JP6352862B2 - Specific gravity separation layer suction / collection tip - Google Patents

Description

  The present invention relates to a chip (specific gravity separation layer suction / collection chip) having a structure suitable for sucking / collecting a specific layer after specific gravity centrifugation.

  When separating lymphocytes from peripheral blood, the blood with anticoagulant added is layered on a solution (separate) that is smaller than the specific gravity of erythrocytes and granulocytes and greater than the specific gravity of lymphocytes, and then centrifuged. The sample is set in the apparatus, and a predetermined centrifugal gravity is applied for a predetermined time, and separated and collected by the difference in specific gravity. Until now, specific gravity separation was performed by using a Pasteur pipette (a pipette with a vertical tube whose tip is opened vertically), etc., with a vertical suction port or a tip of a vertical tubular pipette. Cells are collected. Skilled techniques are required to accurately collect the target cells. It takes time to understand and train this technology, and the quality of the cells collected varies from technician to technician. In other words, the cells below the target layer are taken in, or the results such as the cell death rate due to the change in the suction speed are different, and erroneous information is added to the analysis and inspection results.

This is common in the case of specific gravity separation of biological cells. In particular, separating and collecting lymphocytes from peripheral blood is an indispensable process for analyzing and examining lymphocytes outside the body.
Lymphocytes are isolated and collected in the following process.
(1) Put a separated liquid whose specific gravity is adjusted in a centrifugal sedimentation tube, and overlay blood added with an anticoagulant.
(2) The centrifugal sedimentation tube of (1) is installed in a centrifuge, and a predetermined centrifugal gravity is applied to separate cells according to the difference in specific gravity. (3) As a result of (2), blood has a difference in specific gravity. It is divided into red blood cells / granulocytes, specific gravity separation liquid, lymphocytes, platelets, and plasma, and each layer becomes a layered state.
(4) From the blood in the state of (3), the lymphocyte and platelet layers are aspirated and collected with a pipette or a spoid.
(5) The collected lymphocytes and platelets are transferred to a container filled with physiological saline, placed in a centrifuge, and subjected to centrifugal separation by applying a predetermined centrifugal gravity.
(6) As a result of (5), lymphocytes precipitate in physiological saline, platelets float on the upper layer, and lymphocytes and platelets are separated.
(7) Remove as much of the physiological saline layer containing platelets and plasma from the upper layer of (6) as much as possible, leaving centrifuged lymphocytes.
(8) If necessary, add physiological saline again to (7), apply predetermined centrifugal gravity, centrifuge lymphocytes in the container, and remove the upper layer.
Through the above process, lymphocytes are collected. In the past, cells were collected with a device that had a suction port perpendicular to the layer after centrifugation of the blood, but there is a great possibility that a layer heavier than the specific gravity of the target layer will be sucked. In such a case, it is impossible to remove it in a later process, and erroneous information is added to the analysis and inspection results. Engineers performing work procedures are forced to stay in tension for a long time, and there is a risk that incorrect information will be added to the results of analysis and inspection even if careful work is performed by skilled engineers. It will not be removed.

  For example, as a pipette tip that sucks and accommodates liquid until now, the following Patent Document 1 has a fitting portion that fits into a suction nozzle at the top, and a liquid containing portion that follows the fitting portion. And a tip portion having a suction / discharge port with a diameter smaller than that of the liquid storage portion is provided at the tip thereof through an inclined step portion, and the fitting portion is formed in a tapered shape whose inner surface is reduced in diameter toward the liquid storage portion. In addition, the thickness of the liquid container is smaller than that of the fitting part, and the tip part has a narrow diameter on the inner surface and outer surface, so that the length does not substantially cause bending during the molding process. Is formed.

  However, in the case of the chip described in Patent Document 1, it is possible to collect a layer having a different specific gravity from the purpose, particularly a cell having a higher specific gravity in the lower layer, when collecting cells separated by specific gravity. was there.

Japanese Patent Laid-Open No. 7-284673

  The object of the present invention is to solve the above-mentioned problems and to reliably collect a specific gravity layer for the purpose of collection, and to collect cells with a constant technique regardless of the immaturity of an engineer or human error. It is to provide a specific gravity separation layer suction / collection tip that can be used.

  As a result of various studies, the present inventor has provided a suction port at the tip of a chip that is thin enough to move freely inside a container in which a cell layer separated by specific gravity is allowed to stand, and the suction port sucks the layer When the living cells separated by specific gravity are collected using the chip having the above shape by opening the plate so as to be substantially horizontal, the layers to be collected can be limited and collected accurately. The present invention has been completed by finding that living cells can be collected by a fully automatic device with a constant technique without waiting for skill.

The specific gravity separation layer suction / collection chip of the present invention capable of solving the above-mentioned problems is used by being attached to an automatic suction metering device, and for sucking / collecting a specific layer after specific gravity centrifugation. A fitting connection portion for fitting and connecting to the automatic suction and metering device at the top, and a substantially cylindrical liquid storage portion is formed following the fitting connection portion, and further the liquid storage A tubular tip portion having an inner diameter smaller than the inner diameter of the liquid storage portion and having an inner diameter gradually smaller toward the tip side is formed following the portion, and the length of the tip portion is 30 to 30 mm. in 180 mm, the outer diameter is 1 to 8 mm, the and the lower end of the tubular tip portion is sealed, that the corresponding bottom end an inverted conical or inverted truncated cone shape, the lower end of the peripheral wall of the distal portion the suction and discharge ports are bored, before the suction and discharge ports are provided The outer diameter of the lower end of the tip is 1 to 4 mm, and, of the suction and discharge ports, the opening diameter in the longitudinal direction of the chip, characterized in that a 0.1 to 0.5 mm.

  Further, the present invention is characterized in that the specific gravity separation layer suction / collection chip having the above-described characteristics is provided with a plurality of the suction discharge ports.

  Further, according to the present invention, in the specific gravity separation layer suction / collection tip having the above-described characteristics, the lower end of the tip is an inverted cone shape or an inverted truncated cone shape having a tip angle of 120 ° or less. Is.

  Further, according to the present invention, in the specific gravity separation layer suction / collection tip having the above characteristics, the suction / discharge port forms an angle of 30 ° or less with respect to a plane perpendicular to the central axis of the tip. It is characterized by being perforated.

  In the specific gravity separation layer suction / collection tip having the above-described characteristics, the present invention is provided such that the suction / discharge port is parallel to a plane perpendicular to the central axis of the tip. It is characterized by that.

  According to the present invention, the tip of the specific gravity separation layer suction / collection tip has a length and thinness suitable for efficiently collecting target cells when cells are collected through cell separation by the specific gravity separation method. Since the lower end of the tip is sealed and the suction discharge port is formed in the peripheral wall on the lower end side of the tip, the cell layer to be collected by suction is the uppermost part of the layer separated by specific gravity. Even in the intermediate layer or the lowermost layer, the cells in the collection target layer can be collected and discharged.

It is a figure which shows the external appearance in a preferable example of the chip | tip for specific gravity separated layer suction / collecting | collection of this invention. (A)-(c) is an enlarged sectional view of the tip of the specific gravity separation layer suction / collection tip of the present invention, (a) is a suction discharge port drilled in the peripheral wall near the lower end side. In the case where it is drilled so as to be parallel to the surface orthogonal to the central axis of the tip, (b) shows that the suction / discharge port is 30 ° or less with respect to the surface orthogonal to the central axis of the tip (C) shows that the suction / discharge port has an angle of 30 ° or less with respect to a plane orthogonal to the central axis of the tip. The case where it pierces and inclines below is shown. (A)-(c) is an expanded sectional view of the front-end | tip of the specific gravity separation layer suction / collecting chip | tip of this invention, and the internal structure of the front-end | tip part different from FIG. 2 (a)-(c) is illustrated. Has been.

  As shown in FIG. 1, the specific gravity separation layer suction / collection chip A of the present invention for aspirating / collecting a specific layer after carrying out the specific gravity centrifugation is an automatic suction metering device (not shown). The fitting connection part 1 for fitting connection is provided on the upper part (upper side of the drawing), and this fitting connection part 1 extends slightly in the circumferential direction so as not to be deformed in the vertical direction. It is preferable to provide a plurality of vertical columnar protrusions as shown in FIG.

  In the specific gravity separation layer suction / collection chip of the present invention, a substantially cylindrical (cylindrical) liquid storage portion is provided on the lower side of the fitting connection portion 1 following the fitting connection portion 1. The volume of the liquid container 2 is not particularly limited as long as it is a size that can hold a necessary amount of collected cells or a cell washing solution. It is preferable that the volume is enough to accommodate 2 to 20 ml of solution as a whole. In the present invention, a filter 5 as shown in FIG. 1 is installed in the liquid storage unit 2 in order to prevent the device from being contaminated by droplets of the solution sucked immediately below the fitting portion with the automatic suction metering device. It is preferable to do.

  Further, on the lower side of the liquid storage part 2, following the liquid storage part 2, a tubular tip part 3 having an inner diameter smaller than the inner diameter of the liquid storage part 2 and gradually decreasing toward the tip side. The tubular tip 3 has a length and a thinness that can move horizontally inside a container such as a centrifuge tube or a test tube. In the specific gravity separation layer suction / collection tip of the present invention, The tip 3 has a length of 30 to 180 mm and an outer diameter of 1 to 8 mm. In particular, it is preferable that the outer diameter in the vicinity of the lower end side (near the suction and discharge port) of the tip portion is 3 to 4 mm and the inner diameter is 1.5 to 2.5 mm. The total length of the chip of the present invention is appropriately selected depending on the size of the automatic suction metering device used and the inner diameter and length of a container such as a test tube, but the general total length is 60 to 360 mm.

In addition, the lower end of the tip portion 3 of the chip of the present invention is sealed (closed) as illustrated in FIGS. 2A to 2C or FIGS. 3A to 3C and sealed. The lower end of the tip 3 is tapered so that the interface state of the separated layer is maintained without being disturbed when moving (passing) through the interface of the separated layer. It has a shape. In particular, the lowermost end is preferably rounded. For example, the lower end has an inverted conical shape, and the apex of the inverted conical shape is preferably a rounded hemisphere. At this time, it is preferable that the tip angle θ ₁ of the inverted cone shape or the inverted truncated cone shape portion positioned at the lower end of the tip portion 3 is 120 ° or less, and FIGS. 2A to 2C or FIG. As illustrated in (c), the tip angle θ ₁ may be 90 ° or an acute angle smaller than 90 °.

Further, in the specific gravity separation layer suction / collection chip of the present invention, as shown in FIG. 1, one or more suction discharge ports 4 are formed along the peripheral wall (outer peripheral surface) on the lower end side of the distal end portion 3. The suction and discharge ports 4 may be provided at a plurality of locations on the peripheral wall in the vicinity of the lower end side of the distal end portion 3, and generally at a position that divides the outer periphery on the lower end side of the distal end portion 3 at an equal angle. Provided. In the present invention, the suction discharge port 4 having a structure in which most of the outer peripheral wall on the lower end side of the tip portion 3 is opened (the outer peripheral wall opens close to 270 to 330 °) may be used. The opening shape of the suction / discharge port 4 in the present invention may be circular, square or rectangular, and is appropriately selected according to the object to be sucked and its capacity, but is generally 0.1 mm to 1.2 mm. It has a height (opening diameter in the longitudinal direction of the chip), and preferably opens along the circumference, and particularly has an opening height of 0.3 to 0.5 mm and opens along the circumference. A rectangular mouth is preferred.
In the present invention, since the suction / discharge port 4 is provided as shown in FIG. 1, when the tip 3 of the chip is inserted into the container, the direction parallel to the boundary surface of the separated liquid Thus, the target liquid can be sucked into the chip. For example, when collecting the lymphocyte layer after peripheral blood separation, the suction port is aspirated and collected with the lowest layer of the lymphocyte layer. Thus, not only the specific gravity liquid heavier than the lymphocyte layer, but also lymphocytes can be collected without collecting granulocytes and erythrocytes. Further, in the present invention, the order of sucking into the chip is the cell washing solution, and then the separated and collected lymphocyte layer, so that an effect of flushing the lymphocytes with the cell washing solution when discharging is also generated.

2 (a) to 2 (c) are enlarged sectional views of the tip of the specific gravity separation layer suction / collection tip of the present invention, and the suction / discharge port 4 drilled in the peripheral wall near the lower end side is ( As shown in a), it may be perforated so as to be parallel to a plane orthogonal to the central axis of the tip 3, and as shown in (b), the central axis of the tip 3 angle theta ₂ is 30 ° or less with respect to a plane perpendicular to may be drilled inclined upward so that the (30 ° in the drawing), or, as shown (c), the center of the tip 3 You may perforate and incline below so that angle (theta) ₂ with respect to the surface orthogonal to an axis | shaft may be 30 degrees or less (30 degrees in drawing).
When the angle θ ₂ exceeds 45 °, the possibility of sucking a layer lighter or heavier than the specific gravity of the target layer increases, so the angle θ ₂ is preferably 45 ° or less, more preferably 30 ° or less, 15 A temperature of not more than ° is particularly preferred. The most preferred examples theta ₂ is 5 ° or less, i.e., a chip that has a substantially horizontal with respect to the layer suction and discharge port 4 is taken.
In the present invention, as shown in FIGS. 3 (a) to 3 (c), a conical protrusion 6 may be provided on the bottom surface on the lower end side of the distal end portion 3, and by adopting such an internal structure. The flow of the liquid during suction / discharge can be controlled.

  The specific gravity separation layer suction / collection chip of the present invention is not used repeatedly but is used disposable, and the material thereof is not particularly limited, but is preferably made of plastic, and particularly preferably made of polypropylene resin. In the present invention, as with other medical products, the manufactured chip is subjected to primary packaging in a sterilized space, and then sterilized and then subjected to secondary packaging, whereby sterilization is performed before use. There is no need to do it.

1 has the appearance shown in FIG. 1, and the peripheral wall in the vicinity of the lower end side of the tip end portion is evenly parallel to the plane perpendicular to the central axis of the tip end portion 3 as shown in FIG. A specific gravity separation layer suction / collection chip (made of polypropylene resin) according to the present invention having a structure in which suction / discharge ports were provided at four locations at regular intervals was manufactured by injection molding. The tip of the specific gravity separation layer suction / collection tip (A) is 90.8 mm in length, the outer diameter of the upper end of the tip is 4.8 mm, the inner diameter is 3.0 mm, and the vicinity of the lower end of the tip. The outer diameter is 3.2 mm, the inner diameter is 1.8 mm, the total length of the tip is about 170 mm, and the opening height of the suction / discharge port (4) provided on the peripheral wall near the lower end side of the tip is 0.4 mm. It was.
The above-obtained specific gravity separation layer suction / collection chip of the present invention was attached to an automatic suction metering device, and the layer from which blood lymphocytes had been separated after specific gravity centrifugation was aspirated / collected. Aspirated / collected lymphocyte layers from the direction parallel to the separation interface (lateral direction), and accidentally aspirate the underlying layers (layers with a higher specific gravity than the lymphocyte layers) It was confirmed that the specific gravity separation layer can be sucked / collected without requiring careful work by a skilled technician.

  The specific gravity separation layer suction / collection chip according to the present invention is suitable for efficiently collecting only the target layer after separation by the specific gravity separation method, and other than the work of separating lymphocytes from blood. It can also be widely used for various separation operations (for example, separation of immune-related cells from tissues).

A Specific gravity separation layer suction / collection chip 1 Fitting connection part 2 Liquid storage part 3 Tip part 4 Suction discharge port 5 Filter 6 Conical protrusion part

Claims (5)

A chip that is used by being attached to an automatic suction metering device and for sucking / collecting a specific layer after specific gravity centrifugation,
A fitting connection portion for fitting and connecting with the automatic suction metering device is provided on the upper portion, and a substantially cylindrical liquid storage portion is formed following the fitting connection portion, and the liquid storage portion Subsequently, a tubular tip having an inner diameter smaller than the inner diameter of the liquid container and gradually decreasing toward the tip is formed, and the length of the tip is 30 to 180 mm. in, that the outer diameter of 1 to 8 mm, the and the lower end of the tubular tip portion is sealed, that the corresponding bottom end an inverted conical or inverted truncated cone shape, the suction at the lower end of the peripheral wall of the distal portion A discharge port is formed , an outer diameter of a lower end side of the tip portion provided with the suction discharge port is 1 to 4 mm, and the suction discharge port is opened in a longitudinal direction of the chip. gravity separation layer suction, wherein the aperture is 0.1~0.5mm / Harvesting chip.   The specific gravity separation layer suction / collection chip according to claim 1, wherein a plurality of the suction discharge ports are provided.   The specific gravity separation layer suction / collection tip according to claim 1 or 2, wherein the lower end of the tip has an inverted cone shape or an inverted truncated cone shape with a tip angle of 120 ° or less.   The suction / discharge port is formed so as to form an angle of 30 ° or less with respect to a plane orthogonal to the central axis of the tip portion. The specific gravity separation layer suction / collection tip described.   The specific gravity separation according to any one of claims 1 to 4, wherein the suction / discharge port is perforated so as to be parallel to a plane orthogonal to a central axis of the tip portion. Layer suction / collection tip.

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