Researchers Submit Patent Application, "Apparatus for Isolation of Extracellular Vesicles Comprising Porous Polymer Monolith Microfilter", for Approval
The patent's assignee is
News editors obtained the following quote from the background information supplied by the inventors: "When diagnosed early, most cancers are more apt to be prevented from metastasizing, which gives rise to a higher survival rate for patients. However, high diversity of cancer-related factors contributes to the difficulty of early diagnosis. Active studies have been directed towards biomarkers that are available for the early diagnosis of cancer. One of the most interesting biomarkers is cancer cell RNA. For use in diagnosis, the RNA has been obtained from cancer cells separated from excised cancer tissues or from CTC (circulating tumor cells) in the bloodstream. However, the excision is accompanied by an operation that is invasive to the subject, and the finding of CTC is poor in effectiveness because it is too rare, e.g., one cells per 10.sup.9 blood cells, to detect.
"Most cells shed extracellular vesicles (EV) into body fluid. EV serves as a transporter carrying various materials including proteins, lipids, amino acids, etc. These substances have recently been revealed to be important factors that are indicative of properties of the EV sources, that is, the cells shedding the EV. Thus, newly increasing recognition has been given to the importance of EV. Particularly, cancer cells shed a number of vesicles containing metastasis factors. Cancer cell-shed vesicles are present at a higher level in blood and thus are easier to apply to the diagnosis of cancer, compared to CTC. The vesicles shed from cancer cells can, therefore, be used for early diagnosis of cancer in a non-invasive manner.
"In addition to the small size of vesicles, micron-sized substances and blood cells other than vesicles makes it difficult to obtain information on cancer cells from vesicles. For example, the extraction of information about vesicular nucleic acids of cancer cells by performing PCR (polymerase chain reaction), a powerful tool of analyzing nucleic acids, such as RNA, is obstructed by DNAs of blood cells such as leucocytes, and by pigments of hemoglobin. It is predicted that information derived from vesicles, if isolated with the exclusion of these obstacles, would be useful in the early diagnosis of cancer.
"However, no effective and accurate methods of isolating vesicles from body fluid have been developed, thus far. Centrifugation is representative of the isolation methods of vesicles developed thus far. Primarily, cells are, for example, separated from body fluid on the basis of density difference using a centrifugal separator. Then, centrifugation at higher speeds removes other microparticles and aqueous solutions. However, the centrifugation method is operated in multiple steps, requiring much labor. In addition, the method needs huge facilities as well as a large quantity of body fluid, and thus it is impossible to apply to practical diagnosis in which only a small amount of samples can be utilized. In addition, it takes at least 4 hours to complete the centrifugation method, so that it is not suitable for clinical diagnosis requiring rapidity. A method for isolating microvesicles from body fluid using a microchip onto which an antibody to vesicular proteins is immobilized was developed (US 20120142001 of Skog et al., entitled 'Method for isolation of nucleic acid containing particles and extraction of nucleic acids therefrom'), by which vesicles can be isolated within a short time. However, this method requires a centrifugation procedure for cell removal. A method that is capable of isolating vesicles from a small quantity of body fluid without using complex facilities such as centrifugation separators or without requiring the immobilization of antibodies to collection channels is advantageous in application to clinical diagnosis.
"Leading to the present invention, intensive and thorough research into the isolation of vesicles has resulted in developing a microfilter system that is capable of effectively isolating microvesicles in a non-invasive manner and in finding that the microfilter system is applicable to the clinical diagnosis of cancer."
As a supplement to the background information on this patent application, VerticalNews correspondents also obtained the inventors' summary information for this patent application: "Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and objects of the present invention are to provide an apparatus for the isolation of extracellular vesicles from body fluid, comprising a porous polymer monolith filter, a method for the fabrication thereof, and a method for diagnosing a disease using the same. However, it should be noted that the objects to be achieved by the present invention are not limited to the foregoing objects and that other non-mentioned objects could be apparently understood from the following descriptions by those skilled in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
"The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
"FIG. 1 shows a process of the fabrication of a PPM filter system in which a channel carved in a cross flow pattern on a microchip is filled with a PPM prepolymer solution and only a central region is exposed to UV to form a PPM filter.
"FIG. 2 shows SEM images of a conjunction between the PPM filter and the channel.
"FIG. 3 is a diagram showing a flow path of fluid in a pressure-driven PPM filter system, with vesicles and proteins separated from each other by the PPM filter, wherein the symbol X represents the temporary blockage of one end of each channel path;
"FIG. 4a shows the filtering capacity of a PPM filter prepared from a PPM prepolymer solution with a porogenic solvent content of 60% in which red fluorescent beads with a size of 1 .mu.m are entrapped by the filter (upper panel) whereas green beads with a size of 100 nm pass through the filter (lower panel), FIG. 4b is a graph in which estimated pore sizes are plotted against porogenic solvent contents, with an SEM image insert showing the entrapment of 5-.mu.m polystyrene beads by the filter prepared from a PPM prepolymer solution with a porogenic solvent content of 66%, FIG. 4c is a photographic image showing the formation of large pores in a PPM filter prepared from a solution with a porogenic solvent content of 67%, and FIG. 4d is a photographic image showing the formation of small pores in a PPM filter prepared from a solution with a porogenic solvent content of 58%.
"FIG. 5a is a photographic image of blood cells before passing through a filter, and FIG. 5b is a photographic image of the blood fluid after passing through the PPM filter in a pressure-driven mode, showing that cells are removed from blood.
"FIG. 6a is a graph in which cumulated volumes of the filtered fluid are plotted against filtration time according to porogenic solvent contents, and FIG. 6b is a graph showing the passage rates of fluid through the filters against filtration time.
"FIG. 7 is a graph showing the effect of an electric field as a driving force on the filtration of the protein BSA (bovine serum albumin) and vesicles in an electro-driven PPM filter system, in which the motility of each test sample is calculated by dividing the concentration of the filtrate by the concentration of the introduced sample.
"FIG. 8 is a schematic diagram of an electro-driven PPM filter system showing flow directions of fluid, and the passage of vesicles and proteins through the filter, with an electric field of 6.7 V/cm generated across the filter by applying a voltage of 10 V between two electrodes 1.5 cm apart from each other.
"FIG. 9a is a graph showing that both the pressure- and the electro-driven PPM filter system cause negligible hemolysis as compared to 40-fold diluted blood (2.5% hemolysis), and FIG. 9b is a graph showing that the content of cellular DNA in the filtrate using the pressure- or the electro-driven PPM filter system is too negligible to measure.
"FIG. 10 shows TEM images of vesicles obtained by ultracentifugation (A), a pressure-driven filter system (B), and an electro-driven filter system (C).
"FIG. 11 shows immunoblot images of an experiment group (treated with a primary and a secondary antibody) (left panel) and a control group (treated only with a secondary antibody) wherein, of the three bands visualized in the left panel, two came from IgG present in blood and the other one accounted for the quantity of CD9 expressed in vesicles, and wherein two IgG bands are only visualized in the right panel, demonstrating that the bands in other position of the experimental group represents CD9 protein.
"FIG. 12 is a table showing relative RNA contents to protein in vesicles separated from respective modes, wherein numerals in parentheses are error ranges of six measurements;
"FIG. 13 is a photograph of PCR products that were obtained from vesicles separated in respective modes wherein a band of the housekeeping gene .beta.-actin (upper) is constantly visualized for all groups, whereas a band of the cancer marker melan A (lower) is shown only on the lanes of the vesicles obtained by ultracentrifugation or the filter systems.
"FIG. 14 is a schematic diagram of an apparatus according to one embodiment of the present invention, wherein a microchip is represented by numeral 10, a channel by numeral 20, and a PPM filter by numeral 30."
For additional information on this patent application, see: PARK,
Keywords for this news article include: Antibodies, Cancer, Oncology, Peptides, Immunology, Amino Acids, Blood Cells, Legal Issues, Blood Proteins, Immunoglobulins,
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