Abstract:
A device for mechanically decollating cells from a cell composite, particularly a shear rotor, a cell isolation unit including such a shear rotor and a method for decollating cells from a cell composite. The aforementioned device includes a rotor having a rotor wall, which is concentrically arranged in a receptacle. A rotor seat is connected to a motor and to which the rotor can be fixed in a detachable manner. A rotational movement of the motor can be transmitted to the rotor by means of the rotor seat. The rotor tapers longitudinally towards the bottom of the receptacle so that different circumferential speeds of the rotor can be transmitted to a liquid sample in the receptacle via the rotor wall.

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is based upon and claims priority under 35 USC §119 to PCT/EP2008/000791, filed Jan. 31, 2008 entitled: DEVICE AND METHOD FOR THE MECHANICAL DECOLLATION OF CELLS FROM A CELL COMPOSITE, which is based upon German Patent Application No. 10 2007 005 369.1, filed Feb. 2, 2007 entitled: DEVICE AND METHOD FOR THE MECHANICAL DECOLLATION OF CELLS FROM A CELL COMPOSITE. The entire contents of each above noted application is herein incorporated by reference. 
     FIELD OF THE INVENTION 
     The present invention relates to devices and a method for decollating cells from a cell composite using a shear rotor. 
     BACKGROUND OF THE INVENTION 
     For various experimentation purposes it is necessary to decollate cells present in a cell composite. The state of the art offers different methods for this purpose. Among these methods are for example, the ultrasonic treatment of a cell composite according to the U.S. Pat. No. 5,879,939, multiple filtration through filters of different pore sizes according to the U.S. Pat. No. 5,888,409, the diminution or shredding of a cell composite according to the U.S. Pat. No. 4,028,190, or the shearing of a liquid sample with the cell composite by means of pipetting or a shear rotor according to the German patent DE 32 18 079. 
     The above named methods have different disadvantages. On the one hand, the cell composite present in the liquid sample is frequently overstressed by the energy applied for decollating. This leads to a destruction of the cells, and to a lengthening of the preparation method until intact decollated cells are available. The above disadvantage occurs precisely during mechanical decollating methods, as in the above named diminution device, or during pipetting of the liquid sample. 
     The shear rotor according to DE 32 18 079 has the disadvantage that a liquid sample with the cell composite is subjected everywhere to the same shear forces. In addition, the sample is moved due to turbulent flows within the shear rotor such that a uniform sample processing is not guaranteed. 
     Therefore, it is the objective of the present invention to provide a device and a method for decollating cells from a cell composite, with which, in comparison to the state of the art, samples can be processed more reliably. 
     SUMMARY OF THE INVENTION 
     The above objective is solved by devices and methods according to the claims. Advantageous embodiments and further developments of the present invention are described in the following description, the drawings and the dependent claims. 
     According to one aspect, the present invention discloses a device for mechanical decollating of cells from a cell composite, in particular a shear rotor, which has the following features: a rotor with a rotor wall, where the rotor is disposed concentrically in a receptacle, a motor connected to a rotor seat, to which the rotor can be fastened in a detachable manner and by which a rotational movement of the motor can be transmitted to the rotor, while the rotor tapers in the longitudinal direction towards the bottom of the receptacle, so that different circumferential speeds of the rotor can be transmitted via the rotor wall to a liquid sample in the receptacle. Decollating of cells can be understood as isolating or separating cells from a cell composite, particularly a tissue. 
     The shear rotor described above includes a receptacle in which a liquid sample with a cell composite is received. Within the receptacle, a rotor is disposed rotatably, so that through the rotation of the rotor within the receptacle the cell composite in the liquid sample is sheared, and is decollated into individual cells. Furthermore, the geometry of the rotor guarantees that a circulation of the liquid sample within the receptacle occurs, and thus, also within the gap between the rotor wall and the wall of the receptacle. In this way, the entire volume of the sample is processed and the formation of residues, for example at the bottom of the receptacle, is prevented. For this purpose, the rotor has a structure which tapers downwards, preferably cone-shaped. With a rotation of the rotor, due to its geometry, two imaginary points on the rotor wall move at different speeds if they are disposed with different distances from the bottom of the receptacle. Based on the different rotational speeds which increase with increasing distance from the bottom, pressure differences result in the receptacle and in the gap between the rotor wall and the receptacle wall, which lead to an advantageous circulation and shearing of the liquid sample. 
     According to different embodiments, the receptacle has a cylindrical form or a form of lower conicity compared to the cone-shaped rotor, or widens in the direction of the bottom of the receptacle. 
     According to another embodiment, the rotor and/or receptacle can be produced from a single-use material, for example from plastic or PVC, in order to be able to dispose of this after processing a liquid sample. The use of a rotor and/or receptacle from a single-use material requires that the rotor can be fastened and detached from the rotor seat with minimal effort, for example, automatically. For this purpose, the rotor seat has an outside thread, and the rotor has an inside thread matched to the outside thread. According to another alternative, the rotor seat includes a projection, and the rotor has a snap fit matched to the projection, such that the rotor can also be fastened detachably to the rotor seat. In the case of the simple connection of the rotor seat and the rotor using a snap fit, the rotor seat additionally includes a rotational interlock, with which the rotational movement of the rotor seat can be transmitted to the rotor on the basis of a positive locking and/or non-positive locking connection. 
     In addition, it is preferable, to provide the above mentioned device with auxiliary technical means for performing a turbidity measurement of the liquid sample in the receptacle. For this purpose, the receptacle is produced from a material that can be at least partially penetrated by radiation. According to an alternative, this material can be penetrated by radiation in the visual range, or has a plurality of windows, so that using a light source and an appropriate sensor for recording the quantity of light, a turbidity measurement can be performed on the liquid sample. Furthermore, it is conceivable to perform the turbidity measurement using ultrasound, so that the receptacle must be produced from an ultrasound-permeable material. The turbidity measurement is performed and monitored, for example, by a control unit or a computer, and the data that is collected is appropriately evaluated. 
     The present invention further discloses a cell isolating unit for mechanical decollating of cells from a cell composite, where the unit has the following characteristics: a rotor with a rotor wall where the rotor can be disposed automatically and in a movable manner into a plurality of receptacles with a receptacle wall, a motor connected to a rotor seat, to which the rotor can be fastened in a detachable manner and by which a rotational movement of the motor can be transmitted to the rotor, while the plurality of receptacles is disposed in a receptacle holder, so that via an automatic movement of the rotor and/or the receptacle holder, the rotor can be positioned and rotated concentrically in each one of the receptacles, respectively, of the receptacle holder. 
     The cell isolation unit described above provides the possibility to systematically, successively process a plurality of liquid samples with cell composites with the shear rotor according to the invention. The different liquid samples are disposed in different receptacles within a receptacle holder. Advantageously, the receptacles are arranged regularly such that each individual receptacle position can be selectively reached, for example, using a control module or a computer control. The processing of the liquid sample with the cell composite can occur in the selected receptacle as soon as the rotor is disposed concentrically within the selected receptacle, either due to its own movement, a movement of the receptacle holder, or a combined movement of the rotor seat and receptacle holder. 
     According to a first alternative, the rotor of the cell isolation unit has a cylindrical form. Furthermore, it is preferred that the rotor tapers in its longitudinal direction towards the bottom of the receptacle, and in particular, is preferably designed cone-shaped. These different rotor forms can be combined with receptacles which, in each case, have an opening only on their upper side for filling the receptacle with a liquid sample. According to different embodiments, the receptacles have a cylindrical form, or a form that tapers towards the bottom of the receptacle, in particular, a cone-shaped form. It is also conceivable to design the receptacle such that it expands in the direction of its bottom. 
     According to a further embodiment, the receptacle holder is designed as a circular holder in which the plurality of receptacles is arranged removably in matched openings, equally spaced along a circular shaped track. It is also conceivable to design the receptacle holder as an angular holder so that the plurality of receptacles is disposed along straight lines, for example, equidistant from each other. The specified geometry of the receptacle holder dictates the exact position of the individual receptacles, so that the rotor can be positioned automatically in the individual receptacles with minimal effort, for example, computer controlled. Furthermore, such a design opens up the possibility to fill different receptacles with different samples and/or to process different receptacles with specially adapted decollating methods. 
     According to a further embodiment, the cell isolation unit includes an exchange device, with which the rotor and/or the receptacle can be removed and replaced by a new rotor and/or a new receptacle, respectively. Such an exchange device forms the prerequisite for further automation of the processing of a plurality of liquid samples, if these are supplied, for example, by an automatic dosing device into the newly installed receptacles. 
     Furthermore, it is preferred to equip the cell isolation unit with a temperature control unit so that a liquid sample in one or a plurality of receptacles in the receptacle holder can be temperature controlled in a targeted manner. 
     The present invention also discloses a method for decollating cells from a cell composite that has the following steps: introduction of a liquid sample with a cell composite into a receptacle, the concentric arrangement of the rotor, which tapers in its longitudinal direction towards the bottom, in the receptacle so that different circumferential speeds of the rotor are transmitted via a rotor seat to a liquid sample in the receptacle, and rotation of the rotor, such that the cell composite is decollated into cells. According to a preferred alternative of the method for decollating described above, a rotational speed of the rotor in the receptacle and the geometry of the rotor are matched to each other such that the liquid sample is circulated and sheared within the gap during the rotation of the rotor. This guarantees a processing of the entire sample and prevents unprocessed residues of the liquid sample, for example, on the bottom of the receptacle. 
    
    
     
       DESCRIPTION OF THE ACCOMPANYING DRAWINGS 
       The present invention is explained in more detail in reference to the accompanying drawings. 
         FIG. 1  is a schematic sectional representation of the shear rotor according to the invention, 
         FIG. 2  A, B each depict a perspective representation of an embodiment of the shear rotor according to the invention, 
         FIG. 3  illustrates an embodiment of the cell isolation unit, and 
         FIG. 4  illustrates another embodiment of the cell isolation unit. 
     
    
    
     DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
       FIG. 1  shows a schematic representation of an embodiment of the shear rotor  1  according to the invention. The shear rotor  1  comprises a receptacle  30  and a rotor  20 , which is disposed concentrically and rotatably within the receptacle  30 . The receptacle  30  preferably has a cylindrical interior contour, while the bottom  37  of the receptacle  30  is designed to have a conical or hemispherical shape. It is also preferred that the receptacle  30  is designed to have a conical shape or a shape that widens in the direction of the bottom  37 . The rotor  20  is fixed to a rotor seat  10  (not shown, see below) in a detachable manner. The rotor seat  10 , in turn, is connected to a motor  90  (not shown), so that a rotational movement of the motor  90  can be transmitted to the rotor  20 . 
     The rotor  20  is circumferentially delimited by a rotor wall  25 . The rotor wall  25  is distanced from a rotational axis DA, represented by a dash-dot line, of the rotor  20  by the radius R. The rotor  20  tapers extending in the direction of its longitudinal axis L and towards the bottom  37  of the receptacle  30 . According to a preferred alternative, the rotor  20  is designed cone-shaped. Due to the tapered shape of the rotor  20 , the rotor wall  25  at a larger distance from the bottom  37  is offset from the rotational axis DA by the radius R o , which is greater than a radius R u  at a smaller distance from the bottom  37 . 
     If the rotor  20  is now rotated by the motor about the rotational axis DA, a point on the rotor wall  25  with the offset R o  from the rotational axis DA moves faster than a point on the rotor wall  25  with the offset R u  from the rotational axis DA. Due to the faster circumferential speed in the upper region of the rotor  20  in comparison to its tapered region, there arises a dynamic pressure distribution in the liquid sample  40  in the receptacle  30 . Due to this pressure distribution, the portions of the liquid  40  near the bottom  37  of the receptacle  30  are moved or suctioned into the gap between the rotor wall  25  and the receptacle wall  35  in the direction of surface of the liquid. Within the gap between the rotor wall  25  and the receptacle wall  35 , the liquid sample  40  with the cell composite is subjected to shear forces which lead to a decollating of the cells. Portions of the liquid sample  40  near the receptacle wall  35  sink again towards the bottom  37 , resulting in a circulation of the liquid sample  40 . If the rotational speed and the geometry of the rotor  20  are matched to each other, the circulation and shearing of the liquid sample  40  in the receptacle  30  can be adjusted in a directed way. This guarantees that the liquid sample  40  is moved also in a receptacle  30  with only an upper opening in the gap between the receptacle wall  35  and the rotor wall  25 , without the receptacle  30  having an inlet or a pressure supply near the bottom  37 . In addition, sufficient shear forces are generated in the liquid sample  40  using the planar rotor wall  25  and receptacle wall  35 , in order to decollate the cells. Additionally, it is also conceivable to design the rotor wall  25  with projections and/or profiles, in order to process the liquid sample  40 . 
     The rotor  20  and the receptacle  30  are preferably produced from plastic, in particular PVC. In addition, the rotor  20  and the receptacle  30  can be produced as single-use or multi-use articles, which can be manually or automatically exchanged. 
     According to one embodiment the receptacle  30  is composed of a material that can be permeated by ultrasonic waves or light, in order to be able to perform a turbidity measurement of the liquid sample  40  in the receptacle  30 . Ultrasound or light is directed through the radiation permeable material at the liquid sample  40 . The reflected ultrasonic waves are recorded, for instance, by the ultrasound head that was previously used as the ultrasound source, and are converted into an electrical signal. This electrical signal is supplied for analysis purposes to, for example, a computer or a computing unit. If the turbidity measurement is performed optically, light is radiated through the receptacle wall  35  into the liquid sample  40 , and the occurrence of scattered light, or a portion of the light that penetrated, or the light absorption occurring in the liquid sample  40 , is subsequently recorded using a sensor. Depending on the light signal recorded in the sensor, an electrical signal is generated that in turn, is further supplied to an evaluation unit. The evaluation unit then generates a corresponding signal which represents the result of the turbidity measurement. The result of the turbidity measurement yields information about the decollation of cells and/or the mixing of the liquid sample  40  in the receptacle  30 . 
     It is also conceivable to provide at least one window in the receptacle  30  instead of a radiation permeable material of the receptacle  30 . The turbidity measurement described above can be performed using the at least one window, or with two windows lying across from each other. 
     According to a further embodiment, the receptacle  30  is connected to a temperature-control unit. This unit generates the desired temperature in the liquid sample  40 , so that the desired test conditions can be attained. 
       FIG. 2  shows a preferred embodiment of the rotor  20 , the rotor seat  10 , and the receptacle  30 . The receptacle  30  has a cone-shaped form with an upper rim  32 . The rim  32  serves for supporting and stabilizing the receptacle  30  in a holder, for example in an opening of a circular holder, which is described below in more detail. Based on this geometry of the receptacle  30 , it can be positioned manually or automatically into a receptacle holder, or a circular holder (see below), and can be removed again. 
     According to the first alternative of the rotor seat  10  represented in  FIG. 2A , it includes a circumferential projection  60 , which serves as a snap lock for the rotor  20 . The rotor  20  has an elastic edge region or snap fit  80 , formed in complement to the projection  60 , which acts together in a positive lock or non-positive lock with the projection  60 . So that the rotational movement of the motor connected to the rotor seat  10  can be transmitted to the rotor  20 , the rotor seat  10  includes a rotational interlock  70 . This rotational interlock  70  has the shape of a nub, as indicated in  FIG. 2A . The rotational interlock  70  clamps in a positive locking or non-positive locking manner in the interior of the rotor  20 , for example in a bulge or recess provided for this. In this way, the rotor seat  10 , using the rotational interlock  70 , entrains the rotor  20 , so that the rotor carries out the rotational movement of the motor. 
     According to a further embodiment represented in  FIG. 2B , the rotor seat  10  includes an outside thread  50 . The rotor  20  includes an inside thread  55  matched to the outside thread  50 . Therefore the rotor  20  can be attached with minimal effort to the rotor seat  10  by a threaded connection. 
     Based on the embodiment described above of the rotor seat  10  and the rotor  20 , the rotor  20  can be fastened manually or automatically to the rotor seat  10 , and can be removed from it. 
     The present invention further discloses a cell isolation unit for the mechanical decollation of cells from a cell composite. The cell isolation unit represents a component for the automated dissociation of liquid samples  40  with cell composites. Further components, not represented here, are conceivable, that could be combined with the cell isolation unit in order to provide an automated sample processing. These components serve, for example, for the automated receiving, supplying, and dosing of reagents and liquid samples, the temperature control, and mixing of samples, and separating the sample components, for example by centrifuging. 
     According to a first embodiment represented in  FIG. 3 , the cell isolation unit includes the rotor  20 , already described above, which is connected to a motor  90  via the rotor seat. The motor  90  with the rotor seat and the rotor  20  can be moved automatically along a guideway  120  at least in the vertical direction. It is further conceivable, that the motor  90  is provided to be movable also in the horizontal direction. Using the vertical movement of the rotor  20  along the guideway  120 , the rotor  20  can be lowered into individual receptacles  30 , in order to be disposed there, as described above, and to perform a decollation of cells in a liquid sample. 
     A plurality of receptacles  30  of the cell isolation unit is disposed regularly or irregularly in a receptacle holder  100 . The receptacle holder  100  is preferably designed as a circular holder, in which the receptacles  30  are disposed in openings that are equidistant from each other along a circular track. The openings are matched to the receptacles  30 , so that the receptacles  30  can be placed into the openings, and removed from them, with minimal effort. According to different alternatives, the receptacles  30  have a cylindrical shape, a cone shape, or a shape that widens towards the bottom  37  of the receptacle  30 . According to a first embodiment, the rotor has a cylindrical shape with the attachment possibilities as described above in reference to the rotor  20 . According to a further embodiment, the rotor  20  described above is used. 
     It is additionally preferred to arrange the circular holder  100  on a platform  130  that can be moved by a motor. Using the platform  130 , the circular holder  100  can be moved in a step-wise manner so that individual receptacles  30  can be positioned in a directed manner in reference to the rotor  20 . According to a further alternative, the platform  130  serves as a centrifuge unit, which rotates the circular holder for separating the components of the liquid sample  40 . According to a further embodiment, the platform  130  can be moved also in the vertical direction. This opens up the possibility that the rotor  20  and the motor  90  are disposed in a fixed manner, while the rotor  20  is positioned into the respectively desired receptacle  30  using the vertical and the rotational movement of the circular holder  100 . This positioning can also occur using a combined movement of the rotor  20 /motor  90  and the circular holder  100 /platform  130 . 
     According to a further embodiment, the cell isolation unit includes an exchange device with which the rotor  20  being used, and the receptacles  30 , can be exchanged automatically. This exchange device automatically removes the rotor  20  from the rotor seat  10  and deposits it in a collection container for used single-use articles, or for multi-use articles to be cleaned. Then a new rotor  20  is automatically attached to the rotor seat  10 . In the same manner, a used receptacle  30  is automatically removed and replaced by a new one. 
     According to a further alternative, a temperature control unit (not shown) is used together with the receptacle holder  100 . This temperature control unit brings the liquid sample  40  in at least one of the receptacles  30  of the receptacle holder  100  to the desired temperature, in order to attain the desired processing conditions of the liquid sample  40 . 
     A further embodiment of the cell isolation unit is represented in  FIG. 4 . In contrast to the embodiment of  FIG. 3 , the receptacle holder  100  and a centrifuge unit  110  are disposed next to each other. The motor  90  with the rotor  20  is disposed in an automatically movable manner along a guideway  120 . A sample transfer unit  140  is provided parallel to the motor  90 . Preferably, the sample transfer unit  140  can be rotated about the vertically extending attachment to the guideway  120 , so that the rotor  20  or the sample transfer unit  140  can be positioned automatically over a selected receptacle  30  in the circular holder  100  or in the centrifuge unit  110 . The liquid samples in the receptacles  30  in the circular holder  100  are moved step by step underneath the rotor  20 . The rotor  20 , in turn, is disposed into the receptacle  30  using a vertical movement, and rotated using the motor  90 . In this way, the decollation of a cell composite already contained in a liquid sample can be attained. If a turbidity measurement (see above) is performed at this point on the processed liquid sample in the receptacle  30 , information is obtained whether the mixture is sufficiently mixed and/or whether decollation has occurred in the liquid sample. Depending on the result of the evaluation of the turbidity measurement, the processing of the sample is either continued or ended. After successful decollation, the rotor  20  is retracted from the receptacle  30  by the vertical movement, and is exchanged for a new rotor  20  by the exchange device described above (not shown). The sample transfer unit  140  removes the liquid sample with decollated cells from the receptacle  30 , and transfer it into a receptacle of the centrifuge unit  110 . There, the liquid present in the sample can then be centrifuged, for instance, in order to separate the different components of the liquid sample from each other. 
     REFERENCE LIST 
       1  Device 
       10  Rotor seat 
       20  Rotor 
       25  Rotor wall 
       30  Receptacle 
       32  Projection 
       35  Receptacle wall 
       37  Bottom 
       40  Liquid sample 
       50  Inside thread of the rotor 
       55  Outside thread of the rotor seat 
       60  Projection 
       70  Rotational interlock 
       80  Snap fit 
       90  Motor 
       100  Receptacle holder 
       110  Centrifuge unit 
       120  Guideway 
       130  Platform 
       140  Sample transfer unit 
     L Longitudinal direction 
     DA Rotational axis of the rotor 
     R o  Radius 
     R u  Radius