Centrifuge tube separation system, and methods of use

Abstract

A system for separating biological material includes a centrifuge tube, a separation tube having an open bottom, a cap, and a separation medium disposable within the centrifuge tube and the separation tube. The centrifuge tube and the separation tube sealingly and releasably couples to the cap, such that, when coupled, the separation tube is positioned within the centrifuge tube. The cap is configured to facilitate and/or regulate air- or gas-flow between an area outside of the cap and an interior of the separation tube. When the separation tube is positioned within the centrifuge tube, the open bottom of the separation tube is submersed in the separation medium. The system also includes a hollow needle coupled to a means for regulating a flow of air, gas, or other matter. The needle is insertable through the cap or plug, and is used to facilitate the introduction of matter into the separation tube.

Claims

1. A system for separating biological material, the system comprising: a. a centrifuge tube; b. a separation tube having an open bottom; c. a cap, wherein the centrifuge tube and the separation tube are configured to sealingly and releasably couple to the cap, wherein, when coupled to the cap, a pre-determined length of the separation tube is positioned within the centrifuge tube, wherein an area between an exterior surface of the separation tube and an interior surface of the centrifuge tube is unobstructed along the pre-determined length, wherein a top of the cap comprises an aperture that opens into a cavity of the cap and one or more channels that pass through the top of the cap and into the cavity, wherein, when the cap is coupled to the separation tube, the one or more channels provide open communication, and are configured to facilitate or regulate air- or gas-flow, between an area outside of the cap and an interior of the separation tube, and further comprises a plug that is configured to releasably seal the aperture and one or more channels; and d. a separation medium disposable within the centrifuge tube and the separation tube, wherein a volume of the separation medium is such that, when the separation tube and the centrifuge tube are coupled to the cap, the open bottom of the separation tube is submersed in the separation medium.

2. The system of claim 1, wherein a lower portion of the plug is insertable into the aperture, and wherein the plug includes a flange configured to transition between a closed position and an open position, wherein, in the closed position, the flange is configured to seal the one or more channels, and wherein, when transitioned to the open position, the flange is configured to unseal the one or more channels.

3. The system of claim 2, wherein the lower portion of the plug is releasably retained within the cap by an interference fit.

4. The system of claim 2, wherein an upper portion of the plug is a mechanical actuator configured to transition the flange between the open position and the closed position.

5. The system of claim 1, wherein the separation medium is a density separation medium.

6. An apparatus for separating biological material, the apparatus comprising: a. a centrifuge tube; b. a separation tube having an open bottom; and c. a cap, wherein the centrifuge tube and the separation tube are configured to sealingly and releasably couple to the cap, wherein, when coupled to the cap, a pre-determined length of the separation tube is positioned within the centrifuge tube, wherein an area between an exterior surface of the separation tube and an interior surface of the centrifuge tube is unobstructed along the pre-determined length, wherein a top of the cap comprises an aperture that opens into a cavity of the cap and one or more channels that pass through the top of the cap and into the cavity, wherein, when the cap is coupled to the separation tube, the one or more channels provide open communication, and are configured to facilitate or regulate air- or gas-flow, between an area outside of the cap and an interior of the separation tube, and further comprises a plug that is configured to releasably seal the aperture and one or more channels.

7. The apparatus of claim 6, wherein a lower portion of the plug is insertable into the aperture, and wherein the plug includes a flange configured to transition between a closed position and an open position, wherein, in the closed position, the flange is configured to seal the one or more channels, and wherein, when transitioned to the open position, the flange is configured to open the one or more channels.

8. The apparatus of claim 7, wherein the plug is releasably retained within the cap by an interference fit.

9. The apparatus of claim 7, wherein an upper portion of the plug is a mechanical actuator configured to transition the flange between the open position and the closed position.

10. A method for separating and collecting a target material from a fluid sample using the apparatus of claim 6, the method comprising: depositing a density separation medium into the apparatus, such that a lower portion of the separation tube is submersed in the density separation medium; depositing the fluid sample on top of the density separation medium inside of the separation tube; sealing the cap; centrifuging the fluid sample; uncoupling the centrifuge tube from the cap once centrifugation is complete, wherein the target material is retained within the separation tube; introducing air or gas into the separation tube; and halting air- or gas-flow into the separation tube, wherein the steps of introducing air or gas into the separation tube and halting air- or gas-flow into the separation tube can be repeated as necessary to collect a target material.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) For a more complete understanding of the present invention, the objects and advantages thereof, reference is now made to the ensuing descriptions taken in connection with the accompanying drawings briefly described as follows.

(2) FIG. 1A shows a sectional view of the separation apparatus, according to an embodiment of the present invention;

(3) FIG. 1B shows a perspective view of the separation apparatus, according to an embodiment of the present invention;

(4) FIG. 1C shows a perspective view of the separation apparatus, according to an embodiment of the present invention;

(5) FIG. 1D shows a sectional view of the separation apparatus, according to an embodiment of the present invention;

(6) FIG. 2 shows a perspective view of the centrifuge tube, according to an embodiment of the present invention;

(7) FIG. 3A shows a sectional view of the separation tube, according to an embodiment of the present invention;

(8) FIG. 3B shows a perspective view of the separation tube, according to an embodiment of the present invention;

(9) FIG. 3C shows a perspective view of the separation tube, according to an embodiment of the present invention;

(10) FIG. 4A shows a sectional view of the cap, according to an embodiment of the present invention;

(11) FIG. 4B shows a perspective view of the cap, according to an embodiment of the present invention;

(12) FIG. 4C shows a top plan view of the cap, according to an embodiment of the present invention;

(13) FIG. 4D shows a bottom plan view of the cap, according to an embodiment of the present invention;

(14) FIG. 5A shows a sectional view of the plug, according to an embodiment of the present invention;

(15) FIG. 5B shows a bottom perspective view of the plug, according to an embodiment of the present invention;

(16) FIG. 5C shows a top perspective view of the plug, according to an embodiment of the present invention;

(17) FIG. 5D shows a side elevational view of the plug, according to an embodiment of the present invention;

(18) FIG. 6A shows a sectional view of the plug, according to an embodiment of the present invention;

(19) FIG. 6B shows a perspective view of the plug, according to an embodiment of the present invention;

(20) FIG. 6C shows a side elevational view of the plug, according to an embodiment of the present invention;

(21) FIG. 7A shows a sectional view of the separation tube, the cap, and the plug in an assembled form, according to an embodiment of the present invention;

(22) FIG. 7B shows a perspective view of the separation tube, the cap, and the plug in an assembled form, according to an embodiment of the present invention;

(23) FIG. 7C shows a perspective view of the separation tube, the cap, and the plug in an assembled form, according to an embodiment of the present invention;

(24) FIG. 8A shows a sectional view of the separation tube, the cap, and the plug in operation with the needle and the regulator, according to an embodiment of the present invention;

(25) FIG. 8B shows a perspective view of the separation tube, the cap, and the plug in an assembled form, according to an embodiment of the present invention;

(26) FIG. 8C shows a perspective view of the separation tube, the cap, and the plug in an assembled form, according to an embodiment of the present invention; and

(27) FIG. 9 is a flow chart showing a method of using the separation system, according to an embodiment of the present invention.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

(28) Preferred embodiments of the present invention and their advantages may be understood by referring to FIGS. 1A-9, wherein like reference numerals refer to like elements.

(29) With reference to FIGS. 1A-1D, separation apparatus 5 includes centrifuge tube 10, separation tube 15, and cap 20 (with or without plug 25), each of which are further individually illustrated in FIGS. 2-6C.

(30) With reference to FIG. 2, centrifuge tube 10 has an open top that opens into cavity 30. The bottom of centrifuge tube 10 is closed. Connector 35 is disposed at an upper portion of centrifuge tube 10.

(31) With reference to FIGS. 3A-3C, separation tube 15 has an open top and an open bottom, each of which open into hollow interior 40. Connector 45 is disposed at an upper portion of separation tube 15. With additional reference to FIG. 2, the figures depict connectors 35, 45 as threading that extends around an outer circumference of an upper portion of centrifuge tube 10 and separation tube 15, respectively. However, one skilled in the art would understand and appreciate that alternative connection means could be employed to serve the same function.

(32) With reference to FIGS. 4A-4D, cap 20 has an open bottom that opens into cavity 50. Cavity 50 includes connectors 55, 60 configured to matingly engage connectors 35, 45, respectively, to releasably couple centrifuge tube 10 and separation tube 15 to cap 20. Aperture 62 extends through the top surface of cap 20 and opens into cavity 50. Cavity neck 65 is disposed adjacent to, and is set inward from, connector 55. In an embodiment, retaining lip 70 protrudes from a lower portion of cavity neck 65. In an assembled state, wherein separation tube 15 is coupled to an un-plugged cap 20, cap 20 provides open communication between an area outside of separation apparatus 5 and hollow interior 40.

(33) In an embodiment, one or more channels 75 extend through top surface of cap 20. In an assembled state, wherein separation tube 15 is coupled to cap 20 (plugged or un-plugged), channels 75 provide open communication between the top of cap 20 and cavity 40. In other words, channels 75 serve as a bypass for influent air to travel to cavity 40 when cavity 50 is otherwise sealed. For example, in an embodiment, as shown in FIGS. 1A, 1D and 7A, when separation tube 15 is coupled to cap 20, the interior wall of separation tube 15 is separated from a rear surface of cavity neck 65 by open space 80. Channels 75 pass through cap wall and open into open space 80, allowing air to travel into and through open space 80 without passing through aperture 62.

(34) With reference to FIGS. 5A-6C, plug 25 includes flange 85 that extends from an outer circumference of plug 25. The bottom surface of flange 85 is configured to complement the contour the top surface of cap 20. For example, in a preferred embodiment, the bottom surface of flange 85 is disposed orthogonal to flange's central axis, allowing it to seat onto and create an air-tight seal with the top surface of cap 20. Pull tab 90 extends from an outer edge of flange 85 and facilitates removal of plug 25 from cap 20.

(35) Lower portion of plug 25, i.e., the portion of plug 25 below flange 85, includes cavity 95 and is configured to be received into cavity 50. Upon insertion, lower portion of plug 25 is removably retained within cavity 50 via an interference, i.e., friction, fit between lower portion of plug 25 and the wall surface of cavity neck 65 and/or the inner wall of separation tube 15 to create a sealed, i.e., air-tight, engagement between plug 25 and cavity neck 65 and/or separation tube 15, respectively.

(36) With reference to FIGS. 5A-5D, in an embodiment, groove 87 extends around an outer circumference of plug's lower portion to facilitate a snap fit engagement with cap's retaining lip 70 as plug 25 is inserted into cap 20.

(37) With further reference to FIGS. 5A-5D, in an embodiment, central portion 100 atop plug 25 serves as a mechanical actuator to transition flange 85 between an open position and a closed position. The circumference of central portion 100 is smaller than the circumference of cavity 95, allowing downward displacement of central portion 100. Depression of central portion 100 toward cavity 95 forces flange 85 to invert upward to the open position. Conversely, release of central portion 100 allows flange 85 to relax and return to its native configuration, i.e., the closed position.

(38) With reference to FIGS. 6A-6C, in an embodiment, the circumference of central portion 100 is larger than the circumference of cavity 95, thus preventing downward displacement of central portion 100. By preventing downward displacement of central portion 100, flange 85 is held in the closed position, i.e., it is unable to transition to the open position. This ensures that channels 75 remain sealed to prevent inadvertent spills or leakage during transport, etc.

(39) In an embodiment, hollow needle 102 is used as a means to introduce air or gas into the upper portion of separation tube 15 through plug 25. Needle 102 is connected to regulator 104. Regulator 104 may be a syringe, a pump, or any other device/machine, e.g., an air compressor, configured to regulate a flow of air, gas, or other matter. This allows air or gas to be introduced into the upper portion of separation tube 15 in a regulated, i.e., controlled and calculable, manner.

(40) Plug 25 is constructed of a flexible and/or compressible material with resilient qualities, e.g., plastic, rubber, or silicone. This facilitates the interference fit between lower portion of plug 25 and a wall surface of cavity neck 65 and/or the interior wall of separation tube 15 by allowing the lower portion of plug 25 to undergo a pre-determined degree of deformation, e.g., compression, during engagement/disengagement with cavity neck 65. Further, it facilitates the process of transitioning flange 85 between its open and closed positions. Additionally, it facilitates penetration of needle 102 through top of plug 25 and into cavity 95 for introducing air or gas into separation tube 15.

(41) In an embodiment, a valve or port (not shown) is integrated into plug 25. The valve or port provides an alternative means, whereby needle 102, coupled to regulator 104, may be punctured through valve or port, to inject matter, e.g., air, gas, or biological material, into separation tube 15.

(42) In an embodiment, cap 20 is configured without aperture 62, such that the top of cap 20 is closed, i.e., sealed, and, therefore, cap 20 does not require plug 25 to ensure an a tight seal. In such an embodiment, the interior of separation tube 15 is accessed through cap 20 using one or more valve means. For example, needle 102, coupled to regulator 104, may be punctured through cap 20, or other valve or port (not shown) integrated into cap 20, to inject matter, e.g., air, gas, or biological material, into separation tube 15. At least a portion of cap 20 is preferably constructed of a flexible and/or compressible material with resilient qualities, e.g., plastic, rubber, or silicone, to facilitate penetration of needle 102 through cap 20 and into cavity 95 for introducing matter into separation tube 15.

(43) Referring again to FIG. 1A, assembly of separation apparatus 5 begins with coupling separation tube 15 to cap 20 via engaging connector 45 with connector 60. Once fully engaged, upper edge of separation tube 15 abuts cap ledge 105 to provide a sealed, i.e., air-tight, engagement between cap 20 and separation tube 15. Next, separation tube 15 is received into centrifuge tube 10, and centrifuge tube 10 is coupled to cap 20 via engaging connector 35 with connector 55. Once fully engaged, upper edge of centrifuge tube 10 abuts cap ledge 110 to provide a sealed, i.e., air-tight, engagement between cap 20 and centrifuge tube 10. Next, lower portion of plug 25 is removably inserted into cavity 50. Once fully inserted, lower portion of plug 25 seats into cavity 50, and bottom edge of flange 85 abuts a top surface of cap 20 to provide a sealed, i.e., air-tight, engagement between cap 20 and plug 25. The sealed engagement between cap 20 and plug 25, and cap 20 and separation tube 15, coupled with a means for introducing air or gas into separation tube 15 in a controlled manner, e.g., channels 75 and flange 85, or needle 102 and regulator 104, provides a means for controlled release of liquid from the open bottom of separation tube 15.

(44) With further reference to FIG. 1A, density separation medium 115 is used within separation apparatus 5 to isolate target biological material. Density of density separation medium 115 depends on densities of the material to be separated. For example, to separate target white blood cells from red blood cells, the specific density of density separation medium 115 should be higher than the target white blood cell population. This will allow the higher density red blood cells to sediment through the open bottom of separation tube 15 to the bottom of the centrifuge tube 10. Meanwhile, the lower density white blood cells will remain on top of density separation medium 115 inside of separation tube 15.

(45) Method(s) of Use

(46) FIG. 9 outlines a method of using the separation system, according to an embodiment of the present invention. At step 200, a pre-determined volume of density separation medium 115 is deposited into separation apparatus 5. This can be accomplished, for example, by depositing density separation medium 115 into centrifuge tube 10, followed by inserting separation tube 15, coupled to cap 20, into centrifuge tube 10 and coupling centrifuge tube 10 to cap 20, whereby density separation medium 115 passes into separation tube 15 through open bottom of separation tube 15. As another example, separation tube 15, coupled to cap 20, can be inserted into centrifuge tube 10, followed by coupling centrifuge tube 10 to cap 20 and depositing density separation medium 115 into separation tube 15 through cavity 50, whereby density separation medium 115 flows into centrifuge tube 10 through the open bottom of separation tube 15. The amount of density separation medium 115 deposited into separation apparatus 5 is such that, a lower portion of separation tube 15 is submersed in, and filled with, density separation medium 115.

(47) At step 205, a sample containing biological material, is deposited into separation tube 15 on top of density separation medium 115. This could be accomplished, for example, by puncturing needle 102 through cap 20 and dispensing the sample through needle 102 into separation tube 15 or, alternatively, by depositing the sample into separation tube 15 through cavity 50 via aperture 62. At step 210, in an embodiment in which aperture 62 extends through a top surface of cap 20, plug 25 is engaged with cap 20 to ensure a complete air-tight seal of separation apparatus 5 in preparation for centrifugation. At step 215, separation apparatus 5 containing density separation medium 115 and the sample is placed in a centrifuge and the sample is centrifuged.

(48) With reference to FIGS. 7A-8C, following centrifugation, at step 220, centrifuge tube 10 is removed from cap 20, and separation tube 15 remains connected to cap 20. The sealed, i.e., air-tight engagement between cap 20 and plug 25, and cap 20 and separation tube 15 prevents liquid from flowing out of the lower end of separation tube 15. By sealing the top of separation tube 15, air is prevented from entering and exerting a downward force on liquid inside separation tube 15, leaving only the upward force of atmospheric pressure being exerted on the liquid at the open bottom. As such, the pressure inside separation tube 15 is less than the external atmospheric pressure. Further, the upward force of atmospheric pressure is greater than the force of gravity pulling down on the liquid. Thus, the liquid is held inside separation tube 15.

(49) At step 225, air or gas is introduced into the upper portion of separation tube 15. This increases the pressure inside separation tube 15, such that it is equal to or greater than the external atmospheric pressure. Air or gas can be introduced into separation tube 15 in a number of ways. For example, in an embodiment, pressure is applied to plug 25 to transition flange 85 to its open position, allowing an in-flow of air through channels 75 and into separation tube 15. The degree of inversion is dependent on the amount of pressure applied to plug 25. As such, the volume of influent air can be controlled by adjusting the amount of pressure exerted on plug 25. As air enters the top of separation tube 15, the upward and downward forces of air pressure on the liquid equalizes, leaving gravity as the dominant force causing the liquid to drop out of the bottom of separation tube 15. As air is introduced into separation tube 15, a corresponding volume of liquid is expelled through the lower end of separation tube 15. Therefore, the release of liquid from separation tube 15 can be controlled by adjusting the amount of pressure exerted on cap 20.

(50) In another embodiment, needle 102 is punctured into and through plug 25 and air or gas is introduced into the upper portion of separation tube 15 through needle 102. Needle 102 is connected to regulator 104, such that the air or gas can be introduced into the upper portion of separation tube 15 in a regulated manner. The regulated influx of air or gas defines the release speed of the fluid from the open bottom of separation tube 15. As air or gas is introduced into separation tube 15, a corresponding volume of liquid is expelled through the lower end of separation tube 15. Therefore, the release of liquid from separation tube 15 can be controlled by adjusting the amount of air or gas injected into separation tube 15.

(51) At step 230, air- or gas-flow into separation tube 15 is halted. This is accomplished, for example, by releasing pressure from plug 25, allowing flange 85 to revert to its native configuration, i.e., the closed position, thereby sealing channels 75 and preventing additional air from flowing into separation tube 15. Similarly, air or gas-flow through needle 102 may be shut off. By halting air- or gas-flow into separation tube 15, the pressure inside separation tube 15 drops below the external atmospheric pressure, preventing release of any remaining fluid inside from separation tube 15 until air- or gas-flow resumes.

(52) Optionally, at step 235, the efflux from separation tube 15 is analyzed, based on characteristics including, but not limited to, light absorption, emission, refraction, reflection and diffraction.

(53) While the foregoing method is described and shown in a numerical, step-wise order, it should be understood that the steps are not limited to any specific order. Additionally, some steps may overlap in time, i.e., they may be carried out simultaneously, with other steps.

(54) The invention has been described herein using specific embodiments for the purposes of illustration only. It will be readily apparent to one of ordinary skill in the art, however, that the principles of the invention can be embodied in other ways. For example, one skilled in the art would understand and appreciate that any connection means, e.g., threading, interference fit, snap fit, etc., described herein and/or shown in the drawings, could be substituted with another connection means capable of performing the same function. Similarly, one skilled in the art would understand and appreciate that placement of connection means, e.g., interiorly-located vs. exteriorly-located, could be reversed without deviating from the scope of the present invention. Likewise, it will be readily apparent that the features, functions, and/or elements of the present invention disclosed herein can be used in any combination to produce various embodiments of the present invention. Therefore, the invention should not be regarded as being limited in scope to the specific embodiments disclosed herein, but instead as being fully commensurate in scope with the following claims.