Abstract:
A cell incubating apparatus and a method of culturing a cell in vitro are disclosed, which relates to a medical cell proliferation, pharmaceutical screening and automatic cell analysis in tissue engineering technology. The apparatus establishes an imitating three-dimensional transportation microenvironment by assembling a top plate, a bottom plate and at least one culture plate apparatus for incubating a cell. The apparatus is not only for incubating the tissue specific cell with biological function but also for lowering the cost of culturing personal medical cells.

Description:
BACKGROUND OF THE INVENTION  
       [0001]     1. Field of the Invention  
         [0002]     The invention is related to a cell incubating apparatus and the method of culturing a cell in vitro, and especially, relates to a medical cell proliferation, pharmaceutical screening and automatic cell analysis in tissue engineering technology.  
         [0003]     2. Description of Related Art  
         [0004]     In the field of tissue engineering, cell transplantation will play an important role in the future. Currently, the cell expansion apparatuses, which comprise not only for traditional laboratory cell incubating techniques but also for traditional bioreactors, have limitation for incubating a medical cell source. The shortcoming of the traditional laboratory cell incubating techniques mentioned above is the lack of a mass transporting system and difficult to display the real physiological activities ongoing inside the cell body. The bioreactor is only suitable in protein drug production as in, for example, the production volume of the stromal cells and fibroblasts with better tolerance for the above shortcoming. The bioreactor is excessively large and difficult to operate, has large fluidic shear force, lacks micro-environments similar to the physiological environment, and is impossible to incubate some tissue specific cells so far, for example, hepatocytes. Besides, using these bioreactor to produce personal cells is expensive, therefore, it is difficult industrializing cell production for personal cell therapy.  
         [0005]     Massachusettes Institute of Technology and Massachusetts General Hospital in the United States disclosed a system containing micromaxtrix, an injection unit and sensors in WO9947922. The system is mainly used in pharmaceutical screening and analysis. However, even through the system illustrated above can maintain the cell function in vitro, the cells in the system forms a cell mass which is hard to isolate and expand. In the patent pended by University of California at San Diego, U.S. Pat. No. 20020072116, disclosed a nanoporous silicon bioreactor which is used for pharmaceutical screening such as analysis for the biological activity, metabolism, toxicity, mutagenicity or carcinogenicity of compounds. However, such a bioreactor is only for maintaining the function in vitro and not fit for cell proliferation.  
         [0006]     Therefore, it is desirable to provide an improved method to mitigate the aforementioned problems.  
       SUMMARY OF THE INVENTION  
       [0007]     The cell incubating apparatus of the present invention relates to a cell culture device that can establish an imitating three-dimensional mass transportation microenvironment, which assists the formation of cell cytokine gradient and the heterocellular reaction. The cell incubating apparatus is not only for incubating the tissue specific cells with biological function but also to lower the cost of cell production for personal medical treatment.  
         [0008]     Another purpose of the present invention relates to a method of culturing cell in vitro. This method will establish an imitating three-dimensional transportation microenvironment, which assists to the formation of cell endocrine gradient and the reaction between different types of cells. The cell incubating apparatus of the present invention is not only for incubating the tissue-specific cell with a biological function but also for lowering the cost of culturing cells for personal medication.  
         [0009]     To achieve the purpose above, the cell incubating apparatus of the present invention, which cooperates with a fluid, comprises: a top plate having an input hole; a culture plate having a plurality of incubating units, a receiving part, an output hole, and a fluid tract; and a bottom plate for collecting the fluid form the top plate and the culture plate. Among them, the incubating units and the fluid tract are located on at least one surface of the culture plate. The fluid tract connects the multiple culture units, and the receiving part. The receiving part overlaps the input hole in the top plate to receive the fluid from the input hole. The output hole is located in the incubating unit for outputting the fluid. Moreover, the culture plate is sandwiched between the top plate and the bottom plate as assembled, the fluid flows into the receiving part on the surface of the culture plate from the input hole, passes along the flow path then flows into the culture units, and arrives in the bottom plate from the output hole.  
         [0010]     The method of the present invention comprises following steps: (A) providing an apparatus comprising a top plate having an input hole; a culture plate having a plurality of incubating units, a receiving part, an output hole, and a fluid tract; and a bottom plate for collecting the fluid form the top plate and the culture plate as illustrated above; (B) seeding stromal cells to cover the culture unit; and (C) filling in a fluid into the input hole on the top late, and the fluid flows to the bottom plate by passing the receiving part, the culture unit, flowing path, and the output hole.  
         [0011]     Other objects, advantages, and novel features of the invention will become more apparent from the following detailed description when taken in conjunction with the accompanying drawings. 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0012]      FIG. 1  is an exploded perspective view of the present invention relating to an incubating cell apparatus  
         [0013]      FIG. 2  is a perspective view of an assembly of the present invention relating to an incubating cell apparatus  
         [0014]      FIG. 3A  and  FIG. 3B  are the morphology of HepG2 cells incubated in fluidic condition.  
         [0015]      FIG. 3C  and  FIG. 3D  are the morphology of HepG2 cells incubated in a static condition.  
         [0016]      FIG. 4  is transimission electron microscope image of HepG2 cells cultured in fluidic setting. Microvilli formed have been observed.  
     
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT  
       [0017]     The present invention relates to a cell incubating apparatus preferably comprising a surface of a bottom plate, which is formed with a plurality of micro paths overlapping the output holes for collecting or converging the fluid. More preferably, the bottom plate surface is provided with a collecting trough connected to the micro paths for collecting the fluid. The invention of the incubating apparatus comprises, even more preferably, a transporting tract that connects to the collecting trough and input hole for refluxing the fluid back to the input hole, or the relative location of collecting through the culture plate and the top plate, preferably formed with one spill hole and one over through hole, whereby the collecting trough extends through the through hole, the spill hole and the transporting tract and then connects to the input hole. The present invention discloses an incubating apparatus, wherein the top plate preferably comprises a plurality of apertures overlapping the culture units for testing the interaction between different cells, more preferably, a thin biologically compatible membrane is provided between the top plate and the bottom plate as an interface for interaction between cells. The invention can optionally include at least one micro flowing path. The micro flowing path is formed with a micro path system, which is similar to capillaries. Depending on the requirements, the culture unit also can be seeded and covered by the stromal cell for facilitating the attachment of the cells. The material of the top plate, culture plate and bottom plate of the incubating apparatus of the present invention can be made of any traditional material with machinability. Preferably, the material of the top plate, culture plate and bottom plate are transparent material, more preferably, the material of the top plate, culture plate and bottom plate are acrylic, polycarbonate (PC) or polydimethylsiloxane (PDMS). The invention also relates to a method in incubating a cell in vitro, which comprises, preferably, one transporting tract to connect a collecting hole and an input hole for refluxing the fluid back to input hole. The method of incubating a cell in vitro, more preferably comprises a plurality of apertures overlapping the culture units on the top plate in order to analyze the interaction in different cells. More preferably, the apparatus used in the method of the present invention is provided with a biological membrane located between the apertures and the culture units for providing an interface of cell interaction. The invention relates to the method of incubating a cell in vitro, which even more preferably comprises forming at least one micro path on the culture unit to imitate a micro path system similar to blood capillary. e. The materials of the top plate, culture plate and bottom plate included in the present method are preferably made of transparent material, and more preferably are made of acrylic polymers, PC or PDMS. The method of the present invention comprises preferably a step of using one illuminant on the incubating apparatus in order to observe the cellular activity.  
         [0018]     With reference to  FIG. 1 , the invention of the incubating cell apparatus comprises a top plate  10 , a culture plate  20  and a bottom plate  30 . The bottom plate  30  is sandwiched between the top plate  10  and the bottom plate  30 . The top plate  10 , culture plate  20  and bottom plate  30  are respectively formed with screw holes  15 ,  25 , and  35  in order to fasten them together using securing elements such as screws. The top plate  10  is provided with an input hole  11 , a plurality of apertures  12  and a spill hole  13 . The surface of culture plate  20  is formed with a plurality of culture units  21 , a flow path  22 , which is connected to each culture unit  21  and a through hole  23 . The flow path  22  is provided with a receiving part  221  to receive the fluid from an incubating apparatus and the fluid is injected into the receiving part  221  via the input hole  11 . The culture units  21  each comprise a circular hole  211  for separating the flowing. The circular hole  211  connects to the culture well  213  with a strike hole  212  and the culture well  213  is provided with an output hole  214 . After the fluid is poured into the culture plate  20 , the fluid passes through the output hole  214  and then directly flows down to the bottom plate  30 . The culture well  213  on culture plate  20  overlaps the aperture  12  on the top plate  10 . Respective different cells in aperture  12  and culture well  213  are allowed to culture for testing heterocellular interaction. The culture plate also can be seeded or covered with a biological membrane between the culture well  213  and the aperture  12  to be an interface of cell interaction. The surface of bottom plate  30  is formed with a plurality of micro paths overlapping the output hole  214  and a collecting trough  32 . The output hole  214  connects with the micro path  31 . The micro path  31  can converge the fluid from output hole  214  and collect in the collecting trough  32 . The location of the collecting trough  32  aligns with the spill hole  13  on top plate  10  and the through hole  23  on the culture plate  20 . By using one side of the micro path  31  connecting to the input hole  11  and the other side of that connecting to the transporting tract of the collecting trough (not shown in the figure), which extends through the spill hole  13  and the through hole  23 , the fluid collecting from the collecting trough  32  can be refluxed into the input hole  11 .  
         [0019]     When performing the cell culture, the cells are loaded first. The cell incubating apparatus of the present invention is provided with several possible methods for cell loading, for example, the extracellular matrix is coated on the culture well  213  first and the cells are seeded. Cells are allowed to attach after 24 hours, then top plate  10 , culture plate  20  and bottom plate  30  are assembled and secured with a screw  40  to form a cell incubating apparatus as shown in  FIG. 2 . After the apparatus has been assembled, the culture solution is filled from the input hole  11  into the top plate. The culture solution flows along the flow path  22  and then respectively flows into the micro path, which is similar to capillaries, and the culture well  213 . After the culture plate  20  has been filled with the culture solution, the culture solution vertically flows down to the micro path  31  on the bottom plate  30  and then converges into the collecting trough  32  to form micro fluids that is similar to capillaries, in order to proceed the mass transfer, or build up the cell incubating apparatus first after coated with the extracellular matrix on culture well  213 , after which the filling procedure is begun. The cells circulate in the flowing path and obtain a higher cell seeding density. Then the flowing speed is reduced to zero, and wait for 4-6 hours, and then the filling procedure is carried out at low speed. After cells loading into the present invention of cell incubating apparatus as mentioned above, the filling procedure is started at different speeds and different compositions of each culture plate. After completion of the decided time period, for example, after 1, 2, 5 or 7 days, it can be observed by a microscope if there is any morphological changes occurred in cell structure. As shown in FIGS.  3 A˜ 3 D, HepG2 cells were cultured respectively in the present invention and some prior art of cell incubating apparatus, the result shows that to compare to culture cell in the prior art of stationary culture technique, which forms some circular structure in  FIG. 3C  and  FIG. 3D . The cell incubating apparatus of the present invention displays an island shape of cell structure in the present cell incubating invention and is also very similar to the physical structure.  FIG. 4  is a transmission electron microscopic graph of cells after undergoing the flowing incubating method, wherein microvilli is located in the left bottom circle, and a tight junction is located in the right top circle. It is shown that the cultured cell structure is provided with microvilli and tight junction, which are the specialized structure taken from hepatic tissue. The result shows that the cell-cultured environment supplied by the present invention is provided with the ability of imitating real physical function.  
         [0020]     To perform the analysis procedures, for example, the growth factors in the system keep released when cells are under stimulations, the cell incubating apparatus of the present invention will transport the fluid from the collecting well  32 , pass through the through hole  23 , spill hole  13  and transport tract to the transporting hole  11 . Then the fluid will circulate in the path of the present invention mentioned above. In the meantime, for the preferable example, the material of top plate  10 , culture plate  20  and bottom plate  30  are transparent PC. The cell incubating apparatus of the present invention can be used for testing cellular activity by photon detection system as illustrated by prior arts. On the other hand, the present invention is also provided with a plurality of culture plates between the top plate and the bottom plate; each culture plate can incubate the same or different cells. If the cells are the same, the cell incubating apparatus of the present invention can be expanded for cell proliferation. If the cells are different, the cell incubating apparatus of the present invention can be used for the observation of cell-cell interaction between the cells.  
         [0021]     In reference to tissue engineering, the cell related products need highly personalized; the cell incubating apparatus of the present invention is also needed to combine with biomedica and scale-up technologies beneficial for marketing purpose. The present invention combines the cell physiology knowledge with industrialization purpose, and develops the new generation of bio-mimetic cells incubating system, which is provided with high specification, high density and also mimics the mass transportation system within cells, in order to apply in cell proliferation for medical, pharmaceutical screening and automatic cell analysis. The present invention relates to a novel technique in tissue engineering, which is applied with bio-mimetic techniques in a tissue-specific cell-simulating physiological system. The present invention is in an advantage of the  3 D physical simulating system that can display the real cellular activity in vivo and also help to form cell cytokine/chemokine gradient and hetrocellular interactions, in order to culture the tissue-specific cells for a delicate microenvironment. The cell incubating apparatus of the present invention can also reduce the production cost, provide a manufacture process with an industrialized capability, and in addition can be designed for multiple productions, for disposable productions. The apparatus will proceed to manufacture the reaction system with low production cost and high additional value for future personal medical application.  
         [0022]     Although the present invention has been explained in relation to its preferred embodiment, it is to be understood that many other possible modifications and variations can be made without departing from the spirit and scope of the invention as hereinafter claimed.