Abstract:
A cell incubating apparatus and a method of culturing cells in vitro are disclosed, which relates to medical cell proliferation, pharmaceutical chemical 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 cells. The apparatus is not only for incubating the tissue specific cells with biological function but also for lowering the cost of culturing cells for personal medical use.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The invention is related to a cell incubating apparatus and the method of culturing cells in vitro, and especially, relates to a medical cell proliferation, pharmaceutical chemical screening and automatic cell analysis in tissue engineering technology. 
     2. Description of Related Art 
     In the field of tissue engineering, cell transplantation will play an important role in the future. Currently, the cell propagation apparatuses, which comprise not only for traditional laboratory cell incubating techniques but also for traditional bioreactors, have limitations 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 bioreactors to produce personal cells is expensive; therefore, it is difficult industrializing cell production for personal cell therapy. 
     Massachusettes Institute of Technology and Massachusetts General Hospital in the United States disclosed a system containing micromatrix, 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. 
     Therefore, it is desirable to provide an improved method to mitigate the aforementioned problems. 
     SUMMARY OF THE INVENTION 
     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. 
     Another purpose of the present invention relates to a method of culturing cells in vitro. This method will establish an imitating three-dimensional transportation microenvironment, which assists to the formation of cell cytokine/chemokine gradient and the reactions between different types of cells. The cell incubating apparatus of the present invention is not only for incubating the tissue-specific cells with biological functions but also for lowering the cost of culturing cells for personal medication. 
     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, and a plurality of fluid paths; and a bottom plate for collecting the fluid from the top plate and the culture plate. Among them, the incubating units and the fluid paths are located on the surface of the culture plate faced to the top plate. The fluid paths connect the incubating units and the receiving part. The receiving part overlaps the input hole of the top plate to receive the fluid from the input hole. An output hole is located in each incubating unit for draining away excessive 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 of the top plate, passes along the fluid path then flows into each incubating unit, and arrives in the bottom plate from the output hole in each incubating unit. 
     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, and a plurality of fluid paths; and a bottom plate for collecting the fluid form the top plate and the culture plate as illustrated above; (B) coating extracellular matrix or seeding stromal cells to cover the surface of the incubating unit; and (C) filling in a fluid into the input hole on the top plate, and the fluid flows to the bottom plate by passing the receiving part, the incubating units, the fluid paths, and the output hole of the culture plate. 
     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 
         FIG. 1  is an exploded perspective view of the present invention relating to an incubating cell apparatus 
         FIG. 2  is a perspective view of an assembly of the present invention relating to an incubating cell apparatus 
         FIG. 3A  and  FIG. 3B  are the morphology of HepG2 cells incubated in fluidic condition. 
         FIG. 3C  and  FIG. 3D  are the morphology of HepG2 cells incubated in a static condition. 
         FIG. 4  is transimission electron microscope image of HepG2 cells cultured in fluidic setting. Microvilli formed have been observed. 
         FIG. 5  is an exploded perspective view of another embodiment of the present invention wherein the incubating cell apparatus comprises a biological membrane. 
         FIG. 6  is a perspective view of another embodiment of the present invention apparatus, which further comprises a transporting tract for the fluid collecting. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     The present invention relates to a cell incubating apparatus preferably comprising a bottom plate, which is formed with a plurality of micro paths overlapping the output holes of each incubating unit on the culture plate for converging and collecting 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 of the bottom plate and the input hole of the top plate for refluxing the fluid back to the input hole. Preferably, a spill hole and a through hole is formed on the top plate and the culture plate respectively, which is located at the relative location of collecting trough of the bottom plate, whereby the collecting trough connects to the through hole, the spill hole and the input hole on the top plate with the transporting tract. 
     The present invention discloses an incubating apparatus, wherein the top plate preferably comprises a plurality of apertures which overlap each incubating unit of the culture plate for testing the interaction between different cells, more preferably, a thin biologically compatible membrane is provided between the top plate and the culture plate as an interface for interaction between cells. The invention can optionally include at least one micro fluid path. The micro fluid path is formed in each incubating unit on culture plate to imitate blood capillaries between cells. 
     Depending on the requirements, the incubating units also can be seeded and covered by the stromal cells 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 present invention also relates to a method for incubating cells in vitro, which comprises, preferably, one transporting tract to connect the collecting trough and the input hole for refluxing the fluid back to input hole of the top plate. The method of incubating cells in vitro, more preferably comprises a plurality of apertures on the top plate, which overlap each incubating unit of the culture plate in order to analyze the interaction between 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 incubating units for providing an interface for cellular interaction. The invention relates to the method of incubating cells in vitro, which even more preferably comprises forming at least one micro fluid path on the incubating unit to imitate blood capillaries. 
     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. 
     With reference to  FIGS. 1 ,  2  and  5 , the invention of the cell incubating apparatus comprises a top plate  10 , a culture plate  20  and a bottom plate  30 . The culture plate  20  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 incubating units  21 , a plurality of fluid paths  22 , and a through hole  23 . The fluid paths  22  are connected with each incubating unit  21 . The fluid paths  22  are provided with a receiving part  221  to receive the fluid from the incubating apparatus and a fluid is injected into the receiving part  221  via the input hole  11 . 
     Each incubating unit  21  comprises a circular hole  211  for separating the flowing. The circular hole  211  connects to a 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  50  between the culture well  213  of the culture plate  20  and the aperture  12  of the top plate  10  to be an interface for cell interaction, as shown in  FIG. 5 . 
     The surface of bottom plate  30  is formed with a plurality of micro paths  31  overlapping the output hole  214  of the culture plate  20  and connects to a collecting trough  32  of the bottom plate  30 . 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 . 
     With reference to  FIGS. 1 ,  2  and  6 , 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  when the apparatus of the present invention is assembled. By using a transporting tract  14  connects to the input hole  11 , the spill hole, the through hole  23  and the collecting trough  32 , the fluid can be collected from the collecting trough  32  of the bottom plate  30  into the input hole  11  of the top plate  10 . 
     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 for 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, a culture solution is filled from the input hole  11  into the top plate. The culture solution flows along the fluid path  22  and then respectively flows into the micro fluid path (not shown in all the figures), 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 each culture well  213 , after which the filling procedure is begun. The cells circulate in the fluid paths to 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 a certain 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. 3A˜3D , HepG2 cells were cultured respectively in the present invention and a traditional cell incubating apparatus. The cell culture result of the traditional incubating apparatus with stationary culture technique shows some circular morphologies in  FIG. 3C  and  FIG. 3D . The cell incubating apparatus of the present invention displays an island shape of cell morphology after incubating and is also very similar to the physical cellular structure. 
       FIG. 4  is a transmission electron microscopic graph of cells after undergoing the flowing incubating method of the present invention, 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 contains microvilli and tight junctions, which are the specialized morphology of hepatic tissue. The result shows that the cell-culture environment supplied by the present invention can provide the ability of imitating real physical environment. 
     To perform the analysis procedures, for example, cytokines/chemokines in the system keep released from the cells under stimulations, the cell incubating apparatus of the present invention will transport the fluid from the collecting trough  32 , pass through the through hole  23 , spill hole  13  and transporting tract  14  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 screening cellular activity by a 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 types of cells. If the cells are the same, the cell incubating apparatus of the present invention can be used 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. 
     In reference to tissue engineering, the cell related products need to be highly personalized; the cell incubating apparatus of the present invention is also needed to combine with biomedical 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 chemical 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 3D physical simulating system that can display the real cellular activity in vivo and also help to form cell cytokine/chemokine gradient and heterocellular 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 cost of production, 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. 
     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.