Abstract:
A micro device for cell culture is disclosed, which cooperates with a fluid and includes: a top plate having an inlet port; a orifice plate having a plurality of orifices; a culture plate having a plurality of culture wells and a plurality of injection ports; and a bottom plate having at least one collecting well and at least one collecting flow channel, wherein, the culture plate is placed between the orifice plate and the bottom plate. The collecting flow channel connects to all regulating orifices in the culture wells and guides the fluid from the culture wells, then receives the fluid in the collecting well. The fluid flows into the orifice plate from the inlet port of the top plate, and then diversifies into the culture plate, then arrives at each culture well by way of the injection ports, and finally collects in the collecting well of the bottom plate.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to a micro device for cell culture and, more particularly, to a micro device combining control systems that provide gradient formation of bio-molecules which imitates the physical tissue environment in vivo while culturing cells in vitro. 
     2. Description of Related Art 
     Cell culture techniques are basic in vitro tools used for studying varies biological or biomedical phenomena. During early stage of drug development, for example, cell culture techniques are frequently used to predict the metabolism and toxicology of drug candidates, or xenobiotics, instead of animal model experiments. The advantage of cell culture model is convenient and can used for speeding up the screening process. 
     Generally, current cell culture methods can be classified into several groups: (1) conventional culture without fluidic environments, (2) fluidic culture with single-direction laminar flow. (3) cell culture on 3-D scaffold composed of biomaterials or extra-cellular matrix. Limitation of conventional static culture includes lack of mass transformation system of bimolecular and appropriate micro-architecture. As for single-direction laminar flow, which unable to provide more sophisticated fluidic pattern or gradient formation of bio-molecules that imitate physiological environment in vivo. In summary, a reliable model to represent the physical activities of cells is still required for most predictive study of drug or xenobiotic metabolism and toxicity. 
     A good in vitro model is pivotal to discover the potential of drug candidates as well as to predict xenobiotic metabolism for the studies of environmental toxicology. A cell culture system with micro-architecture cell culture unit which imitating the physical environment, three-dimensional fluidic pattern and dynamic fluidic control system will provide promising platform for predictive study during drug development in vitro. 
     SUMMARY OF THE INVENTION 
     The object of the present invention is to provide a micro device for culturing tissue-specific cells. The micro device of the present invention provides incubating conditions that imitate the physical environment in vivo. Further, the advantages of the present invention are to control the cytokines secretion by control of concentration gradient that mimic microenviroment of liver tissue, create opportunities for cell interaction between different types of cells, hepatocytes and non parenchymal cells, for example. In addition to function as a single culture device, the micro device can be further produced in batches format by disposable materials at low cost, to provide a personal medical device with great benefit. 
     The present invention provides a method for generating pressure and concentration gradients in a cell culture well, comprising introduces a radially-inward fluid or fluids from discrete injection ports on the peripheral of the culture well, and the radially-inward fluid flows toward the central regulating orifices of the culture well to generate pressure and concentration gradients. The term of fluid used in the specification includes liquid or gas or both. 
     The present invention also provides a micro device for cell culture, comprising one or more culture wells; a plurality of discrete injection ports on the peripheral of the culture wells; one or more regulating orifices on the wall of the culture wells; wherein a fluid or fluids were introduced from the discrete injection ports and the fluid(s) radially-inward flow toward the one or more regulating orifices on the wall of the culture wells. 
     The micro device for cell culture of the invention cooperates with a fluid and includes: a top plate having an inlet port; a culture plate having one or more culture wells and one or more injection ports which are able to form a specific fluidic pattern with gradient of nutrients, bimolecular, for example, through a programmed control system connect culture wells with at least one regulating orifice to drain the fluids from the culture wells; and a bottom plate having at least one flow channel. 
     Another embodiment of the micro device for cell culture of the present invention includes: a top plate having at least one inlet port; a orifice plate having a plurality of orifices; a culture plate with a plurality of culture wells and a plurality of injection ports formed on one surface of the culture plate. The injection ports connect the culture wells and orifices on the orifice plate, and the culture wells contain at least one regulating orifice to drain the fluids from the culture wells. A bottom plate has at least one collecting well and at least one flow channel. The culture plate is fixed between the orifice plate and the bottom plate in assembly. The flow channels connect to all regulating orifices in the culture wells, and guide the fluids from the culture wells to the collecting well. Furthermore, the flow channels on the bottom plate can be the collecting well. 
     In the micro device for cell culture of the invention, the fluid flows into the orifice plate from the inlet port, diversifies into a plurality of culture plates via injection ports, and finally arrives at the collecting well of the bottom plate. 
     In this invention, the flow channels and the collecting well can be formed on the surface of the bottom plate which faces the culture plate. Alternatively, the flow channels and the collecting well can be formed on the surface of the culture plate which faces the bottom plate, and the flow channels and the collecting well will be formed when the culture plate is assembled with the bottom plate. 
     The collecting well on the bottom plate can be directly connected with an outlet port in order to drain out the fluid from the collecting well. Alternatively, outlet ports can be defined in the top plate, orifice plate, and culture plate. While assembling the components, each outlet port and the collecting well on the bottom plate are connected so that back-flow of the fluid from the collecting well to the top plate and drain out from the outlet ports on the top plate is facilitated. As a result, the fluid in the collecting well on the bottom plate is circulated, and the objective of full utilization is achieved. 
     In the micro device for cell culture of the invention, a buffer zone is formed to temporarily reserve the fluid from the top plate when connecting the top plate with the orifice plate. This buffer zone can be either on the top plate facing one surface of the orifice plate, or on the orifice plate facing one side of the top plate. 
     The utility of the orifice plate of the present invention depends on requirements, and there is no limitation for the structure of the orifice plate. A better result can be achieved when a plurality of orifices is formed, and each orifice connects with the injection ports and the buffer zone area which is formed when the top plate and the orifice plate are connected, and thus facilitates the fluid in the buffer zone area to diversify to culture plates. 
     A plurality of culture wells is formed on the culture plate with no limitation for the appearance. However, the shape of a circle or polygon is preferred, and more preferably, is hexagonal. The location of the injection ports connecting the culture wells has no limitation. Preferably, the injection ports of the culture plate are formed on the comers of the hexagon-shape culture wells, to receive the fluid from the orifice plate evenly into each culture well. 
     The culture wells in this micro device for cell culture of the invention achieve a very successful result by using bio-compatible material to form micro-patterning, or scaffolds can be applied to each culture well to culture cells with different characteristics. 
     Before using the culture wells of the invention, a matrix can be attached to facilitate cell culture. 
     In order to increase the quantity of cell culture, this micro device can be connected with more than one device to conduct mass cell culture at the same time and in the same environment. No rules are applied when connecting each micro device; however, serial or parallel connection is recommended. 
     A plurality of culture units can also be combined within one single micro device in which the quantity of cell cultured is enlarged. Each culture unit includes a orifice plate and a culture plate placed between the top plate and the bottom plate. Preferably, each cell culture unit includes a dividing plate to separate each culture unit. The aforementioned dividing plate has an outlet hole and a center slot with a plurality of orifices. Each orifice is connected with the injection ports on the culture plate of the culture unit. 
     This micro device performs even better when including a mechanical engineering control system and a biosensor. The mechanical engineering control system can manipulate the variety of fluids, the flow speed, the supply period, and the supply volume. Preferably, micro-patterning is formed inside each culture well. The structure of micro-patterning with at least one recess can be formed with bio-compatible materials by photosensitive procedures. Or, a pattern with at least one recess can be constructed in the culture wells by bio-compatible materials or bio-matrix, to control the growth and arrangements of cells. 
     The orifice plate of the invention decreases the probability of concentration variation and shear force occurring around the inlet port while the fluid flows into an culture well. The cooperation of orifices on the orifice plate and a hexagon-shape culture well achieves the best flow field effect, e.g. the shear force occurs more evenly when fluid flows from the six comers of the hexagon toward the center. Furthermore, the concentration gradient is formed within the cell culture wells by controlling the strength of inputting the fluid. The phenomenon reflects the concentration difference when a cell grows. For example, the concentration gradient of metabolism activity is different from central vein to portal vein of liver cells. The micro device for cell culture of the present invention can imitate the cell growing activity in physical environment in vitro. 
     The present invention also includes a fluid control system which cooperates with a micro device for cell culture including: at least one pump; at least one valve; and one control unit. The micro device for cell culture contains a top plate having at least one inlet port, a orifice plate, a culture plate, and a bottom plate. The valve of the fluid control system connects at least one fluid supply unit and an inlet port on the top plate of the micro device for cell culture. The control unit connects the pump and valve to control the incoming and outgoing fluid. 
     By the fluid control system of this invention, different biomoleculars and drug delivery unit can be connected and automatically controlled to initiate or stop bio signal transmission at a sandwiched time interval. Moreover, a biosensor can also be connected to conduct feedback control on the returned signal. The biological systems can imitate how it transmits the growing factor to control cell behavior, and the long-term cultured cells can be in a more dynamic and varying environment. Therefore, the restraints or changes of the related metabolism system caused by growth factor concentrations degradation or unmorally high are avoided. 
     The present invention also includes a cell culture system comprises the micro device as described above; a microscopy or other image capture unit drug delivery unit; a medium transfer unit; a gas or air supply unit; and a temperature control unit. 
     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 drawing of the micro device for cell culture in example 1; 
         FIG. 2  is a view of the micro device for cell culture in example 2 after assembling; 
         FIG. 3A  is an exploded drawing of the micro device for cell culture in example 2; 
         FIG. 3B  is an exploded drawing of the micro device for cell culture in example 3; 
         FIG. 3C  is an exploded drawing of the micro device for cell culture in example 4; 
         FIG. 3D  is view of the micro device for cell culture after assembling in example 4; 
         FIG. 4  is the imitating result of the fluid field in each culture well of the present micro device for cell culture; 
         FIG. 5  is the pressure change in each culture well of the present micro device for cell culture; 
         FIG. 6A  illustrates the parallel connection of micro devices in example 7; 
         FIG. 6B  illustrates the serial connection of micro devices in example 7; 
         FIG. 6C  illustrates another serial connection of micro devices in example 7; 
         FIG. 7A  shows the explosive drawing of the expanded culture unit of a micro device in example 8; 
         FIG. 7B  shows the explosive drawing of another expanded culture unit of a micro device in example 8; and 
         FIG. 8  shows a diagram of an automatic control system with the present micro device for cell culture. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
     The device in this invention has many kinds of combination as illustrated in the following. 
     EXAMPLE 1 
       FIG. 1  is a detailed diagram of the micro device for cell culture. Four components are included which are: a top plate  10 , a orifice plate  40 , a culture plate  20 , and a bottom plate  30 . Each component has a plurality of screw holes  51  in which screws  50  are used to connect each component. In this example, eight screw holes  51  are formed on each component, to tightly connect with four components by screws  50 . Therefore, cells cultured in the micro device can be prevented from being contaminated through external contacts during the culturing process. 
     An inlet port  11 , an outlet port  12 , and a buffer zone  13  are formed on the surface of the top plate  10 . The buffer zone  13  is formed on the surface of the top plate  10  corresponding to the orifice plate  40 . When the top plate  10  and the orifice plate  40  are assembled, the buffer zone  13  is formed and provides a temporary reserve for the fluid from inlet port  11  on the top plate  10 . 
     A plurality of orifices  41  is formed on the surface of the orifice plate  40 . The orifices  41  receive the fluid from the inlet port  11  on the top plate  10 , then the fluid is evenly guided to the culture plate  20  via the plurality of orifices  41 . Meanwhile, an outlet port  42  is also formed on the orifice plate  40  and corresponds with the outlet port  12  of the top plate  10  to connect each other. 
     A plurality of culture wells  25  is formed on the surface of the culture plate  20 . In the present example, nine hexagon-shape culture wells  25  are formed on the same surface. A injection port  21  is formed at each corner of the hexagon-shape culture well  25 , and connects with the orifice  41  on the orifice plate  40 . 
     At least one regulating orifice  23  is formed in each culture well  25 . The opening of each regulating orifice  23  has a height difference with the bottom of the culture wells  25 . The regulating orifices  23  penetrate the culture plate  20  and connect with bottom plate  30  while assembling. When the fluid surface in culture wells  25  is higher than the regulating orifices  23 , excess fluid is to be removed into the bottom plate  30  by passing through regulating orifice  23 . Meanwhile, an outlet port  22  is also formed on the culture plate  20  at the locations corresponding to the outlet ports  12 ,  42  on the top plate  10  and the orifice plate  40 . 
     At least one collecting well  32  and one flow channel  33  are formed on the bottom plate  30 . When the micro device for cell culture is assembled, the culture plate  20  is placed between the orifice plate  40  and the bottom plate  30 . The flow channel  33  is linked with the regulating orifices  23 , and the fluid from culture wells  25  flows along the flow channel  33  and is gathered in collecting well  32 . 
     When assembling, the screws are tightened in the eight screw holes  51  in the order of top plate  10 , orifice plate  40 , culture plate  20 , and bottom plate  30 ; the configuration of the micro device after assembling is shown in  FIG. 2 . In this example, the orifice plate  40  and the bottom plate  30  are made from soft materials to ensure each component tightly abuts another after assembling. After assembly, the outlet port  12  on the top plate  10  should correspond with the outlet ports  42 ,  22  on the orifice plate  40  and the culture plate  20 , as well as the collecting well  32  on the bottom plate  30 . The excess fluid in collecting well  32  drains out from top plate  10  along outlet ports  42 ,  22 ,  12 . 
     A connecting pipe (not shown) can be utilized to link with inlet port  11  and outlet port  12  on the top plate  10 . The back-flowing fluid can be re-used by flowing into the inlet port  11  via the outlet port  12 , or the excess fluid can drain out of the micro device via the outlet port  12 . 
     In this device, the regulating orifice  23  in culture wells  25  corresponds with flow channel  33  on the bottom plate  30 . Therefore, all the excess fluid from culture wells  25  can be properly collected to avoid overflow on the component and thus contamination is avoided. 
     EXAMPLE 2 
     The other structure of this device is as shown as in  FIG. 3A . The buffer zone  13  formed on the top plate  10  can be formed on orifice plate  40  corresponding to a surface of top plate  10  instead, while the buffer zone  13  can also be formed when the top plate  10  and the orifice plate  40  are assembled. 
     EXAMPLE 3 
     As shown in  FIG. 3B , the flow channel  33  and the collecting well  32  originally formed on the bottom plate  30  can also be formed on the culture plate  20  on a surface facing the bottom plate  30 . The flow channel  33  and the collecting well  32  can thus be formed between the culture plate  20  and the bottom plate  30  when the micro device has been assembled. 
     EXAMPLE 4 
     In  FIG. 3C , the collecting well  32  on the bottom plate  30  is connected with outlet port  12  directly, to drain the fluid in the collecting well  32  out of the micro device without back flow. Therefore, the outlet ports  12 ,  42 , and  22  on the top plate  10 , the orifice plate  40  and the culture plate  20  are not necessary. This type of micro device for culture cell is assembled, and shown in  FIG. 3D . 
     EXAMPLE 5 
     By flow field research, the ability of physical environment imitation is demonstrated by the micro device for cell culture. When analyzing by computer the flow field vector of the cell culture wells, it is discovered that when inputting fluid from the comers of the hexagon-shape culture wells, the pressure on the inlet port does differ from that in the center; the concentration of fluid changes according to the pressure. That is, as shown in  FIG. 4 , the concentration around the inlet port is higher than that in the central position. The difference of pressure can be detected by the gradual concentration relation shown in  FIG. 5 . As shown in  FIG. 4 , the fluid flows from the comers of a hexagon toward the center at the flow rate of 0.075 m/s, the pressure change of 1000(N/m^2) is produced as an imitating result as shown in  FIG. 5 . It is observed that the device of this invention can imitate the gradient effect of molecular diffusion within an organism by concentration difference caused by pressure. 
     EXAMPLE 6 
     To perform cell culture by using the micro device of the invention, cells are loaded first. There are several methods for cell loading. For example, a biological matrix can be attached to cell culture wells  25 , and then cell culture is conducted. The components shown in  FIG. 1  are assembled as shown in  FIG. 2 . After 24 hours of cell attaching, culture media is injected into inlet port  11  for perfusion. The culture media will diversify into culture wells  25  via orifice s  41  in the orifice plate  40 . When the culture wells  25  are full of culture media, the fluid will flow vertically into flow channels  33  on bottom plate  30  via regulating orifice  23  of culture wells  25 , and then gather in collecting well  32 . 
     Alternatively, after the biological matrix is attached to the culture wells  25 , the micro device for cell culture is assembled. Seedling cells are perfused in the micro device. The seedling cells are circulated in the injection ports, and attached to the culture wells with the bio-matrix for propagation. Perfusion may be stopped at this time. After four to six hours, cell culture media is applied again with low flowing rate. 
     After the cells are implanted into the micro device, perfusion can be conducted with different flow rates and different media or solutions. After a predicted culture period, e.g. for 1 day, 3, 5, or 7 days, the cell morphology is observed with a microscope. 
     EXAMPLE 7 
     More than one micro device of the invention can be connected in different ways. As shown in  FIG. 6A  to  FIG. 6C , serial or parallel connection can be applied to connect more than two micro devices  300 ,  301  to perform mass production. 
     Parallel connection of micro devices for cell culture  300 ,  301  is illustrated in  FIG. 6A . A fluid supply unit  110  connects branched diversion pipes  111 ,  112  with the inlet ports  11  on the micro devices  300  and  301 . Moreover, a collecting unit  120  also includes branched diversion pipes  121 ,  122 , and connects with outlet ports  12  on micro devices  300  and  301 . By the way of parallel connection, both micro devices  300  and  301  can conduct cell culture in the same culture media at the same time. 
     When conducting mass production of cells, a cell culture media should be provided in micro devices  300 ,  301  via fluid supply unit  110 . The cell culture is conducted as mentioned in example 3. When the cultured cells are collected, the collecting unit  120  connected with outlet ports  12  can be used to receive the products from independent micro devices  300 ,  301 . 
     Another connection way to join more than two micro devices to perform mass production is by serial connection. As shown in  FIG. 6B , the inlet port  11  on the micro device  300  is connected to a fluid supply unit  110  with a diversion pipe  111 . The inlet port  12  on the same micro device  300  is connected to the inlet port  11  on the micro device  301  with an airtight connecting pipe  130 . The outlet port  12  on the micro device  301  is connected with the collecting unit  120  by the diversion pipe  121 . 
     The culture media is supplied into the micro device  300  via the fluid supply unit  110  as the culturing process mentioned in example  3 . The excess culture media is injected into another serial-connected micro device  301  via the connecting pipe  130 , and the cell culture is then conducted in the second micro device  301 . 
     Alternatively, after conducting cell culture in micro device  300  for a period of time, the connecting pipe  130  is used as a route to output the proliferated cells and media to the serial-connected micro device  301 . Meanwhile, a further cell culture procedure can be conducted to proliferate cells. Then, all the produced cells are gathered in the fluid collecting unit  120  via diversion pipe  121 . 
     Furthermore, another type of serial connection can be used to connect more than two micro devices  300 ,  301  and  302  as shown in  FIG. 6C . In this example, the outlet port  12  on top plate  10  is relocated to the bottom plate  30  and connected with the collecting well  32  on the bottom plate. Each inlet port  11  is connected with the outlet port  12  by different connecting pipe  130  on the three micro devices  300 ,  301  and  302  separately as shown in  FIG. 6C . The inlet port  11  on the first micro device  300  is connected with a fluid supply unit  110  by the diversion pipe  111 , to provide the cell culture media or other required material. Meanwhile, the outlet port  12  on the last micro device  301  is connected to the collecting unit  120  by another diversion pipe  121  to collect product. 
     EXAMPLE 8 
     The micro devices can be connected with more micro devices for mass cell culture as described in example 7. Furthermore, a single micro device for cell culture can also be expanded for mass production while economizing the use of space. 
     Referring to  FIG. 7A , the main components shown are the same as in example 1, and include: a top plate  10 , a orifice plate  40 , a culture plate  20 , and a bottom plate  30 . However, two orifice plates  40 ,  70 , and two culture plates  20 ,  80 , are used in this example instead of using one plate (one orifice plate and one culture plate are one culture unit). A dividing plate  60  is placed between culture plate  20  and the orifice plate  70  for separation of two culture units. 
     In  FIG. 7A , a buffer zone  64  on the dividing plate  60  placed between the culture plate  20  and the orifice plate  70  is formed to receive the excess culture media from culture plate  20 . The excess media flows into the orifice plate  70  via the separating holes  63  formed on buffer zone  64 . The orifices  41  are also used to diversify the excess culture media to each culture unit  25  on the culture plate  80 , and the cell culture is also performed in the second culture unit. 
     If more than one dividing plate  60 , orifice plate  70 , and culture plate  80  are further placed in one micro device, mass cell culture can be performed by the way of serial connection. Also, each culture well in a culture unit is ensured to have the same environment for cell culture, and problems of uncertain hazard are fewer. 
     Further, another way for expanding the use of micro devices for cell culture is illustrated in  FIG. 7B . A buffer zone  13  is formed on the surface which is opposite the surface with culture wells of the culture plate  20 . The buffer zone  13  is formed between the culture plate  20  and the next component (e.g. the second orifice plate  70 . The dividing plate is absent in the present example.), and the fluid from the culture plate  20  can be retained inside. 
     Also, in the present example, the flow channels  33  and the collecting well  32  are formed on one surface of the culture plate  80  instead of on the bottom plate. The flow channels  33  and the collecting well  32  are formed when the culture plate  80  and the bottom plate have been assembled. 
     EXAMPLE 9 
     The micro device for cell culture of the present invention can be combined to other devices for an automatic controlling system. As shown in  FIG. 8 , a controller  100  is connected to multiple fluid supply units  110 A,  110 B,  110 C,  110 D separately. There are four fluid supply units in the present example providing fluids such as media, growth factors or medical reagents. The four fluid supply units  110 A,  110 B,  110 C,  110 D are linked to a pump  200  individually, to pump the fluid into micro device  300 . Meanwhile, the micro device  300  is connected with a product collecting unit  400  and a waste collecting unit  500 . The signals generated inside the micro device  300  are detected by a biosensor  600 , and fed back to controller  100 , to control the variety of the fluid as well as the period or concentration factors. 
     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.