stress inducing apparatus according to one preferred embodiment of this invention.  
         [0025]      FIG. 6  is a diagram showing an automatic shear stress inducing system according to one preferred embodiment of this invention.  
         [0026]      FIG. 7  is a diagram showing the timing and parametric variation of shear stress in an automatic shear stress induction apparatus. 
     
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS  
       [0027]     Reference will now be made in detail to the present preferred embodiments of the invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers are used in the drawings and the description to refer to the same or like parts.  
         [0028]     This invention mainly discloses a method of using a cultured solution to serve as a working fluid for generating shear stress in an apparatus so that the effect of shear stress on the physiological development of biological cells can be studied. The cultured solution serving as a working fluid is normally a Newtonian fluid. In other words, the shear stress is proportional to the shearing rate inside a fluid flow field. The ratio of shear stress over shearing rate is known as viscosity (a), which is a function of temperature. In general, most cellular experiments are carried out at a constant temperature of about 37° C. With an experimental temperature of 37° C., the cultured solution has a viscosity very close to pure water, that is, 6.7×10 −4  kg/m·s.  
         [0029]      FIG. 1  is a diagram showing the principle behind the production of a shear stress. As shown in  FIG. 1 , the lower surface  10  at the bottom of the fluid body is a stationary surface. On the other hand, the fluid layer at the upper surface  12  of the fluid body separated from the lower surface  10  by a distance L is moving at a speed U. The relationship between shear stress and shear strain at various heights within the fluid field is given by the formula:  
               τ   =     μ   ⁢       ⅆ   u       ⅆ   y           ,           (   1   )               
 where τ is the shear stress, u is the horizontal flow speed of the fluid, and y is the vertical distance from the lower surface  10 . For a Newtonian fluid flow, the shear stress is independent of height and hence has a constant value τ=μ(U/L). Therefore, if the upper surface  12  is the surface of the conical disk rotating at an angular speed ω, a point at a radius r from the axis of the spinning disk will move linearly at a speed U=rω. 
 
         [0031]      FIG. 2  is a diagram showing the geometric relationship between a conical disk and a culturing vessel in a shear stress inducing apparatus. As shown in  FIG. 2 , at a radius r from the axis  16  of the conical disk  14 , the surface point A of the conical disk  14  is separated from the bottom surface point B of the culturing vessel  18  by a distance L equal ε+r tan θ. The linear velocity in a direction perpendicular to the paper using the rotational speed N(rpm) of the conical disk  14  is U=rω=2πN/60. Thus, the shear stress τ in the fluid layer between point A and B can be represented by the formula:  
             τ   =       μ   ⁢     U   L       =     μ   ⁢       r2   ⁢           ⁢   π   ⁢           ⁢     N   /   60         ɛ   +     r   ⁢           ⁢   tan   ⁢           ⁢   θ                     (   2   )               
         [0032]     When the distance of separation ε between the top of the conical disk  14  and the bottom surface of the culturing vessel  18  is zero, the shear stress τ is reduced to the equation: 
 
τ=KN  (3), 
 
 where K=μπ/(30 tan θ) is a constant. Hence, the value of shear stress τ varies linearly with the rotational speed of the conical disk  14  and has no direct relationship with the location. If θ=0.5°, the value of K is 0.00804. On the other hand, if ε is not zero, the shear stress τ is a function of the radius r according to the following formula:  
               τ   =         KN   ⁡     (     1   +         ɛ   /     r   0       ⁢   cot   ⁢           ⁢   θ       r   /     r   0           )         -   1       ≡       f   ⁡     (     r   /     r   0       )       ⁢   KN         ,           (   4   )             
 
 where r 0  is the outer radius of the conical disk  14 . According to the function f (r/r 0 ), the smaller the radius r at a particular location, the greater will be the variation in the shear stress generated. 
 
         [0035]      FIG. 3  is a graph showing a function f relating the shear stress with the radius ratio for a conical disk having different distance of separation between the conical disk and a culturing vessel. As shown in  FIG. 3 , when the gap ε is 0.12 mm, the actual shear stress at a radius r equal to 1.5 cm from the center is only half of the expected value. Even at the rim of the conical disk, the actual shear stress is still only about 77% of the expected value. Hence, if an actual shear stress of more than 90% of the expected value in more than 80% of the area of the conical disk is desired, the gap must be carefully controlled to a value below 10 μm. In the following, the aforementioned operating principles are used to design a shear stress inducing apparatus according to one preferred embodiment of this invention.  
         [0036]      FIG. 4  is a diagram showing the basic structural elements of a shear stress inducing apparatus according to one preferred embodiment of this invention.  FIG. 5  is a diagram of an actual shear stress inducing apparatus according to one preferred embodiment of this invention. As shown in  FIGS. 4 and 5 , the shear stress inducing apparatus comprises a platform  43 , a culturing vessel  32 , a conical disk  41 , a stepper motor  44 , an upper cover panel  54 , a bottom base plate  50  and two side base plates  49 . A culturing vessel  32  is placed inside the apparatus in  FIG. 4  but the culturing vessel  32  is removed in  FIG. 5 .  
         [0037]     The culturing vessel  32  is placed on the platform  43 . The conical disk  41  is positioned horizontally inside the culturing vessel  32 . The central axis  58  of the conical disk  41  is positioned vertically relative to the bottom surface of the culturing vessel  32  and aligned roughly with the circular center of the culturing vessel  32 . In this embodiment, the conical disk has a conical slant angle of about 0.5°, for example. Furthermore, the platform  43  also has an opening  45  and a set of four screws  46  for stationing the culturing vessel  32 . The opening  45  facilitates easy access to the culturing vessel  32  and the replenishment of cultured solution to the vessel  32 . The four screws  46  serve to station the culturing vessel  32  to the platform  43 .  
         [0038]     The stepper motor  44  is connected to the conical disk  41  for driving the conical disk  41 . The shaft  40  of the stepper motor  44  is inserted to the hollow central shaft  58  of the conical disk  41  and locked in position using a screw  42  passing through a threaded hole in the wall of the hollow central shaft. When the stepper motor  44  is triggered, the conical disk  41  will rotate accordingly. Besides a stepper motor, other types of motors such as an alternating current or a direct current servomotor can be used instead. A stepper motor is selected in this embodiment mainly because of cost consideration.  
         [0039]     The upper cover panel  51  is set up between the stepper motor  44  and the conical disk  41 . The stepper motor  44  is fastened to the upper cover panel  51 . In this invention, the upper cover panel  51  furthermore comprises a sampling hole  52  that facilitates periodic sampling or replenishment of cultured solution without interfering with an ongoing experiment. In other words, a hole  52  suitable for sampling or adding solution is set up in the upper cover panel  51  between the outer diameter of the conical disk  41  and the culturing vessel  32 . Hence, an operator may sample cultured solution, say, via a syringe, from the vessel  32  or provide additional solution to the vessel  32  without interfering with an ongoing experiment.  
         [0040]     The bottom base plate  50  is set up underneath the platform  43  and the side base plates  49  are set up on each side of the platform  43 . The two side base plates  49 , the bottom base plate  50  and the upper cover panel  51  are joined together to form a base stand. The culturing vessel  32  and the conical disk  41  are housed inside the base stand. The upper cover panel  51  is fixed relative to the two side base plates  49  through a pair of oppositely positioned dowel pins  54   a  and locked to each other through a pair of oppositely positioned large thread-pitch screws  54   b . The bottom base plate  50  and the side base plates  49  are locked up together using four side screws  56 .  
         [0041]     The shear stress inducing apparatus may further include two acrylic observation windows  53  positioned on each side between the bottom base plate  50  and the upper cover panel  51  to facilitate experimental observation by operators. In this embodiment, the observation window  53  extends from a groove on the side base plates  49  and straddles between the bottom base plate  50  and each side of the upper cover panel  51 .  
         [0042]     Note that the conical disk  41  must have a fixed height for better operational stability. To adjust the height of the conical disk  41 , the platform  43  holding the culturing vessel  32  can be raised or lowered to vary the gap between the conical disk  41  and the bottom surface of the culturing vessel  32 . In this embodiment, the height-adjusting mechanism for the platform  43  is a threaded column  47  underneath the rotation platform  43 . The alignment between the central axis of the platform  43  and the conical disk  41  can be improved through increasing the outer diameter of the threaded column  47 . As shown in  FIG. 5 , the outer diameter of the threaded column  47  underneath the platform  43  is almost identical to the inner diameter of the platform  43  holding the culturing vessel  32 . The pitch of the threads in the threaded column  47  is 2 mm, for example. Hence, the platform  43  can be rotated to a topmost dead end position and then wound back 1.8°. With this manipulation, the gap between the conical disk  41  and the bottom surface of the culturing vessel  32  is set to 10 μm or smaller and the tip of the conical disk  41  is also prevented from scratching the bottom surface of the culturing vessel  32 . After setting the gap between the conical disk  41  and the bottom surface of the culturing vessel  32 , a height locking ring  48  underneath the bottom base plate  50  can be used to station the platform  43  at a fixed position.  
         [0043]     The shear stress inducing apparatus of this invention has a very simple structural design. Only a few components require shaping and manufacturing. In this embodiment, aside from the stainless steel conical disk  41 , all the other components are fabricated using aluminum alloy. This not only simplifies and speeds up the production process, but also lowers the production cost. After the fabrication of the components, the aluminum alloy surface is anodized to increase hardness and resist scratching between experiments. The few pieces of dimensionally different setting screws are also fabricated from stainless steel so that the entire apparatus can be used for prolonged periods under a high temperature and moist environment. Obviously, the various components constituting the shear stress inducing apparatus can be fabricated using a material other than aluminum alloy such as stainless steel.  
         [0044]     Since the shear stress inducing apparatus according to this invention has a simple structure that occupies a small volume (typically 15×15×15 cm 3 ), the apparatus has a high degree of operational stability. Furthermore, in-between experiments, the conical disk  41  can be taken out to perform a high-temperature, high-pressure disinfect operation. An operator only has to loosen the four screws  54   a ,  54   b , lift up the stepper motor  44 , upper cover panel  51  and conical disk  41  assembly and removing the conical disk  41  by loosening the setting screw  42 . To perform a detailed cellular analysis of the cultured solution in the culturing vessel  32 , the operator only has to loosen the four setting screws  46 , grasp the vessel  32  through the opening  45  and lift the vessel  32  up from the platform  43 . Conversely, to start a new experiment, the operator only has to reverse the preceding steps.  
         [0045]     In some shear stress experiments, the shear stress inducing apparatus may be required to operate for more than 48 hours and the apparatus may also be required to simulate the complicated shearing state of cells. Hence, the capacity to automate the shear stress inducing apparatus becomes important.  
         [0046]      FIG. 6  is a diagram showing an automatic shear stress inducing system according to one preferred embodiment of this invention. As shown in  FIG. 6 , the automatic shear stress inducing system comprise a temperature-controlled incubator  60 , a plurality of shear stress inducing apparatus  61 , a stepper motor driver  63 , a controller  65  and a multi-channel square-wave generation interface  64 .  
         [0047]     The temperature-controlled incubator  60  provides the apparatus  61  with a high-temperature and high relative humidity environment (for example, a relative humidity up to 97%). Preferably, the temperature-controlled incubator  60  has a volume of 60×60×60 cm 3 . Since each shear stress inducing apparatus  61  occupies a volume of 15×15×15 cm 3  only, a total of 27 shear stress inducing apparatus  61  (only nine is shown for clarity) can be placed inside the temperature-controlled incubator  60  concurrently. Because the shear stress inducing apparatus  61  has been described in detail before, no attempt is made to repeat the explanation here.  
         [0048]     The stepper motor driver  63  is electrically connected to the stepper motor of each shear stress inducing apparatus  61  through stepper motor electric cables  62 . Furthermore, the multi-channel square-wave generation interface  64  is coupled to the stepper motor driver  63  and the controller  65  is coupled to the multi-channel square-wave generation interface  64 . The controller  65  is a micro-controller, a personal computer or a Digital Signal Processor (DSP), for example.  
         [0049]     The automatic shear stress inducing system operates according to a program. The program activates the controller  65  to send square-wave frequency value F (Hz) to the multi-channel square-wave generation interface  64 . Thereafter, the square wave for each channel is transmitted to the stepper motor driver  63  for controlling the rotating speed N=60F/n 0  of the stepper motor in each shear stress inducing apparatus  61 , where n 0  is the number of steps for rotating the stepper motor one revolution. In the meantime, the controller  65  also submits a positive/inverted signal to each channel in the stepper motor driver  63  for controlling the direction of rotation (positive or negative rotation) of the stepper motor in each shear stress inducing apparatus  61 .  
         [0050]     In this embodiment, there are altogether  11  most basic experimental shear stress parameters including 5 parameters for positive rotation in each cycle, 5 parameters for negative rotation in each cycle and a parameter registering the total experimental period. The 5 positive rotation parameters include the maximum rotating speed, the time required to accelerate the motor from a stationary state to the maximum rotating speed, the time stationed at the maximum rotating speed, the time required to decelerate the motor from its maximum rotating speed to a complete stop and the waiting period at the completion of the positive rotation. Similarly, the 5 negative rotation parameters include the same 5 parameters as the positive rotation except for a difference in value and the direction of rotation.  
         [0051]      FIG. 7  is a diagram showing the timing and parametric variation of shear stress in an automatic shear stress induction apparatus. As shown in  FIG. 7 , the shear stress in the positive and the negative direction are τ +  and τ − , and the period in which a constant shear stress is maintained are t c+  and t c−  respectively. The period in which the conical disk starts from a stationary state to one producing a shear stress τ +  is τ t+ . Similarly, the period in which the conical disk decelerates from a state having a shear stress τ +  to a stationary state is τ f+ . In the reverse direction, the rising period and the falling period for generating a shear stress τ −  are t r−  and t f−  respectively. There are two periods for the conical disk to remain at rest at the end of each positive and negative shear stress production, namely, t 0+  and t 0−  respectively. Finally, the total period for the apparatus to carry through the entire experiment is t T .  
         [0052]     Using the shear stress inducing apparatus and the automatic shear stress inducing system of this invention, the shear stress within 80% of the area at the bottom of the culturing vessel is accurately controlled with an experimental error smaller than 10%. In addition, the rotating speed of the conical disk can be programmed to function automatically and the shear stress can be set to within the tolerable range of ±2.0 Pa for normal cells. Hence, the system can be used to simulate various types of stress patterns and find out its effects on the physiologic development of the cells inside a human body. Moreover, to simulate the actual environment inside a human body, the shear stress apparatus is designed to operate inside a temperature-controlled incubator set to a high relative humidity (97% RH) and temperature for long periods. The shear stress inducing apparatus is also designed to occupy a small volume (approximately 15×15×15 cm 3 ) and operate with great stability. Thus, a maximum of 27 sets of shear stress inducing apparatus can be placed inside a temperature-controlled chamber (60×60×60 cm 3 ) to perform a large number of experiments simultaneously. Each shear stress apparatus is also provided with a hole for sampling cultured solution from the vessel periodically to investigate and analyze cellular growth without interfering with the on-going experiment. The apparatus also has such a simple design that it is almost maintenance free. Furthermore, the conical disk can be taken out of the shear stress inducing apparatus for disinfections prior to starting another experiment simply by loosening 5 screws. In other words, the apparatus has all the features necessary for speeding up experimental investigation according to the researchers&#39; need.  
         [0053]     The shear stress inducing apparatus and the automatic shear stress inducing system of this invention has many practical uses in research. For example, the invention can be used for investigating the physiological reaction of cartilaginous cells in a rheumatic state when subjected to various patterns of shear stress and the physiological condition under which the cells start to die. In addition, this invention may combine with DNA micro-array technique to find the controlling genes inside a cell vulnerable to shear stress. The invention can also be used to pre-condition cultured cells and select only those shear-resistant cells prior to a tissue engineering investigation. Furthermore, the shear stress resistant cells selected for carrying out tissue engineering can be thoroughly inspected to serve as quality control and product identification standard. Aside from cartilaginous cells, the invention can be applied to investigate the effect of shear stress due to blood flow on the physiology of cells inside blood vessels.  
         [0054]     In summary, major advantages of this invention includes: 
        1. The shear stress inducing apparatus and automatic shear stress inducing system is able to produce very accurate shear stress so that the experimental error can be reduced to a minimum.     2. The rotating speed of the conical disk inside the apparatus can be programmed to simulate or accelerate the actual shear stress condition inside a human body and find out its effect on the physiological state of the cells.     3. The shear stress inducing apparatus can be put inside a temperature-controlled incubator set to a high relative humidity and temperature to simulate the environment inside the human body.     4. The shear stress inducing apparatus occupies a small volume, has a high operational stability and is cheap to produce. Moreover, a typical temperature-controlled incubator is able to house a considerable number of apparatus so that large number of experiments can be carried out within a short time.     5. The shear stress inducing apparatus is also provided with a sampling hole for sampling cultured solution from the vessel periodically and performing a cellular product inspection or analysis.     6. The shear stress inducing apparatus has a very simple maintenance free structure. Furthermore, the conical disk can be easily taken out of the apparatus for disinfections prior to a fresh round of experiment.        
 
         [0061]     It will be apparent to those skilled in the art that various modifications and variations can be made to the structure of the present invention without departing from the scope or spirit of the invention. In view of the foregoing, it is intended that the present invention cover modifications and variations of this invention provided they fall within the scope of the following claims and their equivalents.