JP2014060996A - Device and process for isolating and cultivating live cells on filter or extracting their genetic material - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a device and a process for isolating live cells on a filter or extracting their genetic material.SOLUTION: The invention comprises steps of attaching, at least temporarily, a filter (108) to a lower opening of a compartment (102) having an air inlet; inserting into the compartment a liquid carrying cells; attaching, in an impermeable manner, a needle, at least temporarily, to a compartment opening, the filter being positioned between the needle and the interior volume of the compartment; perforation, with the needle, of a plug of a vacuum tube with negative pressure relative to ambient pressure; and aspiration, by means of negative pressure from the vacuum tube, of the liquid through the filter, the filter retaining the cells.

Description

  The present invention isolates and / or cultures live or fixed cells on a filter to perform all cell analysis (cytology, immunochemistry, FISH tests, etc.) or simply on a filter. The present invention relates to an apparatus and a method for extracting genetic material amplified as necessary from detached living cells or fixed cells. The invention has particular application in isolating and / or culturing specific cells present in a liquid (especially blood) or extracting the genetic material of these specific cells.

  Certain blood cells (eg, tumor cells or trophoblast cells) are present in very small proportions and must be counted before performing cytological analysis.

  It is known to apply formaldehyde-based binding buffer to a blood sample to immobilize the desired cells and then pass the resulting liquid through a porous filter. The filter is then used to examine the desired cells on the filter under a microscope in the laboratory. However, live cells cannot be obtained using this procedure.

  Therefore, the present inventor can identify a specific marker if a living cell is obtained, and when diagnosing a gene abnormality of a tumor or a trophoblast cell, a molecular biology technique, a cytogenetic technique And the technique of FISH (an acronym for “fluorescence in situ hybridization”) was determined to be applicable under good conditions.

  The present invention addresses these disadvantages by allowing live cells to be collected and subsequently cultured in a suitable medium in the presence of suitable growth factors, under conditions suitable for standard laboratory testing. The purpose is to meet this demand.

  The present invention also relates to the extraction of optionally amplified genetic material from cells isolated on the filter and the detection of variability and gene expression levels related to sensitivity and resistance to target therapy or gene abnormalities.

  The invention is particularly applicable to the recovery and potential homogenous amplification of specific cellular DNA or RNA present in fluids (especially blood).

  For example, from the document PCT / FR2006 / 000562, it is known that a binding buffer based on formaldehyde is applied to a blood sample to fix the desired cells to be sought and then the resulting liquid is passed through a porous filter. ing. The filter is then analyzed under a microscope in the laboratory and cells are detected on the filter. Next, the cells can be collected on a filter and analyzed (eg, gene analysis).

  However, this procedure cannot be reproduced on a large scale at a reasonable cost because of the time, materials and work accuracy required. Therefore, if it is reproduced on a large scale and at low cost, molecular biology analysis can be performed on both tumor cells and trophoblast cells. Furthermore, when cells are fixed with formaldehyde, good quality genetic material cannot be obtained, DNA is partially decomposed to form a cross-link with surrounding proteins, and RNA extraction becomes almost impossible.

  The present invention enables these to be recovered in good condition from the cells under consideration under conditions that are compatible with standard laboratory tests. The purpose of this is to meet this demand. It should also be noted that the present invention makes it possible to isolate fixed cells using fixatives with or without formaldehyde.

To this end, the present invention, according to one aspect, is an apparatus for isolating fixed or live cells on a filter comprising:
A compartment comprising an internal space for containing the liquid containing the cells, a lower opening and an air inlet;
A filter that at least temporarily forms a single unit with the compartment opening for retaining the cells as liquid passes through the filter;
The compartment opening and at least temporarily a needle forming an impermeable single unit, the filter being located between the needle and the interior space of the compartment, the needle comprising: The present invention relates to an apparatus characterized in that a hole is made in a stopper of a vacuum tube that has a negative pressure compared to the ambient pressure in order to suck liquid through the filter.

  Thanks to these features, the device can be used to analyze the fixed living cells of interest, or to analyze cytological characteristics, and cytogenetic experiments for any other cellular test. Living cells that are perfectly suited for culturing to perform can be isolated and collected on a filter, or genetic material can be extracted therefrom.

  This recovery is done directly on the filter and the desired cells are rarely lost. In this way, the majority of the cells under investigation are recovered at low cost and in good condition under conditions suitable for normal laboratory culture. The isolated cells can be used for cell experiments or molecular biology experiments before or after culturing.

With the apparatus constituting the present invention, the cellular material of a specific cell, for example,
A filtration step through which most of the liquid and the other cells are passed through a filter having a micropore with a diameter intermediate between the diameter of the particular cell and the diameter of the other cell;
A lysis step (subsequently with or without DNA and / or RNA amplification in the compartment);
-It is also possible to recover quickly and efficiently after the step of recovering the lysed cellular genetic material on the filter.

The apparatus comprising the present invention has many advantages: 1 / Filtration on the rack or while holding the apparatus in hand can save considerable time and materials (especially vacuum pumps and adapter boxes) ).
2 / Filtration can be done in a sterile hood.
3 / Filtration can be carried out with the device tilted and even approximately horizontal.
4 / conditions are fully standardized and sampling tubes are provided in a standardized manner using a predetermined vacuum suction.
5 / Since the conditions of use include enhanced safety conditions, the device can be fully operated in a sterile hood. Moreover, since the collected blood does not come into contact with the operator, once the vacuum tube is filled it can be removed like a normal blood sampling tube.

  According to a particular feature, the device according to the invention as described above comprises a compartment and a protective cylinder adapted to surround the needle and at least partly surround the vacuum tube while aspirating liquid towards the filter. Further connecting means is provided.

Thanks to such a configuration, the user is protected from accidental needle sticks. Furthermore, the use of the protective cylinder as a guide makes it easier to position the vacuum tube.
Also, the vacuum tube is held firmly in place during aspiration, which causes the vacuum tube to move or shift, preventing air from entering the vacuum tube through the side of the needle in the vacuum tube plug. It is preventing. Since the user does not accidentally touch the needle, the risk of sample contamination by the user is minimized.

  According to a particular feature, the device according to the invention as briefly described above comprises at least temporarily the protective cylinder.

  In this way, protection of the needle, the sample and the user is ensured.

  According to a particular feature, the connecting means is temporary and it is possible to remove the protective cylinder together with the vacuum tube or the needle.

  In this way, the protection of the needle and the user is ensured after separating the protective cylinder from the compartment.

  According to a particular feature, the protective cylinder is provided with a removable film that seals its opening facing the connecting means, through which the vacuum tube is inserted into the protective cylinder.

  Thanks to such a configuration, the vacuum tube is inserted after the user removes the film. This film not only provides better protection from users and needles, but also reduces the risk of sample contamination.

  According to a particular feature, the device according to the invention further comprises a removable filter support made of surgical steel made to be temporarily connected to the lower opening of the compartment.

  Thanks to such a configuration, the filter can be removed with its holder and the cells collected on the filter can be cultured or analyzed or genetic material can be extracted therefrom. Moreover, steel is not toxic to the cells collected on the filter.

  According to a particular feature, the ring is designed with a scannable thickness.

  According to a particular feature, the ring has a discriminator.

  Thus, the identifier, and thus the collected cells, can be associated with the patient. Mistakes are prevented in this way.

  According to a particular feature, the device according to the invention further comprises movement means for the compartment for applying a force to the filter support to release the filter support.

Since the entire filtration module is sterile and manufactured using standards that are commonly applied in molecular biology, any contamination or degradation of live cells or genetic material on the filter can be prevented. The contents of the compartment are kept sterile while operating under a suitable laminar flow hood.
Thus, all steps for isolating and culturing cells or analyzing their genetic material can be performed under aseptic conditions.

  According to a particular feature, the device according to the invention comprises a removable tip attached to the compartment so as to be impermeable and removable. This removable tip is designed to limit the relative movement of the moving means and compartment allowing the force to be applied to release the filter support.

  Thanks to such a configuration, the filter holder is securely held in place. In addition, the tip can protect the filter holder from splashing and contamination.

  Thus, during filtration, the filter is held and then the tip is removed and the filter is released and recovered by releasing the filter support by moving the moving means and compartments respectively and applying force. Can do.

According to a second aspect, the present invention is a process for isolating live cells on a filter or extracting its genetic material,
Attaching a filter at least temporarily to the lower opening of the compartment further comprising an air inlet;
Inserting the liquid containing the cells into the compartment;
Attaching the needle impermeable, at least temporarily to the compartment opening, and placing the filter between the needle and the interior space of the compartment;
Piercing with a needle a vacuum tube plug that has a negative pressure compared to the ambient pressure;
-Suctioning liquid through the filter by negative pressure from a vacuum tube, the filter holding the cells.

According to particular features, the process of the present invention as briefly described above further includes:
-Securing the protective cylinder at least temporarily to the compartment, after which the protective cylinder surrounds the needle;
Inserting a vacuum tube into the protective cylinder during the drilling step.

  The advantages, objectives and specific features of this process are similar to the advantages, objectives and specific features of the apparatus of the present invention as briefly described above and will not be repeated here.

  Other advantages, objects and features of the invention will become apparent from the following description with reference to the accompanying drawings. Note that this description is for the purpose of explanation and is not exhaustive.

It is a schematic perspective view of the assembly of the components of the first embodiment of the apparatus constituting the present invention. It is the schematic sectional drawing which cut | disconnected 1st Embodiment of the apparatus before use. It is the schematic sectional drawing which cut | disconnected 1st Embodiment of the apparatus before use. It is the schematic sectional drawing which cut | disconnected 1st Embodiment of the apparatus before use at the cut surface which cross | intersects the cut surface of FIG. 2A perpendicularly | vertically. It is the schematic sectional drawing which cut | disconnected 1st Embodiment of the apparatus before use at the cut surface which cross | intersects the cut surface of FIG. 2B perpendicularly | vertically. It is a schematic elevation view of the execution step of 1st Embodiment of the apparatus which comprises this invention. It is a schematic sectional drawing of the execution step of 1st Embodiment of the apparatus which comprises this invention. It is a schematic sectional drawing of the execution step of 1st Embodiment of the apparatus which comprises this invention. It is a schematic sectional drawing of the execution step of 1st Embodiment of the apparatus which comprises this invention. It is a schematic elevation view of the execution step of 1st Embodiment of the apparatus which comprises this invention. It is a schematic elevation view of the execution step of 1st Embodiment of the apparatus which comprises this invention. It is a schematic elevation view of the execution step of 1st Embodiment of the apparatus which comprises this invention. It is a schematic sectional drawing of the execution step of 1st Embodiment of the apparatus which comprises this invention. It is a schematic sectional drawing of the execution step of 1st Embodiment of the apparatus which comprises this invention. It is a schematic sectional drawing of the execution step of 1st Embodiment of the apparatus which comprises this invention. It is a schematic sectional drawing of the execution step of 1st Embodiment of the apparatus which comprises this invention. It is a schematic sectional drawing of the execution step of 1st Embodiment of the apparatus which comprises this invention. It is a schematic sectional drawing of the execution step of 1st Embodiment of the apparatus which comprises this invention. It is a schematic sectional drawing of the execution step of 1st Embodiment of the apparatus which comprises this invention. It is a schematic sectional drawing of the execution step of 1st Embodiment of the apparatus which comprises this invention. Figure 3 is a flow chart of the steps performed in the first specific embodiment of the process making up the present invention. FIG. 6 is a schematic perspective view of the assembly of parts of a second specific embodiment of the apparatus constituting the present invention. It is the schematic sectional drawing which cut | disconnected 2nd Embodiment of the apparatus before use. It is the schematic sectional drawing which cut | disconnected 2nd Embodiment of the apparatus before use by the cut surface which cross | intersects the cut surface of FIG. 6A perpendicularly | vertically. It is the schematic sectional drawing which cut | disconnected 2nd Embodiment of the apparatus before use. It is the schematic sectional drawing which cut | disconnected 2nd Embodiment of the apparatus before use by the cut surface which cross | intersects the cut surface of FIG. 6C perpendicularly | vertically. FIG. 6 is a schematic elevation view of the steps performed in the second embodiment of the apparatus making up the present invention. It is a schematic sectional drawing of the step performed by 2nd Embodiment of the apparatus which comprises this invention. It is a schematic sectional drawing of the step performed by 2nd Embodiment of the apparatus which comprises this invention. It is a schematic sectional drawing of the step performed by 2nd Embodiment of the apparatus which comprises this invention. FIG. 6 is a schematic elevation view of the steps performed in the second embodiment of the apparatus making up the present invention. FIG. 6 is a schematic elevation view of the steps performed in the second embodiment of the apparatus making up the present invention. FIG. 6 is a schematic elevation view of the steps performed in the second embodiment of the apparatus making up the present invention. It is a schematic sectional drawing of the step performed by 2nd Embodiment of the apparatus which comprises this invention. It is a schematic sectional drawing of the step performed by 2nd Embodiment of the apparatus which comprises this invention. It is a schematic sectional drawing of the step performed by 2nd Embodiment of the apparatus which comprises this invention. It is a schematic sectional drawing of the step performed by 2nd Embodiment of the apparatus which comprises this invention. It is a schematic sectional drawing of the step performed by 2nd Embodiment of the apparatus which comprises this invention. It is sectional drawing of the filter support body integrated in 2nd Embodiment of the apparatus which comprises this invention. Figure 6 is a flow chart of the steps performed in the second specific embodiment of the process making up the present invention. FIG. 2 is a schematic view of a particular embodiment of the device prior to use. FIG. 11 is a schematic view of the embodiment of the apparatus shown in FIG. 10 after removing the protective film and before inserting the vacuum tube. FIG. 12 is a schematic view of the embodiment of the apparatus shown in FIGS. 10 and 11 after insertion of a vacuum tube. FIG. 13 is a schematic view of the embodiment of the apparatus shown in FIGS. 10-12 when the vacuum tube and protective cylinder are removed. Figure 5 is a flow chart of the steps performed in a particular embodiment of the process making up the present invention.

  In the following description of the drawings, the system assumes the filtration of liquids (especially blood), which comprises means for removing the filter support. However, the invention is not limited to these preferred embodiments, but a compartment for containing a liquid, a filter that is at least temporarily attached to the opening of the compartment, and an opening of the compartment at least temporarily. And a needle that is attached so that it does not leak, and while the hole in the pre-loaded vacuum tube is pierced, the needle pumps the liquid sucked through the filter, and the target cells are retained on this filter. Extends to any system.

  The needle has a very fine tip (see FIGS. 3B-3D) designed to penetrate the tube stopper and a wider aperture designed to accommodate the lower end of the compartment or filter support. The negative pressure of the vacuum tube is quite large and the space is larger than the volume of liquid to be filtered.

  In a preferred embodiment in which the needle can be removed from the compartment, once the compartment is filled with a liquid containing the cells of interest, the needle aperture is aligned with the opening of the compartment to recover the cells, and then the needle is thin. Make a hole in the stopper of the vacuum tube with the tip. Since negative pressure is applied, liquid filtration usually occurs automatically within 60 seconds.

  In this way, filtration can be performed while holding the apparatus in hand. In particular, this saves a lot of time and material since there is no longer any need to have a vacuum pump or an adapter box for the compartment in the pump.

  Furthermore, since the filtration can be performed in a sterile hood, the device can be tilted or even placed horizontally.

  Next, an embodiment is described having a removable filter support and means for removing the removable filter support from the compartment without touching it.

  FIG. 1 shows a reservoir or compartment 102, a tip 104, a seal 106, a filter and its support 108, a moving means 110, a seal 112, and a plug 114 with an air inlet (not shown).

  The compartment 102 is cylindrical. Its upper end can be sealed with a plug 114 so that it is impermeable except for the air inlet. The lower end of the compartment 102 has a discontinuous ring on the outer surface that guides the protrusion of the moving means 110, and the ring guides the body of the moving means 110.

  The moving means 110 is generally cylindrical and has two protrusions extending toward the tip 104. These protrusions narrow toward the tip 104 while being separated from each other by a distance smaller than the diameter of the filter support 108. As will be explained later, this special shape, particularly the shape in which the protrusions 116 are curved toward each other, causes the moving means 110 to apply pressure to the filter support 108 after removal from the tip 104. So that the filter support is released from the compartment 102 along with the filter while the moving means 110 is moving toward the filter support 108.

  The end of the protrusion 116 of the moving means 110 and the lower end of the compartment 102 are designed to be inserted into a cell culture box or cell culture well. On the other hand, the discontinuous ring at the end of the compartment 102 has a diameter such that the compartment can be supported on the edge of a cell culture box or cell culture well.

  A tip or adapter 104 is located in the lower opening of the compartment 102 so that the compartment is impermeable, sterile and removable. The tip 104 tightens the outer wall of the compartment 102 and has a tapered lower opening with a diameter smaller than the diameter of the compartment 102.

  This tapered lower opening of the tip or adapter 104 is long enough to allow mechanical attachment without leakage of the needle aperture (see FIGS. 3B-3D).

  In a shape that matches the shape of the lower end of the compartment 102 having the side projections 118, the tip 104 has rotation locking means for tightening the projections in a known manner. In this manner, the tip 104 securely holds the filter holder in place during the filtration step. Furthermore, the tip 104 protects the filter from splashing and contamination.

  When attached, the lower end of the compartment 102 has an opening that opens toward the filter held by the filter support 108. One end of the filter support itself is held in place by the lower end of the compartment 102 and the other end by the tip 104.

  In the first embodiment, the filter support 108 has a ring-shaped washer shape. The filter has fine holes. The filter is attached to the bottom of the filter support 108 and then inserted into the lower end of the compartment 102 together with the washer.

  The removable filter support 108 is preferably ring-shaped and made of surgical steel. In fact, steel is not toxic to the cells collected on the filter. Furthermore, the filter support made of surgical steel is easier to remove and stronger than if it is made of plastic. The ring is preferably designed with a scannable thickness. This ring preferably has a discriminator. In this way, the discriminator and thus the collected cells can be associated with the patient. Mistakes are prevented in this way.

  Alternatively, the filter support 108 is made of PVC and has a thickness of 0.4 mm or less, preferably less than 0.3 mm. The outer diameter is 12.6 mm, for example. The diameter of the filter to which the filter support 108 is attached is, for example, 5.9 mm.

  The compartment 102, the tip 104, and the moving means 110 are made of, for example, polypropylene. The seals 106 and 112 are made of silicone, for example.

  2A and 2C are vertical axis cross-sectional views of a first embodiment of the apparatus constituting the present invention with the components shown in FIG. 1 assembled. 2B and 2D are enlarged views of the components of FIGS. 2A and 2C, respectively. 2A and 2D, the elements described with respect to FIG. 1 can be seen.

  FIG. 3A is an elevational view when the device is in a stored configuration. FIG. 3B shows the tip 104 being inserted into the aperture 181 of the needle 180. The needle has another very thin, slanted end 182 that makes it easier to puncture the tube stopper. The aperture 181 of the needle 180 is preferably made of plastic. The end 182 of the needle 180 is preferably made of metal. Needle 180 may be placed over tip 104 before or after liquid (eg, blood) (not shown) is introduced into compartment 102 through its upper opening.

  FIG. 3C shows the needle 180 being imperviously connected to the tip 104 and starting to open a hole in the plug 186 of the vacuum tube 185.

  FIG. 3D shows that the hole is completely penetrated through the plug 186 by the needle 180, and the inside of the vacuum tube 185 having a negative pressure is connected to the space of the compartment 102 containing the liquid containing the target cell through the filter 108. Show. The internal space of the vacuum tube 185 is larger than the volume of liquid to be filtered.

  During aspiration, some of the larger diameter cells in the liquid present in the compartment 102 are retained by the filter 108, whereas the majority of the liquid and the contents of the lysed cells and (if necessary) Cells that are smaller in size than the walls and cells to be collected are drawn through the filter 108 into the vacuum tube 185.

  Next, as shown in FIGS. 3E and 3F, the tip 104 is rotated to release from the protrusion 118 and removed. Next, as shown in FIGS. 3G and 3H, the tip of the compartment 102 is inserted into a cell culture box or culture well 130.

  As described above and shown in FIG. 3I, the end of the moving means 110 near the protrusion 116 and the lower end of the compartment 102 are designed to be inserted into a cell culture box or culture well. In contrast, the discontinuous ring at the end of the compartment 102 has a diameter sufficient to support the ring at the edge of the culture box or culture well 130.

  More precisely, as shown in FIGS. 3J and 3K, the moving means 110 can still move parallel to the axis of the compartment 102 at this position.

  As shown in FIGS. 3N and 3M, during this movement, the protrusion 116 of the moving means moved by the operator's finger exerts a vertical force on the filter support 108 from top to bottom, The filter support is released from the lower end of the compartment 102. The filter and filter support 108 then fall into the cell culture box or culture well 130.

  Finally, as shown in FIG. 3O, the compartment 102 and the moving means 110 are removed from the cell culture box or cell culture well 130.

  FIG. 4 summarizes the execution of these steps.

  In step 202, the parts of the device are assembled. In step 203, the stopper 114 is removed. In step 204, the tip 104 is inserted into the aperture 181 of the needle 180. In step 205, a liquid (eg, blood) containing cells to be isolated and optionally cultured is introduced from the top of compartment 102.

  In step 206, the tip 182 of the needle 180 is placed near the center of the plug 186, pressure is applied to the compartment 102, and the needle is plugged until the end of the needle reaches the internal space under vacuum or vacuum of the vacuum tube 185. 186 penetrates.

  In step 210, filtration is performed by sucking cells smaller than the target cell, lysed cells, and most of the liquid present in the compartment 102 into the vacuum tube 185, so that the target cell is placed on the filter 108. Hold.

  In step 212, the vacuum tube 185 and the needle 180 are removed. In step 214, the tip 104 is removed. In step 216, the end of compartment 102 is inserted into a cell culture box or cell culture well.

  In step 218, the moving means and compartment are each moved to apply force to the filter support 108 to release it and drop it with the filter into the cell culture box or cell culture well 130. In step 220, the compartment 102 and moving means 110 are removed from the cell culture box or cell culture well 130.

  In step 222, cell culture is performed in the cell culture well 130 by a known method. Note that the presence of support 108 around the top surface of the filter (on which surface holds cells isolated on the filter) prevents the cells from leaving the filter.

  In step 222, where the live cells of interest are cultured on the filter, the filter is coated (or contains live cells of interest), eg, a thin layer of Matrigel containing factors suitable for growth of the cells of interest. Contact with a layer of Matrigel® previously placed in the bottom of the plate well and / or culture flask).

  If it is desired to observe the cell or its genetic material in step 224, the filter support 108 is removed using tweezers. This is facilitated by the presence of a cylindrical hole or indentation on the side of the top surface of the filter support 108.

  The filter support 108 can then be placed on a glass slide and the filter can be covered with a disk-shaped cover glass of a diameter suitable for the top surface where the filter is not used. Analysis of cells or extraction of genetic material thereof is performed by a known method.

  FIG. 5 shows reservoir or compartment 302, tip 304, seal 306, filter support 308, moving means 310, seal 312, and plug 314.

  Compartment 302 is generally cylindrical. Its upper end can be sealed with a plug 314 except for an air inlet (not shown). The lower end of the compartment 302 has a cylinder 350 that is separated from the body of the compartment 302 except when mechanically connected to the outer surface in the form of side ribs 352. The cylinder 350 has an outer diameter corresponding to the inner diameter of the main body of the moving means 310, and can guide the moving means during movement. The cylinder 350 is provided with an opening 354 designed to allow the protrusion 316 of the moving means 310 to be inserted and slid in the longitudinal direction.

  The moving means 310 is entirely cylindrical and has two protrusions 316 extending toward the tip portion 304. These protrusions 316 narrow toward the tip 304 in a state of being separated from each other by a distance smaller than the diameter of the filter support 308. As will be explained later, this special shape, in particular the shape in which the protrusions 316 are curved towards each other, causes the moving means 310 to apply pressure to the filter support 308 after removal from the tip 304. So that the filter support is released from the compartment 302 along with the filter while the moving means 310 is moving toward the filter support 308.

  In a shape that matches the shape of the lower end of the section 302 having the side projections 318, the tip 304 has a rotation locking means for tightening the projections in a known manner. In this manner, the tip 304 securely holds the filter holder in place during the filtration step. Further, the tip 304 protects the filter from splashing and contamination.

  When attached, the lower end of the compartment 302 has an opening that opens toward the filter held by the filter support 308. The filter support itself is held in place, one at the lower end of the compartment 302 and the other at the tip 304.

  As shown in FIG. 8, the filter support 308 in the second embodiment has a shape suitable for the shape of the Eppendorf tube.

In particular,
The upper inner side of the support 308 has a shape similar to the upper inner side of the Eppendorf tube, so that the upper opening of the support can be closed with an Eppendorf tube plug;
The filter support 308 can be inserted at the top of the Eppendorf tube because the upper outer side of the support 308 matches the shape of the upper outer side of the Eppendorf tube.

  In addition, the filter support 308 functions as a column that lyses the cells retained on the filter and allows the cell lysate and genetic material to be transferred from the filter support to the Eppendorf tube by centrifugation.

  Filter support 308 is preferably made of polycarbonate plastic. The compartment 302, the tip 304, and the moving means 310 are made of, for example, polypropylene. The seals 306 and 312 are made of silicone, for example.

  6A and 6C are cross-sectional views of a second embodiment of the device according to the invention with the components shown in FIG. 5 assembled, cut along mutually perpendicular axes. 6B and 6D are enlarged views of the components of FIGS. 6A and 6C, respectively. 6A-6D, the elements described with respect to FIG. 5 can be seen.

  FIG. 7A is an elevational view when the device is in a stored configuration. 7B to 7D are the same as FIGS. 3B to 3D except that the reference numeral 304 is the tip.

  As shown in FIGS. 7E and 7F, the tip 304 is rotated to release from the protrusion 318 and removed. Next, as shown in FIGS. 7G, 7H, and 7I, the tip of the compartment 302 is inserted into the Eppendorf tube support 328 to which the Eppendorf tube 330 is attached.

  The moving means 310 is then lowered parallel to the axis of the compartment 302 as shown in FIGS. 7J and 7K. As the protrusion 316 of the moving means 310 is moved by the operator's finger, it exerts a downward force on the filter support 308, releasing the filter support from the lower end of the compartment 302. The filter and filter support 308 then falls into the Eppendorf tube 330.

  Finally, as shown in FIG. 7L, the section 302 and the moving means 310 are removed.

  FIG. 9 summarizes the execution of these steps.

In step 402, the parts of the device are assembled. In step 403, the stopper is removed.
In step 404, the plug 304 is inserted into the aperture 181 of the needle 180. In step 405, a liquid (eg, blood) containing cells to be filtered and optionally cultured is introduced from the top of compartment 302.

  In step 406, the tip 182 of the needle 180 is positioned near the center of the plug 186 and a force is applied to the compartment 302 until the end of the needle reaches the internal space of the vacuum tube 185 at negative or vacuum pressure. The needle is passed through the plug 186.

  In step 410, filtration is performed by sucking cells smaller than the target cells present in compartment 302, lysed cells, and most of the liquid into vacuum tube 185.

  In step 412, the vacuum tube 185 and the needle 180 are removed. In step 414, the tip 304 is removed. In step 416, the tip of the compartment 302 is inserted into the Eppendorf tube support.

  In step 418, the moving means and compartment are each moved, applying force to the filter support to release it so that it falls into the Eppendorf tube. Finally, in step 420, compartment 302 and moving means 310 are removed and the Eppendorf tube is plugged again.

  The Eppendorf tube is then used in a known manner, e.g. for the steps of lysis, centrifugation and recovery of genetic material whose whole genome has been pre-amplified or not amplified.

  In steps 422 and 424, the genetic material (especially DNA and RNA derived from a desired cell) collected at the bottom of the Eppendorf tube after centrifugation is analyzed. The amplified DNA is used as a matrix to detect changes in sensitivity or resistance to the target treatment. In addition or alternatively, gene expression levels relating to sensitivity or resistance to the target treatment using cDNA obtained from RNA by RT conversion and amplified as a matrix ("c" means complementary) Is detected. Using genetic material obtained from trophoblast cells filtered from blood collected from pregnant women, potential genetic abnormalities can be identified.

  Using a pair of forward and reverse primers and a probe pair in quantitative real-time PCR ("polymerase chain reaction"), a predetermined volume of amplified genetic material (especially DNA) is collected and sensitive to the target treatment Or, a change in resistance is detected.

The principle of the study on variability in sensitivity and resistance to the targeted therapy performed in the illustrated embodiment is as follows. Allele discrimination or “SNP genotyping assays” (SNP is an acronym for single nucleotide polymorphism) can collect information about the presence or absence of incidental mutations in a gene. The first step 422 of allele discrimination is a quantitative real-time PCR reaction performed using two primers for amplifying the sequence of interest and two probes (eg, TaqMan®). One of these probes recognizes the mutated sequence and the other recognizes the normal sequence. These two probes are associated with different fluorescent dyes (eg, “VIC” fluorescent dyes for probes that hybridize to normal sequences and “FAM” fluorescent dyes for probes that hybridize to mutated sequences). . The second step 424 uses an allele discrimination program that measures the initial and final fluorescence produced by the FAM and / or VIC fluorochrome. This program makes it possible to distinguish the various sequences present in each sample.
-An increase in VIC fluorescence alone indicates a homozygous profile of normal sequence.
-An increase in FAM fluorescence alone indicates a homozygous profile of the mutated sequence.
-An increase in both VIC and FAM fluorescence indicates a heterozygous profile.

  These probes are paired between two primers and reveal the presence or absence of mutations based on their associated fluorescent color.

  In other embodiments, for example, in 50 cycles of quantitative real-time PCR (an acronym for “polymerase chain reaction”), using forward and reverse primer pairs and probes, the sensitivity and resistance to target therapy is related. In order to detect the level of gene expression, a predetermined volume of genetic material is collected, in particular RNA that is converted to cDNA and amplified by RT (an acronym for “reverse transcription”).

  In each of the above embodiments, the filter is preferably made from polycarbonate plastic and has a hydrophilic surface treatment. Using such a filter increases the retention of certain cells and reduces the attachment of other cells or their contents, especially when cells are lysed.

  The filter has a pore diameter that is preferably centered around a value that is, for example, 1 μm smaller than the corresponding value used for the same cells that are fixed (ie, hardened).

  For example, if the fixed cell pore diameter is centered at 7.5 μm, the filter diameter is centered at a smaller value (eg, 6.5 μm). Since the diameter is dispersed, there are few pores having a diameter of more than 7 μm.

For applying the present invention to blood cells, the filter has a density of 50,000 to 200,000 pores / cm ² , preferably about 100,000 pores / cm ^2. .

  Thanks to the use of polycarbonate plastic filters, the required negative pressure is much smaller than that of prior art systems, on the order of a quarter. Thereby, the deterioration of the cells to be collected on the filter is prevented.

  If another variation is present, the filter support 108 or 308 is mechanically moved to the moving means until the compartment is moved toward the culture well or Eppendorf tube to apply force to release the filter support from the moving means. Attached. In the latter case, pressure is applied to the upper end of the compartment 102 or 302 by reversing the role of the moving means 110 or 310 and holding the filter support in or in front of the glass slide, culture well or Eppendorf tube. It is a change that can be easily carried out by those skilled in the art based on the description of the above-described embodiment.

  FIGS. 10-14 relate to particular embodiments of the present invention and the processes that make up the present invention, which employ a protective guide cylinder for a vacuum tube.

  These particular embodiments supplement any of the above embodiments, but have been shown in FIGS. 10-14 in conjunction with the embodiments shown in FIGS.

  10-13 show the protection cylinder 502 attached to the compartment 102 by the compartment 102, the moving means 110 and the two connecting means 504 and 520. FIG.

  The protective cylinder comprises a connecting means component 504, a matted component 506, a transparent component 508, and a film 510 in the opening facing the compartment 102.

  Part 520 is formed inside the end of compartment 102. FIG. 13 shows a particular embodiment of a part 520 comprising four protrusions disposed on the sides of the cylindrical part in a coaxial relationship with the compartment 102. In this embodiment, the component 504 is composed of four grooves having an outer shape corresponding to the grooves of these protrusions. These grooves 524 extend elliptically from the opening made to fit the protrusion 522 toward the inside of the protective cylinder 502, and each of the grooves 524 is rotated by rotating the protective cylinder 502 as shown by the arrows in FIG. The protrusion 522 enters the corresponding groove 524 so that the protective cylinder 502 is fastened to the compartment 102.

  The protective cylinder 502 is attached to the distal end portion having the needle 180 as follows. The needle 180 is embedded in the lower part of the part 524 (this is the part facing the compartment 502). The part 524 is attached to the part 506 by power with four spokes. These spokes are on the surface of the part 504 and are inserted into four grooves on the surface of the part 506.

  The role of the part 506 is to hide the needle 180. The transparent component 508 allows the user to confirm the filtration status and the end of filtration.

  A film 510 is attached that covers and seals the entire lower opening of the cylinder 502, and its outer portion extends a short distance from the cylinder 502 (shown in FIG. 10). Thus, the film 510 can be easily removed by being on the outer side.

  The film 510 protects the user from touching the needle 180. Film 510 also protects against the risk of needle 180 becoming clogged and / or contaminated.

  The cylinder 502 is color-coded, for example, blue, green, or yellow, depending on the intended use of the filtration device (respectively, cytological research, molecular biology research, and culture research).

  Note that in FIG. 11, after the liquid to be filtered is inserted into the compartment 102 and the film 510 is removed, a vacuum tube 185 fitted with a plug 186 is inserted into the protective guide cylinder 502. A force is then applied to the vacuum tube 185 and the needle 180 punctures the plug 186 as described above.

  In the assembly shown in FIG. 12 thus produced, the liquid present in the compartment 102 is filtered by the negative pressure originally present in the vacuum tube 185.

  Note that in FIG. 11, after the liquid to be filtered is inserted into the compartment 102 and the film 510 is removed, the vacuum tube 185 fitted with its stopper 186 is inserted into the protective guide cylinder. Next, a pressing force is applied to the vacuum tube 185 and the needle 180 punctures the plug 186 as previously described.

  In the assembly shown in FIG. 12 thus produced, the liquid present in the compartment 102 is filtered by the negative pressure originally present in the vacuum tube 185.

  As shown in FIG. 13, when filtration is complete, the protective cylinder 502 and the needle 180 therein are removed together.

  FIG. 14 summarizes the execution of these steps.

  In step 602, the parts of the device are assembled. In step 603, the stopper 114 is removed. In step 604, a liquid (eg, blood) containing cells to be isolated and optionally cultured is introduced from the top of compartment 102.

  In step 605, the vacuum tube 185 is inserted into the protective cylinder 502, which has the effect of placing the tip 182 of the needle 180 near the center of the plug 186. In step 606, the needle is pierced through the plug 186 until the end of the needle reaches the internal space of the vacuum tube 185 at a negative or vacuum pressure by applying an antagonistic force.

  In step 610, filtration is performed by aspirating vacuum cells 185 with cells smaller than the target cells, all lysed cells, and most of the liquid present in the compartment 102, and filtering the target cells with the filter 108. Hold on.

  In step 612, the protection cylinder 502, the vacuum tube 185, and the needle 180 are removed.

  The subsequent steps have already been described with respect to other embodiments and depend on these embodiments. They are therefore not described here again.

  From the foregoing it can be seen that the user is protected from accidental needle sticks while the protective cylinder is attached to the compartment. Furthermore, the positioning of the vacuum tube is facilitated by the guidance provided by the protective cylinder. The vacuum tube is held firmly in place during suction. As a result, the vacuum tube is moved or displaced, thereby preventing air from entering the vacuum tube through the side of the needle in the stopper of the vacuum tube. Since the user does not accidentally touch the needle, the risk of sample contamination by the user is minimized.

  Preferably, as shown in FIGS. 10 to 13, the apparatus constituting the present invention is provided with the protective cylinder at least temporarily. In other embodiments, the assembly takes place immediately before use.

  Preferably, as shown in FIGS. 10-13, the connecting means is temporary, allowing the protective cylinder to be removed together with the vacuum tube. If another variation exists, these two parts are removed in two successive steps.

In an embodiment, the device constituting the present invention is presented in the form of a kit comprising two inner bags in an outer bag,
The first inner bag comprises an assembled device as shown in FIGS. 2A, 6A and 10;
The second inner bag contains needles, vacuum tubes, culture wells and circular slides and / or Eppendorf tubes or other useful accessories for using the device.

  By using the present invention, it is possible to avoid high-risk cell collection (for example, amniotic fluid cells) and simultaneously perform cell culture for amniocentesis, for example. Furthermore, the fact that the filter support 108 is shaped like a reservoir allows immunocytochemical reactions and fluorescence in situ hybridization (“FISH”) reactions to be performed directly in this support.

Claims (12)

An apparatus for isolating fixed or live cells on a filter or extracting genetic material from said live cells,
A compartment having an internal space for containing the liquid containing the cells, a lower opening, and an air inlet;
Movable means configured to move in each of the compartments;
The filter maintained by the opening of the compartment and / or the movable means is designed to hold the cells as liquid passes through the filter;
The compartment opening, at least temporarily, a needle forming a single impermeable unit, and the filter is located between the needle and the internal space of the compartment, the needle passing through the filter Designed to penetrate vacuum tube plugs with negative pressure related to ambient pressure to aspirate liquid,
The connecting means between the compartment and a protective cylinder surrounding the needle is configured to surround at least a portion of the vacuum tube while aspirating the liquid through the filter;
The apparatus characterized in that the movable means and the compartment are each configured to move to apply force to the filter and to release the filter after suction of the liquid through the filter.   The apparatus of claim 1, comprising at least temporarily the protective cylinder.   The device according to claim 1, wherein the connecting means is temporary and allows the protective cylinder to be removed together with the vacuum tube.   The protective cylinder includes a movable film that seals an opening of the protective cylinder facing the connection means, and an opening configured to insert the vacuum tube into the protective cylinder after the movable film is removed. The apparatus as described in any one of Claims 1-3 provided.   The apparatus according to claim 1, further comprising a surgical steel movable filter support configured to be temporarily connected to the lower opening of the compartment.   6. The apparatus of claim 5, wherein the thickness of the filter support is adapted to allow the filter support to be scanned.   The apparatus according to claim 5, wherein the filter support comprises a discriminator.   A removable end portion that is impermeable and removably attached to the compartment, the end portion being configured to limit relative movement of the movable means and the compartment. Item 8. The apparatus according to any one of Items 1 to 7. A method for isolating live cells on a filter or extracting genetic material of said live cells, the method comprising:
A step for temporarily attaching the filter to the lower opening of the compartment further comprising an air inlet, or for attaching to the movable means configured to operate in each of said compartments;
Inserting a liquid containing the cells into the compartment;
Attaching a needle impermeable, at least temporarily to the compartment opening, and placing the filter between the needle and the interior space of the compartment;
Securing a protective cylinder to the compartment at least temporarily, after which the protective cylinder surrounds the needle;
Using the needle to pierce a vacuum tube plug that has a negative pressure relative to ambient pressure, and to insert the vacuum tube into the protective cylinder during the piercing step;
Aspirating the liquid through the filter by the negative pressure from the vacuum tube, the filter holding the cells;
Operating the movable means and each of the compartments to apply a force to the filter and release the filter.   The method of claim 9, comprising removing the protective cylinder along with the vacuum tube.   The protective cylinder includes a movable film that seals an opening of the protective cylinder facing the connecting means, and further includes the step of removing the movable film before inserting the vacuum tube into the protective cylinder. The method according to any one of 10 above.   A step for removing a removable end attached to the compartment so as to be impervious and removable between the step of sucking and operating each of the compartments for releasing the movable means and the filter is further provided. 12. A method as claimed in any one of claims 9 to 11, wherein the distal end is configured to limit associated movement of the movable means and the compartment.

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