Patent Grant
8039249
The present invention relates to a cell separation device, and in particular to a compact disk based platform for separating and detecting cells labeled with immunomagnetic beads.
Detection and quantification of cancer cells or rare cells present in body fluids are regarded as a potential indicator for clinical diagnoses, prognostication, and biomedicine research. For example, circulating tumor cells (CTC) are rare in the blood of patients with metastatic cancer, and it is possible to monitor the response of CTC to adjuvant therapy. To detect and quantify the rare cells present in fluids, the rare cells must be first separated. Thus, cell separation techniques are developed.
Various cell separation techniques are now available, including fluorescence activated cell separation (FACS), dielectrophoresis (DEP) cell separation, separation techniques that employ massively parallel microfabricated sieving devices, magnetically activated cell separation (MACS), and other techniques that uses optics and acoustics. Among these cell separation techniques, FACS and MACS are most often used.
Although it is often used, FACS suffers several drawbacks, including high cost, difficult disinfection, and consuming a great amount of sample. Contrary to FACS, MACS is efficient to obtain a major quantity of the target cells in a short period and reduces the consumption of the sample. However, these cells must be transferred to a slide or an observation platform before they can be observed with a microscope. Such a process of transfer often leads to a great cell loss.
U.S. Pat. No. 5,565,105 discloses a magnetocentrifugation method, wherein charged particles are deposited in a rotor board and a magnetic field is vertically applied to the rotor board, whereby when the rotor board is brought into rotation, the charged particles contained in the rotor board are moved within the magnetic field, and are thus acted upon by Lorentz force to separate from non-charged particles.
U.S. Pat. No. 6,297,062 discloses a method for separating at least one species of biological entities from a sample solution. Each species being a first member of a pair forming group, from a sample solution by contacting the sample solution with a matrix of magnetic particles wherein each magnetic particle in the matrix is coupled to the second member of the pair forming group. The matrix should contain magnetic particles, coupled to several different species of second members of the pair forming groups. When the sample is contacted with said matrix, and each species of biological entities, binds to its specific second member of the pair forming group which is present in a discrete location, from the other entities, and due to the magnetic properties of the magnetic particles, each species may be obtained separately.
U.S. Pat. No. 6,723,510 discloses a method for separating particles with minimized particle loss, wherein a detergent containing matrix beads are bound with a sample containing target particles so as to reduce the loss of the target particles in the separation processes.
U.S. Pat. No. 7,094,354 discloses a microfluidic device provides separation of particles in a liquid sample, particularly, separation of a sample of whole blood into its components for further analysis. Separation into red blood sample has been transferred into a separation chamber with the application of centrifugal force of less than about five times gravity. When blood in the sample, a separation chamber for receiving the sample and separating it into its fractions using low gravitational forces, and vents for removing the air displaced by blood and its fractions.
In view of the above described, FACS needs a high cost system and requires extended time in operation and MACS is effective in obtaining a major portion of target cells and thus reduces the operation time for sampling in doing analysis. However, MACS may suffer the disadvantage of high cell loss.
Thus, an objective of the present invention is to provide a compact disk based platform for separating and detecting cells labeled with immunomagnetic beads, which is low cost, is easy to perform detection and observation, and has reduced cell loss, and which is applicable to separate at least two cells that are respectively labeled and not labeled with the immunomagnetic beads.
The solution adopted in the present invention to overcome the problems of the conventional techniques is a disk-like carrier board and a magnetic attraction unit, wherein the disk-like carrier board forms at least one flow channel structure, which comprises an inner reservoir, an outer reservoir, and a plurality of micro flow channels. The inner reservoir is arranged adjacent to a geometric center of the disk-like carrier board for receiving a sample fluid, which contains at least two cells that are respectively labeled and not labeled with immunomagnetic beads. The outer reservoir is arranged adjacent to an outer circumferential rim of the disk-like carrier board. The plurality of micro flow channels is arranged between and in fluid communication with the inner and outer reservoirs. The magnetic attraction unit is arranged above the disk-like carrier board and adjacent to the inner reservoir to generate a magnetic force having a predetermined distribution of intensity.
The solution adopted in the present invention is effective in maintaining the cells labeled with immunomagnetic beads in the inner reservoir by using the magnetic force and allowing the cells that are not labeled with immunomagnetic beads to move to the outer reservoir by means of a centrifugal force so as to separate the cells that are labeled with the immunomagnetic beads.
Further, the compact disk based platform for separation and detection of immunomagnetic bead labeled cells can be easily manufactured by means of for example laser machining, CNC machining, micromachining, or injection molding and the material that is used to make the platform can be easily acquired, thereby offering an advantage of low cost manufacturing.
Further, the cells that are not labeled with immunomagnetic beads can be directly collected in collection bins to allow an operator to carry out direct observation without transferring the cells to a slide or an observation platform. This makes the observation easy and, due to no transfer of cell needed, cell loss can be minimized.
The present invention will be apparent to those skilled in the art by reading the following description of the preferred embodiments of the present invention, with reference to the attached drawings, in which:
With reference to the drawings and in particular to
Also referring to
The flow channel structure 2 is formed in the flow channel structure layer 15 of the disk-like carrier board 1. In the instant embodiment, the base layer 14 and the flow channel structure layer 15 are made of acrylic resins, such as polymethylmethacrylate (PMMA) and the cover layer 16 is comprised of a layer of a thin film. The flow channel structure layer 15 is processed by CO2 laser machining to form the flow channel structure 2 and is then bound to the base layer 14. Thereafter, the cover layer 16 is applied atop the flow channel structure layer 15 to completely cover and enclose the flow channel structure 2. This way is advantageous by being easy to manufacture, using low cost materials, and reducing manufacturing costs.
Apparently, the flow channel structure layer 15 can alternatively be formed as a multiple-layered structure including multiple plates stacked and bound together. Further, the disk-like carrier board 1 can be alternatively made a single-layered structure and the material used is not limited to acrylic reins. The flow channel structure 2 can alternatively formed by employing machining techniques other than laser machining, such as CNC machining, micromachining, and injection molding.
Also referring to
The inner reservoir 21 has an inner bank 211, which is adjacent to the geometric center 11 of the disk-like carrier board 1, and an outer bank 212, which is in fluid communication with the micro flow channels 22. In the inner bank 211 of the inner reservoir 21, a fluid inlet opening 213 is defined and extends in a direction toward the geometric center 11 of the disk-like carrier board 1.
The outer reservoir 23 also has an inner bank 231, which is in fluid communication with the micro flow channels 22, and an outer bank. 232, which is adjacent to the outer circumferential rim 12 of the disk-like carrier board 1. In the instant embodiment, the inner bank 231 of the outer reservoir 23 forms at least one vent hole 233. The outer bank 232 defines a plurality of collection bins 24.
In the instant embodiment, the magnetic attraction unit 3 comprises a magnetic ring; alternatively, it can be replaced by an array of magnets that are properly arranged. Various magnets, such as permanent magnets and electromagnets, can be used. The magnetic attraction unit 3 is arranged above the disk-like carrier board 1 and close to the central hole 13 of the disk-like carrier board 1 to generate a magnetic force Fb having a predetermined distribution of intensity.
In the instant embodiment, the driving device 4 is arranged below the disk-like carrier board 1 and the spindle 41 is operatively coupled to the central hole 13 of the disk-like carrier board 1 for spinning the disk-like carrier board 1.
Referring to both
A sample fluid 5 that is subjected to cell separation is introduced through the fluid inlet opening 213 into the inner reservoir 21. In the instant embodiment, the sample fluid 5 contains two cells, one being Jurkat cell (J), which is a human lymphoma cell and the other being MCF7 cell (M), which is a human breast cancer cell. The Jurkat cells J are labeled with immunomagnetic beads C that contain CD45 anti-body A. The MCF7 cells M are not labeled. It is apparent that a sample fluid containing more than two cells, of which one is labeled with immunomagnetic beads, can be used.
The disk-like carrier board 1 is driven by the driving device 4 to spin in a given spinning direction I. The sample fluid 5 is acted upon by the centrifugal force Fc induced by the spinning of the disk-like carrier board 1 and flows from the inner reservoir 21 through the micro flow channels 22 to the outer reservoir 23.
Since the Jurkat cells J contained in the sample fluid 5 are labeled with the immunomagnetic beads C, and since the immunomagnetic beads C are attracted the magnetic force Fb generated by the magnetic attraction unit 3, the Jurkat cells J will remain in the inner reservoir 21. The MCF7 cells M that are not labeled with immunomagnetic beads C are driven by the centrifugal force Fc to flow with the sample fluid 5 from the inner reservoir 21 through the micro flow channels 22 into the outer reservoir 23 to be collected in the collection bins 24.
Referring to
In practical applications, the multiple flow channel structures 2 formed in the disk-like carrier board 1 allows for simultaneously performing separation, as well as subsequent detection/observation, for multiple sample fluids, whereby operation efficiency can be enhanced. In respect of detection and observation, cells to be detected/observed, such as MCF7 cells M in the instant embodiment, can be labeled with fluorescence substance in advance to enhance the observation.
Referring to
Referring to
Referring to both
When the disk-like carrier board 1 is driven by the driving device 4 to spin, the Jurkat cells J that are labeled with immunomagnetic beads C are attracted by the magnetic force Fb′ generated by the magnetic attraction unit 3′. In case that some of the Jurkat cells J are not attracted and securely held by the magnetic ring 31 that is closet to the geometric center 11 of the disk-like carrier board 1, they will still be attracted and held by the magnetic rings 32, 33, 34 that are located outside the magnetic ring 31. In this way, the Jurkat cells J that are labeled with immunomagnetic beads C will be step-by-step acted upon by the magnetic forces individually generated by the sequentially arranged magnetic rings to ensure that the Jurkat cells J that are labeled with immunomagnetic beads C will be retained in the inner reservoir 21 thereby enhancing the result of separation.
Although in the preferred embodiments of the present invention, MCF7 cells M and Jurkat cells J are taken as examples for discussion purposes, the present invention is also applicable to for example fetal cell separation, cell separation for whole blood sample, separation of endothelial colony forming cells (ECFC) from umbilical cord blood (UCB).
Although the present invention has been described with reference to the preferred embodiments thereof, it is apparent to those skilled in the art that a variety of modifications and changes may be made without departing from the scope of the present invention which is intended to be defined by the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 97148520 A | Dec 2008 | TW | national |
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| Number | Date | Country | |
|---|---|---|---|
| 20100151560 A1 | Jun 2010 | US |
