Patent Application
20130130236
1. Field of the Invention
The present invention relates to separation methods and uses thereof, and more particularly, relates to a separation method for fluorescent magnetic nanodiamond-labeled cells and a use thereof.
2. Description of Related Art
In recent years, a variety of functional nanomaterials have been developed for applications in the biomedical field, such as the use of nanosomes for carrying drugs in cancer therapies (Batist G. et al., J. Clin Oncol. 2001, March 1; 19(5):1444-54; Chen Y., et al., Mol. Ther. 210 April; 18(4):828-34); the use of a fluorescent-bound nanoscale magnet to perform dynamic tracking and cell labeling (Maxwell D. J., et al., Stem Cells. 2008 February; 26(2):517-24; Ruan J. et al., Int J Nanomedicine, 20116:425-35.); the use of a combination of nanoscale quantum dots and silicon oxide in biological imaging (Erogbogbo F., et al., ACS Nano. 2011 Jan. 25; 5(1):413-23); the use of a combination of nanoscale quantum rods and phospholipid in tumor imaging and tacking (Yong K. T., et al., ACS Appl Mater Interfaces. 2009 March; 1(3):710-9); and the use of nanotubes for carrying drugs in cancer therapies (Liu Z. et al., Cancer Res. 2008 Aug. 15; 68(16):6652-60).
The limitations of the applications of nanomaterials in the biomedical field include the difficulty to track the positions and movements of the nanomaterials in vivo, the toxicity of quantum dots to organisms and the uncertain biocompatibility of nanoscale carbon materials such as carbon nanotubes and fullerenes.
As substitutes for the aforesaid nanomaterials, a nanodiamond (ND) is a nanomaterial applicable in the biomedical field, due to its excellent biocompatibility and low oxidative pressure induction (i.e., lower toxicity) as compared with other nanoscale carbon materials. It has been corroborated that ND can be used in many cell lines without obvious cytotoxicity (Liu K. K., et al., Biophys J. 2007 Sep. 15; 93(6):2199-208; Yu S. J., et al., J Am Chem. Soc. 2005 Dec. 21; 127(50):17604-5; Vaijayanthimala V., et al., Nanotechnology. 2009 Oct. 21; 20(42):425103; Huang H., et al., Nano Lett. 2007 November; 7(11):3305-14; Schrand A. M., et al., Nanoscale. 2011 February; 3(2):435-45). At the same time, ND does not lead to obvious abnormality in cell division, differentiation and morphological changes in embryonic development (Liu K. K., et al., Biomaterials 2009; 30(26):249-59; Mohan N., et al., Nano Lett. 2010; 10(9):3692-3699).
In terms of applications in the biomedical field, a ND has many advantages due to its biocompatibility, electrochemical and optical properties. (For example, biological molecules or therapeutic agents can be provided, either by chemical modification of the ND, or by allowing physical attachment to the ND, which acts as a convenient binding platform for chemical agents). The NDs with functionalized surface can bind to fluorescent molecules (Schrand A. M., et al., Nanoscale 2011; 3(2):435-445; Chang I. P., et al., J Am Chem Soc 2008; 130(46):15476-15481); lysozyme (Chao J. I., et al., Biophys J 2007; 93(6):2199-2208); growth hormones (Cheng C. Y., et al., Appl Phys Lett 2007; 90(16):3); DNA (Zhang X. Q., et al., ACS Nano 2009; 3(9):2609-2616); cytochrome c (Huang L. C., et al., Langmuir 2004; 20(14):5876-5884); proteins (Hartl A., et al., Nat Mater 2004; 3(10);736-742); and anti-cancer agents (Huang H., et al., Nano Lett 2007; 7(11):3305-3314; Liu K. K., et al., Nanotechnology 2010; 21(31):14); A ND can emit bright fluorescence, and does not bring about photobleaching and light scintillation (Yu S. J., et al., J Am chem. Soc 2005; 127(50): 17604-17605; Chao J. I., et al., Biophys J 2007; 93(6):2199-2208). The fluorescent property can be introduced to a ND by chemically modifying the ND with a fluorescent molecule, or by binding a fluorescent molecule to the ND (Schrand A. M., et al., Nanoscale 2010; 3(2):435-445; Chang I. P., et al., J Am Chem Soc 2008; 130(46):15476-15481; Hens S. C., et al., Diamond Relat Mater 2008; 17(11):1858-1866). In addition to the fluorescent property, a ND with magnetic property can be used in magnetic resonance imaging (MRI) detection. It has been reported that ion implantation of isotopes (15N) and (12C) can create magnetism in ND particles by generating sp2/sp3 defects (Talapatra S, et al., Phys Rev Lett 2005; 95(9):097201). Further, magnetic nanoparticles can be fixed onto the surfaces of NDs by using a metal-containing compound, to thermally damage a hot mixture of mineral oil and ND (Gubin S. P., et al., Diamond Relat Mater 2007; 16(11):1924-1928).
It has been reported that by using a microwave-arcing process, iron nanoparticles (ferrocence) is chemically bonded to the surface of an ND via a grapheme layer, to form a ND with fluorescence and magnetism (so-called fluorescent magnetic nanodiamond (FMND)) (Chang I. P., et al., J Am Chem Soc 2008; 130(46):15476-18481). Magnetic nanodiamond (MND) is chemically modified by surface grafting using polyacrylic acid, so as to allow the fluorescent portion to be covalently bonded to the surface of MND, and thereby converting MND to FMND. The fluorescent portion used in FMND is fluorescein O-methylacrylate, which emits bright green fluorescence at a wavelength of 488 nm, and collects the emitted light at a wavelength of 510 nm to 530 nm.
Although a theory postulating that endocytic ND clusters can be segregated during cell division and remain as a single ND cluster, there is currently no report on methods for separating ND-labeled cells and whether such labeled cell lines survive or have the ability to be sub-cultured.
The present invention provides a method for separating labeled cells and a use of the labeled cells. More specifically, the present invention relates to a method for separating cells labeled with FMNDs and a use thereof.
In an aspect of the present invention, a method for separating labeled cells is provided, wherein the method comprises the following steps: culturing target cells; co-culturing the target cells with the solution, so as to allow the FMNDs in the solution to label the target cells; and separating the FMND-labeled target cells from the co-culture of target cells and solution by means of the fluorescence and magnetism of the labeled target cells.
Further, the FMND-labeled target cells are separated by means of the difference between the fluorescence intensity of the FMND-labeled target cells and that of the unlabeled cells. In an embodiment, a flow cytometer is used to screen the FMND-labeled target cells.
Preferably, the method further comprises steps of washing of the co-culture of target cells and the FMND solution prior to use of the flow cytometer, so as to collect the FMND-labeled target cells and the unlabeled target cells; the method also comprises the subsequent concentration of the FMND-labeled target cells and the unlabeled target cells by centrifugation.
In another embodiment, a magnetic device is used to separate the FMND-labeled target cells. Preferably, the method further comprises the steps of washing of the co-culture of target cells and FMND solution prior to the use of a magnetic device, so as to collect the FMND-labeled target cells and the unlabeled target cells; the method further comprises concentrating the FMND-labeled target cells and the unlabeled target cells by centrifugation; and suspending the FMND-labeled target cells and the unlabeled target cells in a container with a buffer solution, so as to allow the magnetic device to adhere the FMND-labeled target cells onto the tube walls of the container.
The separation method of the present invention further comprises the step of cryopreserving the sorted FMND-labeled target cells.
In yet another aspect of the present invention, a separated cell is provided, wherein the cell is one that is separated according to the separation method of the present invention.
Further, the labeled and separated cell is used for standardization, detection, imaging and tracking of cells, the analysis of biomolecular activity and the screening of drug activity.
Further, the cell is an animal cell, including a cancer cell and a stem cell.
The present invention uses FMND-labeled target cells, and utilizes the fluorescence and magnetism of the FMND to separate the cells labeled by the labeling method. The labeled cells can continue to survive, be stored and be further cultured. The labeled cells are suitable for applications such as standardization, detection, imaging and tracking of cells, analysis of biomolecular activity and screening of drug activity.
The present invention is further exemplified by the following examples, but the examples should not be construed as intending to limit the scope of the present invention.
FMNDs were prepared as described using a published method (Chang I. P., et al., Am Chem Soc 2008; 130(46):15476-18481). Specifically, magnetic nanodiamonds (MNDs) were composed of pristine NDs and iron nanoparticles (ferrocene) via a microwave-arcing process. The ferrocene particles and NDs formed MNDs by chemically bonding. To introduce fluorescence in MNDs, MNDs were converted into FMNDs by covalent surface grafting with polyacrylic acids and fluorescein o-methacrylate. FMND particles were dissolved in distilled de-ionized water or phosphate-buffered saline (PBS), before the treatment of cells using FMND.
To examine the size distribution of FMNDs dissolved in DDW and PBS, the concentration of FMND particles in DDW or PBS (0.5 mg/ml) was prepared and analyzed by DLS (BI-200SM, Brookhaven Instruments Co., Holtsville, N.Y.). In a particular suspension, when a beam of laser hits the particle, the particles scattered some of the laser. The measured data were subjected to the BIC dynamic light scattering software (Brookhaven Instruments Co.). The scattered light changed over time, and the average particle size was calculated by the variation of scattered light.
HFL-1 cells (ATCC #CCL-153) were normal lung fibroblasts derived from a Caucasian fetus. The A549 lung epithelial cell line (ATCC #CCL-185) was derived from the lung adenocarcinoma of a 58 year-old Caucasian male. RKO was a human colon carcinoma cell line. BFTC905 cells were derived from human bladder carcinoma. MCF-7 was a human breast cancer cell line. HeLa was a c human cervical cancer cell line. HFL-1 and HeLa cells were maintained in DMEM medium (Invitrogen Co., Carlsbad, Calif.). A549, BFTC905 and MCF-7 cells were maintained in RPMI-1640 medium (Invitrogen). The complete media contained 10% fetal bovine serum (FBS), 100 unit/ml penicillin and 100 mg/ml streptomycin. These cells were incubated at 37° C., and maintained in 5% CO2 in a humidified incubator (310/Thermo, Form a Scientific, Inc., Marietta, Ohio).
The cells were plated in 96-well plates at a density of 1×104 cells/well for 16 to 20 hours. The cells were treated with or without FMNDs for 24 hours in complete medium. Subsequently, the medium was replaced and the cells were incubated with 0.5 mg/ml of 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma Chemical Co., St. Louis, Mo.) in complete medium for 4 hours. The survived cells converted MTT to formazan, which generated a blue-purple color when dissolved in dimethyl sulfoxide (DMSO). The intensity of formazan was measured at a wavelength of 565 nm using a plate reader (VERSAmax, Molecular Dynamics Inc., CA) for enzyme-linked immunosorbent (ELISA) assays. Cell viability was calculated by dividing the absorbance of the cells treated with FMNDs by that of the cells not treated with FMNDs.
A549 cells were seeded at a density of 1×106 cells per 100-mm Petri dish in complete medium for 24 hours. Then, the cells were treated with or without FMNDs (50 μg/ml) for 24 hours. Subsequently, the cells treated or untreated with FMNDs were re-cultured in fresh medium for counting the total cell number every 2 days, for a total of 10 days.
A549 cells were plated at a density of 7×105 cells per 60-mm Petri dish in complete medium for 16 to 20 hours. After treatment in medium with or without 10 to 100 μg/ml FMNDs, the cells were washed twice with PBS. The cells were trypsinized and collected by centrifugation at 1500 rpm for 5 minutes. The cell pellets were re-suspended in PBS. To avoid cell aggregation, the cell suspension was filtered through a nylon mesh membrane. Finally, the samples were analyzed by a flow cytometer (FACSCalibur, Becton-Dickinson, San Jose, Calif.). A minimum of 10,000 cells were analyzed. The fluorescence of FMNDs was triggered and emitted at a wavelength of 488 nm, and was collected in the green light signal range. The fluorescence intensity, particle complexity and cell size were quantified using a minimum of 10,000 cells by CellQuest software (BD, Biosciences).
The cells were cultured on cover slips, and kept in a 35-mm Petri dish for 16 to 20 hours before treatment. After treatment with or without FMNDs, the cells were washed with isotonic PBS (pH 7.4), and then were fixed with 4% paraformaldehyde solution in PBS for 1 hour at 37° C. Thereafter, the cover slips were washed three times with PBS, and non-specific sites were blocked in PBS containing 10% FBS and 0.3% Triton X-100 for 1 hour. The β-tubulin and nuclei were stained with anti-β-tubulin Cy3 (1:100) and Hoechst 33258 (Sigma Chemical co., St. Louis, Mo.) for 30 minutes at 37° C., respectively. Thereafter, the samples were examined under an OLYMPUS confocal microscope (FV500, OLYMPUS, Japan) that was equipped with an UV laser (405 nm), an Ar laser (488 nm) and a HeNe laser (543 nm).
The cells were plated at a density of 2×106 cells per 100-mm Petri dish in complete medium for 24 hours. After treatment with FMNDs (50 μg/ml) for 24 hours, the cells were washed twice with PBS. The washed cells (including the FMND-labeled A549 cells and unlabeled A549 cells) were trypsinized, and collected by centrifugation at 1500 rpm for 5 minutes to collect cell pellets. The cell pellets were re-suspended in 1 to 2 ml ice-cold sorting buffer, which contained 1 mM EDTA, 25 mM HEPES and 2% FBS in PBS. To avoid cell aggregation, the cell suspensions were filtered through a nylon mesh membrane. The fluorescence-activated cell sorting analyses were performed with a FACSCalibur sieve (Becton-Dickinson). The FMDN-bearing cells, which displayed green fluorescence intensity in a flow cytometer, were selected for separation. The separated cells were collected in a 50 ml centrifuge tube that had been coated with 10% FBS on the wall and contained 15 to 20 ml of complete medium inside. After separation, the cell suspensions were centrifuged at 1000 rpm for 10 minutes. Then, the cell pellets were re-suspended in complete medium. The cells were incubated at 37° C., in 5% CO2 in a humidified incubator, or added 10% DMSO for storage in liquid nitrogen.
The cells were plated at a density of 2×106 cells per 100-mm Petri dish in complete medium for 24 hours. After treatment with 50 μg/ml FMNDs for 24 hours, the cells were washed twice with PBS. The washed cells (including the FMND-labeled A549 cells and unlabeled A549 cells) were trypsinized, and collected by centrifugation at 1500 rpm for 5 minutes to collect cell pellets. The cell pellets were re-suspended in 1 ml PBS and transferred to 1.5 ml eppendorf tubes. The eppendorf tubes were placed onto a magnetic rack (Magna GrIP Rack, Millipore, Bedford, Mass.) for at least 3 minutes, until the cell pellets were absorbed on to the tube walls (Think OK, check). Then, the suspensions were removed, and the cell pellets were dissolved in complete medium. The cell suspensions within the eppendorf tubes were repeatedly placed onto the magnetic rack 5 times. Finally, the FMND-bearing cells were incubated at 37° C., in 5% CO2 in a humidified incubator, or added 10% DMSO for storage in liquid nitrogen.
To compare the total protein expression profiles between the parental and FMND-bearing cells, the cells were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The separated FMND-bearing cells were lysed in the ice-cold cell extraction buffer (pH 7.6), which contained 0.5 mM DTT, 0.2 mM EDTA, 20 mM HEPES, 2.5 mM MgCl2, 75 mM NaCl, 0.1 mM Na3VO4, 50 mM NaF and 0.1% Trinton-X10. The protease inhibitors included 1 μg/ml aprotinin, 0.5 μg/ml leupeptin and 100 μg/ml 4-(2-aminoethyl) benzenesulfonylfluoride were added to the cell suspension. The cell extracts were gently rotated at 4° C. for 30 minutes. After centrifugation, the pellets were discarded, and the protein concentrations of the supernatants were determined by a BCA protein assay kit (Pierce, Rockford, Ill.). Equal amounts of proteins (40 μg) were subjected to electrophoresis by 12% SDS-PAGE. After electrophoresis, the gel was stained with a coomassie blue buffer (0.1% coomassie blue, 10% acetic acid and 45% methanol) for 1 hour.
Data were analyzed using Student's t test, and a p value of <0.05 was considered as statistically significant in the experiments.
To measure the size distribution of FMNDs, 0.5 mg/ml of FMND solution prepared in DDW or PBS was analyzed by DLS. As shown in
To examine cell cytotoxicity following treatment with FMNDs in human lung cells, HFL-1 normal fibroblasts and A549 human lung carcinoma cells were used. The cells were treated with FMNDs, and analyzed by MTT assays. As shown in
A549 cells were treated with 50 μg/ml of FMND for 24 hours, and then further cultured for another 10 days. The total cell number was analyzed every 2 days.
To examine the uptake ability of FMNDs in A549 cells, the cells were treated with FMNDs (50 μg/ml), and analyzed by confocal microscopy. The FMND particles exhibited green fluorescence in A549 cells at a wavelength of 488 nm, and emission was collected in the range of 510 to 530 nm. The cytoskeleton of β-tubulin protein was stained with the Cy3-labeled mouse anti-β-tubulin. The nuclei were stained with Hoechst 33258 that presented with blue color. As shown in
Detection of A549 Lung Cancer Cells with FMNDs by a Flow Cytometer
The fluorescence intensity of FMNDs in A549 cells was examined by the flow cytometer. A549 cells were treated with FMNDs (10 to 100 μg/ml, 24 hours), and then analyzed by the flow cytometer.
The fluorescent and magnetic properties of FMNDs provided for (contributed to) the separation of ND-bearing cells by FACS-purified function of the flow cytometer with a sieve and magnetic device. To separate the ND-bearing cells, A549 cells were treated with FMND (50 μg/ml, 24 hours), and collected by the flow cytometer with a sieve.
After separation, the FMND-bearing cells were immediately examined under a live-cell imaging system of the fluorescent and phase contrast microscope. Comparison with the parental cells shows that the separated FMND-bearing cells exhibited significant fluorescence intensity under a fluorescent microscope (
A549 cells were treated with or without 50 μg/ml of FMND for 24 hours. The FMND-bearing cells were separated by the magnetic device as described previously. The cell morphology, viability and growth ability of the FMND-bearing cells of different generations were similar to the parental cells (
The separating ratio of the generations of the FMND-bearing cells was calculated by dividing the separated FMND-bearing cell number by the total cell number counted before separation. The separating ratio of the first generation had an average of 75.84%, and the separating ratio in the fourth generation had an average of 60%. The first generation of the FMND-bearing cell lines was stored in liquid nitrogen. After re-thawing of the FMND-bearing cells, the cell morphology and viability were still similar to the parental cells (
According to the present invention, a method for labeling cells using fluorescent magnetic nanodiamonds is provided. This invention also allows the use of the flourescent and magnetic properties to separate the cells which had been pre-labeled. According to the method of the present invention, the labeled or separated cells have cell morphology, viability and growth ability similar to those of the parent cells after sub-culturing or Cryopreservation. The method provided by the present invention is useful in the standardization, detection imaging or tracking of cells, the analysis of biomolecular activity and screening of drugs in the biomedical field.
The above examples only exemplify the principles and the effects of the present invention. They are not used to limit this invention. It is possible for those skilled in the art to modify and/or alter the above examples (whilst/before) carrying out this invention without contravening its spirit and scope. Therefore, the protection scope of this invention should be indicated as stated by the following claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 100141978 | Nov 2011 | TW | national |
