Patent Publication Number: US-9850459-B1

Title: Method and apparatus for primary cell isolation

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to an isolating apparatus, particularly to a primary cell isolating apparatus. The present invention also relates to a method of use, particularly to a method for using the primary cell isolating apparatus. 
     2. Description of the Prior Arts 
     In the recent years, the technology of primary cell isolation has been maturely developed, and the primary cell culture has been applied in various fields, so the isolation of the primary cells is urgently needed for mass production and the manufacture standards need unification. 
     The isolation of the primary cell of the prior art has multiple steps, and these multiple steps comprising obtaining the tissue, slicing, centrifugation, enzyme reaction, washing, culturing, etc in one laboratory need to be completed by many technicians. The sizes of the tissues after slicing differ from one technician to another, and also some of the steps need to be repeated. Isolation of the primary cell often takes half of the working day from obtaining the tissue to starting cell culture, and when a large amount of primary tissue needs to be isolated, the technicians needed to accomplish the task is further increased. 
     However, the large number of technicians needed will not only increase the cost in manpower, but also substantially increase the probability of contaminating the primary cells since some of the isolation steps of the primary cell are not always done in the germ-free room or in the laminar flow cabinet, and the overall isolation process lacks efficiency. 
     Therefore, an isolation apparatus for saving manpower, reducing tissue isolation error by different technicians, and reducing contamination of the primary cells is needed, and the disadvantage of the prior art should be resolved. 
     SUMMARY OF THE INVENTION 
     According to the above description, the objective of the present invention is to provide a primary cell isolating apparatus, such that manpower can be saved, the efficacy of tissue slicing degree can be unified, and the contamination can be reduced. 
     To achieve the above objective of the present invention, the invention provides the primary cell isolating apparatus comprising a slicing space, a working space, a picking space, and a sliding device. The slicing space comprises a slicing device located in the slicing space. The working space comprises a first gate, a heater, a rotating holder, a centrifuge, an open-close cap device, a tube rack, and a liquid transferring device. The first gate is adjacent to the slicing space and the working space is adjacent to the slicing space. The heater is proximal to the first gate. The rotating holder is adjacent to a side of the heater that is opposite the first gate. The centrifuge is adjacent to a side of the rotating holder that is opposite the heater. The open-close cap device is proximal to the first gate and is opposite the heater. The tube rack is adjacent to a side of the open-close cap device that is opposite the first gate. The liquid transferring device is adjacent to a side of the tube rack that is opposite the open-close cap device. The picking space comprises a second gate adjacent to the working space and the picking space is adjacent to the working space. The sliding device is mounted through the slicing space, the working space, and the picking space, and is located below the first gate of the working space and the second gate of the picking space; the sliding device has a cart slidable on the sliding device. 
     Preferably, the rotating holder is a robotic arm. 
     Preferably, the liquid transferring device comprises a shelf, at least one pipette, a tip rack, a rotating plate, and a collecting station. The at least one pipette is located at the top of the shelf. The tip rack is movably located at a side of the shelf. The rotating plate is rotatably located in the shelf, and the rotating plate has multiple containers on the rotating plate. The collecting station is adjacent to the shelf and is located below the at least one pipette. 
     More preferably, the number of the at least one pipette is two, and the two pipettes are adjacent to each other. 
     Preferably, the sliding device has two opposite sides; the heater, the rotating holder, and the centrifuge are located at one of the two opposite sides of the sliding device, and the open-close cap device, the tube rack, and the liquid transferring device are located at the other side of the sliding device. 
     In another aspect, the present invention relates to a method of using the primary cell isolating apparatus as abovementioned, comprising the steps as follows: 
     ( 1 ) placing a tissue into the slicing device of the slicing space; 
     ( 2 ) holding a tube, located at the tube rack by the rotating holder, and transferring the tube onto the cart of the sliding device; moving the cart loaded with the tube to the open-close cap device and opening a cap of the tube; after opening the first gate, moving the cart loaded with the tube without the cap from the working space to the slicing space, and closing the first gate; 
     ( 3 ) slicing the tissue by the slicing device and then flushing the tissue into the tube by a buffer; opening the first gate, moving the cart loaded with the tube containing the tissue to the open-close cap device of the working space, and closing the first gate; 
     ( 4 ) closing the cap onto the tube containing the tissue by the open-close cap device, holding the tube containing the tissue by the rotating holder and transferring the tube to the centrifuge, and then forming a first supernatant and a first precipitate in the tube after centrifugation; transferring the tube after centrifugation back to the cart; 
     ( 5 ) moving the cart loaded with the tube after centrifugation from the open-close cap device to the liquid transferring device, discarding the first supernatant, retaining the first precipitate in the tube, and adding an enzyme to mix with the first precipitate in the tube via the liquid transferring device; moving the cart loaded with the tube containing the enzyme to the open-close cap device, and closing the cap of the tube by the open-close cap device; 
     ( 6 ) shaking the tube containing the enzyme by the rotating holder and transferring the tube to the heater for enzyme reaction; 
     ( 7 ) holding the tube after enzyme reaction by the rotating holder, transferring the tube to the centrifuge, and then forming a second supernatant and a second precipitate in the tube after centrifugation; transferring the tube after centrifugation to the cart by the rotating holder, and opening the cap of the tube by the open-close cap device; 
     ( 8 ) moving the cart loaded with the tube after centrifugation from the open-close cap device to the liquid transferring device, and discarding the second supernatant, retaining the second precipitate in the tube, and adding a wash buffer to mix with the second precipitate in the tube via the liquid transferring device; moving the cart loaded with the tube containing the wash buffer to the open-close cap device, and closing the cap of the tube by the open-close cap device; 
     ( 9 ) shaking the tube containing the wash buffer by the rotating holder, transferring the tube to the centrifuge, and then forming a third supernatant and a third precipitate in the tube after centrifugation; transferring the tube after centrifugation back to the cart, and opening the cap of the tube by the open-close cap device; 
     ( 10 ) moving the cart loaded with the tube after centrifugation from the open-close cap device to the liquid transferring device, and discarding the third supernatant, retaining the third precipitate in the tube, and adding a medium to mix with the third precipitate in the tube via the liquid transferring device, wherein the tube containing the medium has primary cells; moving the cart loaded with the tube containing the medium to the open-close cap device, and closing the cap of the tube by the open-close cap device; 
     ( 11 ) shaking the tube containing the medium and transferring the tube to the cart by the rotating holder; opening the second gate, and moving the cart loaded with the tube containing the medium from the working space to the picking space; and 
     ( 12 ) closing the second gate and culturing the primary cells located in the tube containing the medium. 
     Preferably, before steps ( 5 ), ( 8 ), and ( 10 ), the method further comprises moving the tip rack of the liquid transferring device below the two pipettes, which are a first pipette and a second pipette, combining tips of the tip rack with the two pipettes, moving the tip rack away from the two pipettes, and then transferring the liquid by the two pipettes. 
     More preferably, after discarding the supernatant via the first pipette in the steps ( 5 ), ( 8 ), and ( 10 ), the method further comprises moving the cart away from the liquid transferring device, rotating the rotating plate loaded with a waste container below the first pipette containing the supernatant and discharging the supernatant into the waste container; sucking liquid in one of the containers except the waste container by the second pipette, moving the cart loaded with the tube below the second pipette containing the liquid, and discharging the liquid into the tube and mixing the precipitate with the liquid by the second pipette; moving the cart loaded with the tube containing the liquid to the open-close cap device, and closing the cap of the tube by the open-close cap device; moving the tip rack below the pipettes, and the tip rack assisting in removing the tip on the first pipette and the tip on the second pipette to the collecting station; wherein in the step ( 5 ), one of the containers of the rotating plate is an enzyme container, and the liquid is enzyme; wherein in the step ( 8 ), one of the containers of the rotating plate is a wash buffer container, and the liquid is wash buffer; wherein in the step ( 10 ), one of the containers of the rotating plate is a medium container, and the liquid is medium. 
     The advantage of the present invention is that the primary cells can be automatically isolated by the slicing space, the working space, the picking space, and the sliding device of the primary cell isolating apparatus during different processes, such that manpower can be saved, the efficacy of tissue slicing degree can be unified, and the contamination can be reduced. 
     Other objectives, 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 a perspective view of a primary cell isolating apparatus of the present invention; 
         FIG. 2  is a top view of the primary cell isolating apparatus of the present invention; 
         FIG. 3  is a perspective view of the slicing space of the primary cell isolating apparatus of the present invention; 
         FIG. 4  is a perspective view of a part of the working space of the primary cell isolating apparatus of the present invention; 
         FIG. 5  is a perspective view of another part of the working space of the primary cell isolating apparatus of the present invention; 
         FIG. 6  is a perspective view of a picking space of the primary cell isolating apparatus of the present invention; 
         FIG. 7  is a flow chart of step  1  (S 1 ) to step  6  (S 6 ) of the method of using the primary cell isolating apparatus of the present invention; 
         FIG. 8  is a flow chart of step  7  (S 7 ) to step  12  (S 12 ) of the method of using the primary cell isolating apparatus of the present invention. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     As shown in  FIG. 1  and  FIG. 2 , a primary cell isolating apparatus  1  of the present invention is a laminar flow cabinet, and the primary cell isolating apparatus  1  comprises a slicing space  10 , a working space  20 , a picking space  30 , and a sliding device  40 ; wherein the slicing space  10 , the working space  20 , and the picking space  30  are sequentially adjacent; wherein the sliding device  40  is mounted through the slicing space  10 , the working space  20 , and the picking space  30 . 
     As shown in  FIG. 2  and  FIG. 3 , the slicing space  10  comprises a slicing device  11  located in the slicing space  10 . The slicing device  11  is a laminar flow cabinet having UV light for sterilization to achieve a sterile operating space. 
     As shown in  FIG. 2 ,  FIG. 4 , and  FIG. 5 , the working space  20  comprises a first gate  21 , a heater  22 , a rotating holder  23 , a centrifuge  24 , an open-close cap device  25 , a tube rack  26 , and a liquid transferring device  27 . The first gate  21  is adjacent to the slicing space  10 . The heater  22  is proximal to the first gate  21 . The rotating holder  23  is adjacent to a side of the heater  22  that is opposite the first gate  21 ; in a preferred embodiment, the rotating holder  23  is a robotic arm. The centrifuge  24  is adjacent to another side of the rotating holder  23  that is opposite the heater  22 . The open-close cap device  25  is proximal to the first gate  21  and is opposite the heater  22 . The tube rack  26  is adjacent to a side of the open-close cap device  25  that is opposite the first gate  21 . The liquid transferring device  27  is adjacent to a side of the tube rack  26  that is opposite the open-close cap device  25 . The liquid transferring device  27  comprises a shelf  271 , at least one pipette  272 , a tip rack  273 , a rotating plate  274 , and a collecting station  275 . The shelf  271  is cubical. The at least one pipette  272  is located at the top of the shelf  271 ; in a preferred embodiment, the number of the at least one pipette  272  is two, i.e., a first pipette and a second pipette, and the two pipettes  272  are adjacent to each other. The tip rack  273  is movably located at a side of the shelf  271 . The rotating plate  274  is rotatably located in the shelf  271 , and the rotating plate  274  has multiple containers on the rotating plate  274 ; wherein the containers are waste containers, enzyme containers, wash buffer containers, medium containers, etc. The collecting station  275  is adjacent to the shelf  271  and is located below the at least one pipette  272 . In a preferred embodiment, the sliding device  40  has two opposite sides; the heater  22 , the rotating holder  23 , and the centrifuge  24  are located at one of the two opposite sides of the sliding device  40 , and the open-close cap device  25 , the tube rack  26 , and the liquid transferring device  27  are located at the other side of the sliding device  40 . 
     As shown in  FIG. 6 , the picking space  30  comprises a second gate  31  adjacent to the working space  20 , so the picking space  30  is adjacent to the working space  20 . 
     As shown in  FIG. 2  and  FIG. 6 , the sliding device  40  is located below the first gate  21  of the working space  20  and the second gate  31  of the picking space  30 ; the sliding device  40  has a cart  41  slidable on the sliding device  40 . 
     As shown in  FIG. 7 , the present invention provides a method for using the primary cell isolating apparatus  1  as abovementioned, comprising the following steps that: 
     ( 1 ) a tissue is placed into the slicing device  11  of the slicing space  10 ; 
     ( 2 ) a tube, having a cap, located at the tube rack  26  is held by the rotating holder  23 , and the tube is transferred onto the cart  41  of the sliding device  40 . The cart  41  loaded with the tube is moved to the open-close cap device  25  and the cap of the tube is opened. After the first gate  21  is opened, the cart  41  loaded with the tube without the cap is moved from the working space  20  to the slicing space  10 , and the first gate  21  is closed; 
     ( 3 ) the tissue is sliced by the slicing device  11  and then is flushed into the tube by a buffer. The first gate  21  is opened, the cart  41  loaded with the tube containing the tissue is moved to the open-close cap device  25  of the working space  20 , and the first gate  21  is closed; 
     ( 4 ) the cap is closed by the open-close cap device  25  onto the tube containing the tissue, and the tube containing the tissue is held by the rotating holder  23  and is transferred to the centrifuge  24 . The condition of the centrifugation is 200×g, 4 minutes at room temperature, and then a first supernatant and a first precipitate are formed in the tube after centrifugation. The tube after centrifugation is transferred back to the cart  41 ; 
     ( 5 ) the tip rack  273  of the liquid transferring device  27  is moved below the two pipettes  272 , which are the first pipette and the second pipette, the tips of the tip rack  273  are combined with the pipettes  272 , and the tip rack  273  is moved away from the pipettes  272 . The cart  41  loaded with the tube after centrifugation is moved from the open-close cap device  25  to the liquid transferring device  27 , the first supernatant is discharged by the first pipette, and the first precipitate is retained in the tube. The cart  41  is moved away from the liquid transferring device  27 , the rotating plate  274  loaded with a waste container is rotated below the first pipette containing the first supernatant, and the first supernatant is discharged into the waste container. Enzyme in an enzyme container is sucked by the second pipette, the cart  41  loaded with the tube is moved below the second pipette containing the enzyme, and the enzyme is ejected into the tube and the first precipitate is mixed with the enzyme by the second pipette. The cart  41  loaded with the tube containing the enzyme is moved to the open-close cap device  25 , and the cap of the tube is closed by the open-close cap device  25 . The tip rack  273  is moved below the pipettes  272 , and the tip rack  273  assists in removing the tip on the first pipette and the tip on the second pipette to the collecting station  275 ; the tips of the tip rack  273  are combined with the pipettes  272 , and the tip rack  273  is moved away from the pipettes  272 ; 
     ( 6 ) the tube containing the enzyme is shaken by the rotating holder  23  and the tube is transferred to the heater  22  for enzyme reaction; the condition of the enzyme reaction is proceeded at 37° C., and the tube is shaken once per 10 minutes for 6 times by the rotating holder  23 ; 
     ( 7 ) the tube after enzyme reaction is held by the rotating holder  23 , and the tube is transferred to the centrifuge  24 . The condition of the centrifugation is 200×g, 4 minutes at room temperature, and then a second supernatant and a second precipitate is formed in the tube after centrifugation. The tube after centrifugation is transferred to the cart  41  by the rotating holder  23 , and the cap of the tube is opened by the open-close cap device  25 ; 
     ( 8 ) the present step is similar to step ( 5 ), but the difference is that the enzyme container on the rotating plate  274  is replaced with a wash buffer container. The details are as follows: 
     the cart  41  loaded with the tube after centrifugation is moved from the open-close cap device  25  to the liquid transferring device  27 , the second supernatant is discharged by the first pipette, and the second precipitate is retained in the tube. The cart  41  is moved away from the liquid transferring device  27 , the rotating plate  274  loaded with the waste container is rotated below the first pipette containing the second supernatant, and the second supernatant is discharged into the waste container. Wash buffer in the wash buffer container is sucked by the second pipette, the cart  41  loaded with the tube is moved below the second pipette containing the wash buffer, and the wash buffer is ejected into the tube and the second precipitate is mixed with the wash buffer by the second pipette. The cart  41  loaded with the tube containing the wash buffer is moved to the open-close cap device  25 , and the cap of the tube is closed by the open-close cap device  25 . The tip rack  273  is moved below the pipettes  272 , and the tip rack  273  assists in removing the tip on the first pipette and the tip on the second pipette to the collecting station  275 ; the tips of the tip rack  273  are combined with the pipettes  272 , and the tip rack  273  is moved away from the pipettes  272 ; 
     ( 9 ) the tube containing the wash buffer is shaken by the rotating holder  23 , and the tube is transferred to the centrifuge  24 . The condition of the centrifugation is 200×g, 4 minutes at room temperature, and then a third supernatant and a third precipitate are formed in the tube after centrifugation. The tube after centrifugation is transferred back to the cart  41 , and the cap of the tube is opened by the open-close cap device  25 ; 
     ( 10 ) the present step is similar to step ( 5 ), the difference is that the enzyme container on the rotating plate  274  is replaced to a medium container. The details are as follows: 
     the cart  41  loaded with the tube after centrifugation is moved from the open-close cap device  25  to the liquid transferring device  27 , the third supernatant is discharged by the first pipette, and the third precipitate is retained in the tube. The cart  41  is moved away from the liquid transferring device  27 , the rotating plate  274  loaded with the waste container is rotated below the first pipette containing the third supernatant, and the third supernatant is discharged into the waste container. Medium in the medium container is sucked by the second pipette, the cart  41  loaded with the tube is moved below the second pipette containing the medium, and the medium is ejected into the tube and the third precipitate is mixed with the medium by the second pipette, wherein the tube containing the medium has primary cells. The cart  41  loaded with the tube containing the medium is moved to the open-close cap device  25 , and the cap of the tube is closed by the open-close cap device  25 . The tip rack  273  is moved below the pipettes  272 , and the tip rack  273  assists in removing the tip on the first pipette and the tip on second pipette to the collecting station  275 ; the tips of the tip rack  273  is combined with the pipettes  272 , and the tip rack  273  is moved away from the pipettes  272 ; 
     ( 11 ) the tube containing the medium is shaken and the tube is transferred to the cart  41  by the rotating holder  23 . The second gate  31  is opened, and the cart  41  loaded with the tube containing the medium is moved from the working space  20  to the picking space  30 ; and 
     ( 12 ) the second gate  31  is closed, and the primary cells in the tube containing the medium are cultured. 
     Even though numerous characteristics and advantages of the present invention have been set forth in the foregoing description, together with details of the structure and features of the invention, the disclosure is illustrative only. Changes may be made in the details, especially in matters of shape, size, and arrangement of parts within the principles of the invention to the full extent indicated by the broad general meaning of the terms in which the appended claims are expressed.