US20120199241A1 - Devices and methods for overlaying blood or cellular suspensions - Google Patents
Devices and methods for overlaying blood or cellular suspensions Download PDFInfo
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- US20120199241A1 US20120199241A1 US13/207,323 US201113207323A US2012199241A1 US 20120199241 A1 US20120199241 A1 US 20120199241A1 US 201113207323 A US201113207323 A US 201113207323A US 2012199241 A1 US2012199241 A1 US 2012199241A1
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- fluid
- open
- layering device
- ended container
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
- B01L3/5082—Test tubes per se
- B01L3/50825—Closing or opening means, corks, bungs
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/483—Physical analysis of biological material
- G01N33/487—Physical analysis of biological material of liquid biological material
- G01N33/49—Blood
- G01N33/491—Blood by separating the blood components
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/02—Adapting objects or devices to another
- B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/06—Fluid handling related problems
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/06—Fluid handling related problems
- B01L2200/0684—Venting, avoiding backpressure, avoid gas bubbles
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/04—Closures and closing means
- B01L2300/046—Function or devices integrated in the closure
- B01L2300/047—Additional chamber, reservoir
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/02—Devices for withdrawing samples
- G01N1/10—Devices for withdrawing samples in the liquid or fluent state
- G01N1/12—Dippers; Dredgers
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/04—Investigating sedimentation of particle suspensions
- G01N15/042—Investigating sedimentation of particle suspensions by centrifuging and investigating centrifugates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/04—Investigating sedimentation of particle suspensions
- G01N15/05—Investigating sedimentation of particle suspensions in blood
Definitions
- This invention relates to a thermoplastic device for overlaying blood or cellular suspension over a volume of Ficoll contained in a 50 ml or 15 ml conical centrifuge tube.
- WBC's white blood cells
- Ficoll density gradient protocol is efficient at yielding a high percentage of WBC's from a sample, it is time consuming, messy, and requires hours of hands-on training.
- the most time consuming part of the Ficoll density protocol is the “overlay”, where blood or cellular suspension is carefully poured over the surface of the Ficoll as to prevent any mixing of the two liquids. Two separate layers must be formed with minimal mixing in order to yield a sufficient number of cells.
- the overlay is completed by holding two conical centrifuge tubes together, one with Ficoll and one with blood or cellular suspension, and slowly pouring the blood or cellular suspension over the Ficoll liquid.
- the protocol depends on the lab technician to judge how fast or slow to pour the blood or cellular suspension onto the Ficoll liquid. This dependence on human technique frequently results in spills, mixing the blood/cellular suspension with Ficoll, or total loss of sample.
- the overlay method is time consuming and tedious. The majority of the time spent isolating WBC's is spent on the overlay method. Larger experiments that require a lot of WBC's are split into multiple experiments because there is simply not enough time in the day to overlay a large volume of blood or cellular suspension.
- Described and claimed herein are devices and processes for overlaying a fluid, such as blood or a cellular suspension over a volume of a base material, such as Ficoll.
- the base material is initially poured into a conical centrifuge tube (e.g., standard 50 ml or 15 ml tubes).
- the device at least partially defines a reservoir for temporarily storing the overlay fluid, a fluid barrier separating the reservoir and the container, one or more fluid channels across the fluid barrier, and an exhaust regulator.
- Certain features of the exhaust regulator and channels allow an adequately regulated, steady flow of blood or cellular composition into the conical centrifuge tube and onto the Ficoll liquid, without substantially disturbing a surface layer of the Ficoll regardless of the level of skill or care of the clinician.
- the fluid layering device requires no skill, thus eliminating prior training from others, mixing, spills, or ruined experiments.
- the device dispenses blood/cellular suspension in an ordered, uniform fashion so that a perfect overlay occurs in the minimal amount of time. Multiple devices can be used at once to dramatically reduce the total time spent overlaying in an experiment.
- the device is sterile, disposable, and affordable—an economical and feasible alternative to the mainstream overlay technique in PBMC isolation.
- a fluid layering device is configured to control a flow of fluid into an open-ended container.
- the device includes a fluid barrier configured to prevent passage of fluid from a proximal reservoir toward a distal end of the open-ended container.
- a peripheral seal extends along an outer perimeter of the fluid barrier and is configured for sealing engagement along an interior surface of the open-ended container.
- the device further includes at least one groove formed in the peripheral seal. The at least one groove is configured to provide a controlled flow of fluid across the fluid barrier and along the interior surface of the open-ended container.
- the device also includes an exhaust vent configured to vent from the open-ended container gas displaced by the controlled flow of fluid.
- the device includes a proximal reservoir defined at least partially by a proximal surface of the fluid barrier.
- the reservoir is open-ended, defined by an elongated cylindrical wall extending proximally from the fluid barrier.
- the device includes a coupling arrangement adjacent to the open end of the reservoir.
- the device includes the coupling arrangement comprises a thread.
- the device includes the fluid barrier comprises at least one drain port in fluid communication with a proximal end of each of the at least one grooves.
- the device includes a proximal handle allowing for insertion and removal of the fluid layering device with respect to the open-ended container.
- the exhaust vent terminates in an exhaust port disposed along an outer surface of the fluid layering device.
- each of the at least one grooves extends helically along a cylindrical surface defining the peripheral seal.
- the device includes a shoulder positioned to abut at least a portion of a rim of the open-ended container when the fluid layering device is inserted therein, the shoulder position with respect to a proximal end of the fluid layering device to control height of the fluid barrier along a longitudinal axis of the open-ended container.
- the device is sterilized.
- the device includes is formed from material selected from the group consisting of: plastics; polymers; resins; glass; ceramics; metals; and combinations thereof.
- fluid stored within the fluid layering device is observable through a sidewall of the device.
- At least a portion of the fluid layering device is translucent or transparent.
- a process for controlling a flow of fluid into an open-ended container includes positioning a fluid barrier above a surface of a base material disposed in a distal end of the open-ended container.
- a first fluid is added into a reservoir positioned proximal to the fluid barrier.
- a flow of fluid is directed from the reservoir across the fluid barrier and into the distal end of the open-ended container, fluid passing the fluid barrier flowing toward the base material along an interior surface of the open-ended container. Gas displaced by the controlled flow of fluid is exhausted from the distal end of the open-ended container, such that a layer of the first fluid is deposited over the base material without substantially disturbing a surface of the base material.
- the process includes positioning the fluid barrier comprises inserting at least a proximal portion of a fluid layering device into an open end of the open-ended container.
- the process includes removing the fluid barrier from the open-ended container.
- the process includes centrifuging the layered material.
- the base material is Ficoll.
- the fluid is a cellular suspension.
- the cellular suspension is blood.
- the device also includes means for adding a first fluid into a reservoir positioned proximal to the fluid barrier and means for directing a flow of fluid from the reservoir across the fluid barrier and into the distal end of the open-ended container, wherein passing the fluid barrier flowing toward the base material occurs along an interior surface of the open-ended container.
- the device also includes means for exhausting from the distal end of the open-ended container gas displaced by the controlled flow of fluid.
- a fluid layering device configured to control a flow of fluid into an open-ended container containing a base material includes a longitudinally extending cylindrical side wall open at its proximal end and a fluid barrier disposed across a distal end of the cylindrical side wall, a proximal surface of the fluid barrier and an interior surface of the cylindrical side wall forming an open-ended reservoir.
- the device also includes an insertable portion extending distally from the fluid barrier.
- the insertable portion includes a longitudinally extending sealing wall positioned to form a fluid-tight seal along a peripheral interior surface of a proximal portion of the open-ended container and at least one groove extending along the sealing wall and terminating in a peripheral fluid port configured to ensure fluid flowing into the open-ended container flows along an interior surface of the open-ended container.
- the device also includes at least one drain in fluid communication between the reservoir and a proximal end of each of the at least one grooves and an exhaust vent in fluid communication with the open-ended container. The vent regulates the flow of gas displaced by the controlled flow of fluid, thereby contributing to a rate of fluid flow.
- the at least one groove extends helically along the sealing wall.
- the device further includes a threaded coupling engagement along a distal portion of the cylindrical wall.
- FIG. 1 illustrates a side view of one embodiment of a fluid layering device.
- FIG. 2 illustrates a longitudinal cross-section of FIG. 1 .
- FIG. 3A illustrates a proximal end view of the fluid layering device of FIG. 1 and FIG. 2
- FIG. 3B is a distal end view of the fluid layering device of FIG. 1 and FIG. 2 .
- FIG. 4A is a side view of the fluid layering device of FIG. 1 aligned for insertion into an open ended conical container.
- FIG. 4B illustrates the fluid layering device after insertion into the conical container.
- FIG. 5A illustrates a cross-section of the conical open ended container with the fluid layering device of FIG. 1 and FIG. 2 inserted into its open end.
- FIG. 5B illustrates in more detail, engagement of an insertable portion of the fluid layering device with an interior surface of the open ended conical container.
- FIG. 6A-6D illustrate operation of an embodiment of a fluid layering device in controlling flow of a fluid into an open ended conical container containing a base material.
- FIG. 7A-7D illustrate an alternative embodiment of a fluid layering device.
- FIG. 8A-8C illustrate insertion of the embodiment of the fluid layering device illustrated in FIG. 7A-7D into an open end of the conical container and its direction of a fluid flow into the container.
- FIG. 9 illustrates yet another embodiment of a fluid layering device.
- FIG. 10A illustrates an open ended container comprising a base material and a blood sample layered according to one embodiment of the invention.
- FIG. 10B illustrates contents of the open ended container illustrated in FIG. 10A after applied to a centrifuge process.
- FIG. 10C illustrates the open ended container contents of FIG. 10B after further processing.
- FIG. 11 illustrates an embodiment of a process for directing fluid flow into an open ended container.
- a first fluid such as blood or a cellular suspension onto a base material.
- the base material can include, but is not limited to, sugar density gradients such as Ficoll, Percoll, Isopercoll, and isopycnic sucrose density gradient.
- the cellular suspension can include, but is not limited to, monocyte cultures; Tc clones; islets of Langerhans from pancreatic tissue (e.g., Dellê et al., The Use of Iodixanol for the Purification of Rat Pancreatic Islets, T RANSPLANTATION P ROCEEDINGS, Vol. 39, No.
- neural cells from brain tissue e.g., Sims & Anderson, Isolation of mitochondria from rat brain using Percoll density gradient centrifugation, N ATURE P ROTOCOLS, Vol. 3, 2008, pages 1228-1239
- ovarian follicles e.g., Martinez-Madrid et al., Ficoll density gradient method for recovery of isolated human ovarian primordial follicles, F ERTILITY AND S TERILITY, Vol. 82, No.
- spermatozoa from epididymis e.g., Haldar et al., Ficoll Gradient Isolation of Immaturesperm of High Purity and Intactness From Goat Epididymis, S YSTEMS B IOLOGY IN R EPRODUCTIVE M EDICINE, Vol. 24, No. 2, 1990, pages 125-128); and plant cells (e.g., Attree & Sheffield, An evaluation of Ficoll density gradient centrifugation as a method for eliminating microbial contamination and purifying plant protoplasts, J OURNAL P LANT C ELL R EPORTS, Vol. 5, No.
- the fluid layering device can be coupled through a coupling extension to an open end of a container, such as a conical centrifuge tube, already including the base material. Once attached, the device regulates flow of the first fluid from the reservoir into the conical tube so that a suitable overlay layer is formed without substantially disturbing a surface of the base material, regardless of the skill and care of the user.
- a container such as a conical centrifuge tube
- FIG. 1 A side view of an embodiment a fluid layering device 100 is illustrated in FIG. 1 .
- the device 100 includes an elongated cylindrical housing 102 defining an open ended reservoir 104 at one end and an insertable coupling portion 106 at an opposite end.
- One or more helical channels 108 are defined along a peripheral side wall 144 of the coupling portion 106 .
- the helical channels 108 extend from a top or proximal end of the coupling portion 106 to a bottom or distal end of the coupling portion 106 .
- the number of triangle openings 110 and helical channels 108 depend on the size/model of the invention.
- There is a circular exhaust vent 118 which allows gas to be vented from the inside of the conical centrifuge tube to the
- the entire device 100 can be made of a common material.
- the device is made from a moldable material, such as a hard, translucent, non-flexible plastic such as polystyrene (PS), acrylonitrile butadiene styrene (ABS), or polycarbonate (PC).
- PS polystyrene
- ABS acrylonitrile butadiene styrene
- PC polycarbonate
- FIG. 2 details the cross-section of the device 100 and illustrates the exhaust vent 112 and an interior barrier wall 114 .
- the barrier wall is a conical divider 114 .
- the exhaust vent is composed of a rectangular tunnel 116 that leads from the conical centrifuge volume to a circular tunnel 118 that exits a proximal side wall of the device.
- the cross-section of this tunnel 116 , 118 determines the rate of gas exiting the conical tube and thus the rate of the blood/cellular suspension entering the conical tube via the helical channels 108 .
- the conical divider physically separates the contents of the cylindrical reservoir from the conical centrifuge tube. Its conical shape assists in the complete evacuation of the reservoirs contents into the centrifuge tube.
- FIG. 3A illustrates a proximal end view of the fluid flow device illustrated in FIG. 1 and FIG. 2 .
- a proximal end or rim of the reservoir wall 120 defines an open end of the reservoir 104 .
- a proximal surface 122 of the conical divider 114 defines a bottom of the reservoir 104 .
- the proximal surface 122 follows the contour of the conical divider 114 terminating in a conical peak 124 centrally located along a base surface of the reservoir 104 .
- An annular trough 126 extends between a base portion of the proximal surface 122 and the base of the interior surface of the reservoir walls 120 .
- One or more openings 128 a , 128 b , 128 c are located along the annular trough 126 .
- three triangular openings 128 are disposed about the annular trough.
- a proximal portion of an exhaust vent housing 130 is also visible.
- the exhaust vent exit tunnel 118 is illustrated in phantom, exiting the reservoir wall 120 at the exhaust vent outlet port 132 .
- a fluid placed in the reservoir 104 will be directed by the conical peak 124 toward the annular trough 126 . The fluid is able to exit the reservoir 104 through the triangular openings 128 .
- FIG. 3B Illustrated in FIG. 3B is a distal end view of the fluid layering device 100 .
- a proximal extension 106 defines along its outer peripheral surface a peripheral sealing wall 140 .
- An annular shoulder 142 is defined along a base portion of the reservoir wall 120 extending in radial direction beyond the peripheral sealing wall 144 .
- One or more fluid ports are defined along a proximal end of the proximal extension 312 .
- three fluid ports 146 a , 146 b , 146 c (generally 146 ) are disposed evenly about the circumference of the proximal extension 106 .
- a distal surface 148 of the conical divider 114 extends across an opening of the proximal extension 106 .
- An inner conical peak 150 is shown centrally to the distal surface 148 .
- the distal surface 148 includes an exhaust vent inlet port 152 .
- the exhaust vent inlet port 152 is in fluid communication with the exhaust vent exit tunnel 118 allowing gas to exit through the exhaust vent outlet port 132 .
- the fluid layering device 100 is positioned in axial alignment with an elongated open end conical container 200 .
- the fluid layering device 100 is positioned such that a distal end of the insertable portion 106 faces an open end 202 of the open ended container 200 . Coupling of the two devices is accomplished by advancing the fluid layering device 100 towards the open end 200 advancing the insertable portion 106 within the open end 202 of the container 200 .
- the fluid layering device 100 is advanced axially until the annular shoulder 142 abuts an annular rim 204 of the open end 202 .
- An illustration of the fluid layering device 100 fully inserted within the open ended container 200 is shown in FIG. 4B . Also visible is the exhaust vent outlet port 132 positioned proximally to the annular shoulder 142 such that the exhaust outlet port 132 remains unobstructed after insertion.
- FIG. 5A a Illustrated in FIG. 5A a is a lateral cross-section of the open ended container 200 with the fluid layering device 100 fully inserted into its open end 202 .
- FIG. 5B illustrates in more detail arrangement of the peripheral sealing wall 144 , the helical groove 108 and an inner wall surface 210 .
- a fluid channel 220 is formed along the helical groove 108 being defined by the helical groove 108 and an adjacent portion of the inner wall surface 210 .
- the abutting arrangement of the shoulder 142 against the annular rim 204 Also shown in more detail.
- FIG. 6A Operation of an embodiment of a fluid layering device in controlling flow of a fluid into an open ended conical container is illustrated in the series of FIG. 6A through FIG. 6D .
- a base material such as Ficoll is inserted into an open end of the upright container 200 .
- the base material 230 pools along the conical bottom end, such that a surface of the base material is spaced apart from the open end 202 by a distance h 1 .
- the fluid layering device 100 is coupled to the container 200 as described above and illustrated in FIG. 4B and FIG. 5A .
- a distal end of the insertable portion 106 resides at a height h 2 above the surface of the base material.
- the reservoir 104 extends proximally from the open end 202 to a height h 3 .
- a first fluid in this instance a cellular suspension—blood
- a first fluid is poured into an open end 105 of the reservoir 104 .
- the container-fluid layering device arrangement is positioned in upright or vertically, such that gravity will drive the flow of blood into the container 200 .
- the arrangement can be positioned at an angle, but preferably not much more than about 30 degrees measured from vertical.
- gravity induces a downward flow, forcing blood 240 from the reservoir 104 into the channels formed between helical grooves 108 and the inner surface of the container 210 .
- the fluid 240 exits the grooves 108 along the interior walls 210 of the substantially upright container 200 .
- Gravity continues to drive blood flow 242 downward towards the exposed surface of the base material 230 .
- Surface tension keeps the blood flow 242 substantially directed along the inner side walls 210 .
- air pressure within the chamber 244 increases.
- the exhaust vent 115 allows air to bleed off from the chamber 244 , thereby reducing the pressure and allowing for continued blood flow 242 .
- the exhaust vent 115 can be used to control flow of blood 242 into the container 200 .
- a substantially narrow exhaust vent 115 restricts the flow of gas similarly restricting flow of blood 240 into the chamber 244 .
- the fluid layering device 100 can be removed carefully as shown in FIG. 6C . Care must be exercised when separating the device 100 from the container so as not to disturb the layered arrangement of fluid 240 and base material 230 (e.g., blood and Ficoll).
- the fluid layering device 100 is preferably sterilized. Although it may be possible to re-sterilize (e.g., autoclave) the device 100 , it is generally anticipated that it will be disposable based on the relative simplicity of design and minimum costs.
- FIG. 7A-7D An alternative embodiment of a fluid layering device 300 is illustrated in FIG. 7A-7D .
- the device 300 includes a barrier wall 302 defining a perimeter conforming to a lateral cross section of the open ended container 330 .
- the perimeter is substantially circular.
- the barrier wall 302 defines a proximal surface 303 facing the fluid reservoir and a distal surface 305 facing the base material.
- a peripheral sealing wall 304 extends between the proximal surface 303 and the distal surface 305 and along the outer perimeter of the barrier wall 302 . When inserted into an open end of the container 330 , the barrier wall prevents fluid flow from the reservoir toward the base material.
- the fluid layering device 300 also includes one or more grooves 306 formed in the peripheral sealing wall 304 , configured to allow a controlled flow of fluid from the reservoir toward the base material.
- six such grooves 306 are evenly distributed around the circular perimeter of the barrier wall 302 .
- Each groove is formed as rectangular groove 306 .
- the rectangular shape of the groove 306 is not meant to be limiting in any way.
- Other shaped grooves are contemplated, such as triangular, elliptical, circular, polygons, random shapes, and combinations of any such shaped.
- the grooves are directed parallel to a longitudinal axis of the device.
- the grooves may be angled, and/or curved, for example, in a helix arrangement. It is not necessary that all of the grooves 306 be identical in size, shape, or orientation.
- the fluid layering device 300 also includes one or more exhaust vents.
- the device 300 includes two such vents.
- Each exhaust vent 307 ′, 307 ′′ (generally 307 ).
- Each vent 307 includes an elongated exhaust vent lumen 314 defined by a proximal handle 310 and extending between an exhaust vent inlet port 308 and an exhaust vent exit port 316 .
- the handle 310 extends proximally away from the proximal surface of the barrier wall 302 .
- the handle 310 extends axially for a length sufficiently longer than any intended insertion depth, such that at least a proximal portion of the handle 310 extends beyond an open end of the container 330 .
- the handle includes an open area to facilitate removal of the fluid layering device 300 from the container by providing a surface upon which a finger, fingers, or suitable instrument may apply an axial removing force to remove the device 300 from the container 330 .
- FIG. 8A-8C Operation of an embodiment of the alternative embodiment of the fluid layering device illustrated in FIG. 7A-7D and described above is illustrated in the series of FIG. 8A-8C .
- a base material such as Ficoll is inserted into an open end of the upright container 330 .
- the base material pools along the conical bottom end 331 , such that a surface 333 of the base material 319 is spaced apart from the open end 332 by a distance h 1 .
- a distal portion of the fluid layering device 300 including the barrier wall 302 is inserted into the open end of the container 330 .
- a distal surface of the barrier wall 302 When fully inserted, a distal surface of the barrier wall 302 resides at a height h 2 above the surface 333 of the base material 319 as illustrated in FIG. 8B .
- a reservoir 332 extends proximally from a proximal surface of the barrier wall 332 to the open end 332 to a height h 3 .
- a proximal portion of the handle remains exposed, extending to a height h 5 measured from the open end 332 .
- a sufficient portion of the open area 312 remains exposed to allow for insertion of a finger or suitable instrument during removal process.
- a first fluid in this instance a cellular suspension blood
- a first fluid is poured into the open end 332 of the reservoir 30 .
- the container-fluid layering device arrangement is preferably positioned upright or vertically, such that gravity will drive the flow of blood 350 into the container 330 .
- the arrangement can be positioned at an angle, but preferably not much more than about 30 degrees measured from vertical.
- the fluid layering device 300 can be removed carefully by puling the exposed portion of the handle 310 . Care must be exercised when separating the device from the container so as not to disturb the layered arrangement of blood 352 and Ficoll 319 .
- FIG. 9 illustrates yet another embodiment of a fluid layering device 380 .
- the device 380 includes a proximal reservoir 382 and an insertable base portion 394 .
- the reservoir 382 includes an open ended fluid chamber 384 and an elongated side wall 385 extending along a cord dissecting a cylindrical wall of the reservoir 382 . Fluid poured into the open end portion 384 flows toward a barrier wall 392 forming a base portion of the reservoir 382 .
- One or more drain apertures 390 are disposed along an outer periphery of the barrier wall 392 , allowing fluid stored within the container to enter one or more longitudinally extending channels 398 defined along a peripheral sealing wall 396 .
- Fluid flows along the longitudinal channels 398 between the channels 398 and an interior wall 210 of the open-ended container 200 .
- Gas displaced by the inflowing fluid exits the container 200 through an exhaust vent 388 .
- the exhaust vent directs expelled gas (e.g., air) into a vent portion 386 defined between an opposite side of the elongated separating wall 385 and an opposing portion of the cylindrical side wall, such that exiting exhaust air is not interfered with by fluid stored within the reservoir 382 .
- FIG. 10A illustrates an open ended container 400 comprising a layered arrangement of blood 402 over Ficoll-paque PLUS 404 .
- the layered arrangement 402 , 404 can be accomplished using any of the devices and processes described herein.
- the layered arrangement 402 , 404 can be centrifuged using standard techniques to separate various blood components.
- FIG. 10B illustrates contents of the container illustrated in FIG. 10A after applied to a centrifuge process.
- a bottom layer 406 of granulocytes and erythrocytes is formed along the very bottom of the container 400 .
- Disposed above this layer 406 is a first intermediate layer 408 of Ficoll-paque PLUS.
- first intermediate layer 408 is a second intermediate layer 410 of lymphocytes, and above that is a top layer 412 of plasma and platelets.
- the top layer 412 can be removed, for example by simply pouring it out of the container as illustrated in FIG. 10C .
- FIG. 11 illustrates an embodiment of a process 500 for directing fluid flow into an open ended container.
- an open-ended container is partially filled with a base material. This is generally accomplished as part of protocols known to those skilled in the art, such as a protocol for PBMC isolation.
- the fluid layering device is then coupled to the container from its open end at 502 .
- the coupled arrangement defines a fluid reservoir for temporarily storing a first fluid during the layering process. Also defined are one or more fluid channels providing fluid communication between the reservoir and an interior region of the container in a space or chamber formed between a barrier wall and a surface of the base material.
- a fluid is added to the reservoir at 504 .
- the fluid flows under the influence of gravity through the one or more fluid channels into the chamber above the surface of the base material at 506 .
- the fluid flow is directed along an inner surface of the container wall, avoiding any free-falling droplets that would otherwise disturb the surface tension of the base material.
- the side-wall flow continues under the influence of gravity until it reaches the surface of the base material.
- the fluid then begins to pool along the surface.
- pressure of any gas within the chamber such as air is raised.
- Gas (air) is exhausted from the chamber at 508 by pressure induced by the fluid flow. Exhausted air reduces the pressure and allows for continued fluid flow.
- an equilibrium can be reached between the inflow of fluid and outflow of exhaust gas.
- the rate of flow can be controlled by at least one of the dimension of the fluid channels (e.g., length, shape, direction, diameter) and dimensions of one or more exhaust vents.
- the fluid layering device is removed from the chamber at 510 .
- the device is removed after all of the fluid has flowed from the reservoir into the chamber. Removal of the device allows for further processing, such as centrifuging at 510 (optional).
Abstract
A device is described that overlays a first fluid, such as blood or a cellular suspension onto a base material, such as a Ficoll liquid density gradient. In some embodiments, the fluid layering device includes a cylindrical reservoir, a fluid barrier, a coupling extension, one or more helical channels in fluid communication with the reservoir, and an exhaust vent. The fluid layering device can be coupled through its coupling extension to an open end of a container, such as a conical centrifuge tube, including the base material. Once attached, the device regulates flow of the first fluid from the reservoir into the conical tube so that a suitable overlay is formed without substantially disturbing a surface of the base material, regardless of the care and skill of the user.
Description
- This invention relates to a thermoplastic device for overlaying blood or cellular suspension over a volume of Ficoll contained in a 50 ml or 15 ml conical centrifuge tube.
- The isolation and preparation of leukocytes, more generally referred to as “white blood cells” (WBC's), from whole blood or cellular suspension using a Ficoll density gradient is generally the first technique to be carried out in any immunological experiment. WBC's are the main components of our immune system and thus are the main target for experimentation.
- While the Ficoll density gradient protocol is efficient at yielding a high percentage of WBC's from a sample, it is time consuming, messy, and requires hours of hands-on training. The most time consuming part of the Ficoll density protocol is the “overlay”, where blood or cellular suspension is carefully poured over the surface of the Ficoll as to prevent any mixing of the two liquids. Two separate layers must be formed with minimal mixing in order to yield a sufficient number of cells. The overlay is completed by holding two conical centrifuge tubes together, one with Ficoll and one with blood or cellular suspension, and slowly pouring the blood or cellular suspension over the Ficoll liquid.
- There are several major drawbacks to the overlay method in the Ficoll density gradient protocol. Primarily, the protocol depends on the lab technician to judge how fast or slow to pour the blood or cellular suspension onto the Ficoll liquid. This dependence on human technique frequently results in spills, mixing the blood/cellular suspension with Ficoll, or total loss of sample. In addition, the overlay method is time consuming and tedious. The majority of the time spent isolating WBC's is spent on the overlay method. Larger experiments that require a lot of WBC's are split into multiple experiments because there is simply not enough time in the day to overlay a large volume of blood or cellular suspension.
- Clearly, then, there is a need for a device used to overlay blood or cellular suspension that can be used by any lab technician without prior training and that can speed up the overlay process. Such a device would virtually eliminate prior training or human error in the overlay process, dramatically reduce the time it takes to overlay, and increase yield by reducing spills.
- Further, such a needed device would be relatively easy to manufacture on a large scale, use, dispose, and would allow much larger experiments to be performed. The present invention fulfills these needs and provides further related advantages.
- Described and claimed herein are devices and processes for overlaying a fluid, such as blood or a cellular suspension over a volume of a base material, such as Ficoll. In some applications, the base material is initially poured into a conical centrifuge tube (e.g., standard 50 ml or 15 ml tubes). The device at least partially defines a reservoir for temporarily storing the overlay fluid, a fluid barrier separating the reservoir and the container, one or more fluid channels across the fluid barrier, and an exhaust regulator. Certain features of the exhaust regulator and channels allow an adequately regulated, steady flow of blood or cellular composition into the conical centrifuge tube and onto the Ficoll liquid, without substantially disturbing a surface layer of the Ficoll regardless of the level of skill or care of the clinician.
- The devices and processes described and claimed herein offer certain advantages over existing overlay techniques. First, the fluid layering device requires no skill, thus eliminating prior training from others, mixing, spills, or ruined experiments. Next, the device dispenses blood/cellular suspension in an ordered, uniform fashion so that a perfect overlay occurs in the minimal amount of time. Multiple devices can be used at once to dramatically reduce the total time spent overlaying in an experiment. The device is sterile, disposable, and affordable—an economical and feasible alternative to the mainstream overlay technique in PBMC isolation. Other features and advantages of the present invention will become apparent from the following more detailed description, taken in conjunction with the accompanying drawings, by way of example, the principles of the invention.
- In one embodiment, a fluid layering device is configured to control a flow of fluid into an open-ended container. The device includes a fluid barrier configured to prevent passage of fluid from a proximal reservoir toward a distal end of the open-ended container. A peripheral seal extends along an outer perimeter of the fluid barrier and is configured for sealing engagement along an interior surface of the open-ended container. The device further includes at least one groove formed in the peripheral seal. The at least one groove is configured to provide a controlled flow of fluid across the fluid barrier and along the interior surface of the open-ended container. The device also includes an exhaust vent configured to vent from the open-ended container gas displaced by the controlled flow of fluid.
- In some embodiments, the device includes a proximal reservoir defined at least partially by a proximal surface of the fluid barrier.
- In some embodiments, the reservoir is open-ended, defined by an elongated cylindrical wall extending proximally from the fluid barrier.
- In some embodiments, the device includes a coupling arrangement adjacent to the open end of the reservoir.
- In some embodiments, the device includes the coupling arrangement comprises a thread.
- In some embodiments, the device includes the fluid barrier comprises at least one drain port in fluid communication with a proximal end of each of the at least one grooves.
- In some embodiments, the device includes a proximal handle allowing for insertion and removal of the fluid layering device with respect to the open-ended container.
- In some embodiments, the exhaust vent terminates in an exhaust port disposed along an outer surface of the fluid layering device.
- In some embodiments, each of the at least one grooves extends helically along a cylindrical surface defining the peripheral seal.
- In some embodiments, the device includes a shoulder positioned to abut at least a portion of a rim of the open-ended container when the fluid layering device is inserted therein, the shoulder position with respect to a proximal end of the fluid layering device to control height of the fluid barrier along a longitudinal axis of the open-ended container.
- In some embodiments, the device is sterilized.
- In some embodiments, the device includes is formed from material selected from the group consisting of: plastics; polymers; resins; glass; ceramics; metals; and combinations thereof.
- In some embodiments, fluid stored within the fluid layering device is observable through a sidewall of the device.
- In some embodiments, at least a portion of the fluid layering device is translucent or transparent.
- In another embodiment, a process for controlling a flow of fluid into an open-ended container includes positioning a fluid barrier above a surface of a base material disposed in a distal end of the open-ended container. A first fluid is added into a reservoir positioned proximal to the fluid barrier. A flow of fluid is directed from the reservoir across the fluid barrier and into the distal end of the open-ended container, fluid passing the fluid barrier flowing toward the base material along an interior surface of the open-ended container. Gas displaced by the controlled flow of fluid is exhausted from the distal end of the open-ended container, such that a layer of the first fluid is deposited over the base material without substantially disturbing a surface of the base material.
- In some embodiments, the process includes positioning the fluid barrier comprises inserting at least a proximal portion of a fluid layering device into an open end of the open-ended container.
- In some embodiments, the process includes removing the fluid barrier from the open-ended container.
- In some embodiments, the process includes centrifuging the layered material.
- In some embodiments of the process, the base material is Ficoll.
- In some embodiments of the process, the fluid is a cellular suspension.
- In some embodiments of the process, the cellular suspension is blood.
- In some embodiments a fluid layering device configured to control a flow of fluid into an open-ended container includes means for positioning a fluid barrier above a surface of a base material disposed in a distal end of the open-ended container. The device also includes means for adding a first fluid into a reservoir positioned proximal to the fluid barrier and means for directing a flow of fluid from the reservoir across the fluid barrier and into the distal end of the open-ended container, wherein passing the fluid barrier flowing toward the base material occurs along an interior surface of the open-ended container. The device also includes means for exhausting from the distal end of the open-ended container gas displaced by the controlled flow of fluid.
- In yet another embodiment, a fluid layering device configured to control a flow of fluid into an open-ended container containing a base material includes a longitudinally extending cylindrical side wall open at its proximal end and a fluid barrier disposed across a distal end of the cylindrical side wall, a proximal surface of the fluid barrier and an interior surface of the cylindrical side wall forming an open-ended reservoir. The device also includes an insertable portion extending distally from the fluid barrier. The insertable portion includes a longitudinally extending sealing wall positioned to form a fluid-tight seal along a peripheral interior surface of a proximal portion of the open-ended container and at least one groove extending along the sealing wall and terminating in a peripheral fluid port configured to ensure fluid flowing into the open-ended container flows along an interior surface of the open-ended container. The device also includes at least one drain in fluid communication between the reservoir and a proximal end of each of the at least one grooves and an exhaust vent in fluid communication with the open-ended container. The vent regulates the flow of gas displaced by the controlled flow of fluid, thereby contributing to a rate of fluid flow.
- In some embodiments of the device, the at least one groove extends helically along the sealing wall.
- In some embodiments the device further includes a threaded coupling engagement along a distal portion of the cylindrical wall.
- The foregoing and other objects, features and advantages of the invention will be apparent from the following more particular description of preferred embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of the invention.
-
FIG. 1 illustrates a side view of one embodiment of a fluid layering device. -
FIG. 2 illustrates a longitudinal cross-section ofFIG. 1 . -
FIG. 3A illustrates a proximal end view of the fluid layering device ofFIG. 1 andFIG. 2 -
FIG. 3B is a distal end view of the fluid layering device ofFIG. 1 andFIG. 2 . -
FIG. 4A is a side view of the fluid layering device ofFIG. 1 aligned for insertion into an open ended conical container. -
FIG. 4B illustrates the fluid layering device after insertion into the conical container. -
FIG. 5A illustrates a cross-section of the conical open ended container with the fluid layering device ofFIG. 1 andFIG. 2 inserted into its open end. -
FIG. 5B illustrates in more detail, engagement of an insertable portion of the fluid layering device with an interior surface of the open ended conical container. -
FIG. 6A-6D illustrate operation of an embodiment of a fluid layering device in controlling flow of a fluid into an open ended conical container containing a base material. -
FIG. 7A-7D illustrate an alternative embodiment of a fluid layering device. -
FIG. 8A-8C illustrate insertion of the embodiment of the fluid layering device illustrated inFIG. 7A-7D into an open end of the conical container and its direction of a fluid flow into the container. -
FIG. 9 illustrates yet another embodiment of a fluid layering device. -
FIG. 10A illustrates an open ended container comprising a base material and a blood sample layered according to one embodiment of the invention. -
FIG. 10B illustrates contents of the open ended container illustrated inFIG. 10A after applied to a centrifuge process. -
FIG. 10C illustrates the open ended container contents ofFIG. 10B after further processing. -
FIG. 11 illustrates an embodiment of a process for directing fluid flow into an open ended container. - Described herein are examples of devices and processes configured to overlay a first fluid, such as blood or a cellular suspension onto a base material. The base material can include, but is not limited to, sugar density gradients such as Ficoll, Percoll, Isopercoll, and isopycnic sucrose density gradient. The cellular suspension can include, but is not limited to, monocyte cultures; Tc clones; islets of Langerhans from pancreatic tissue (e.g., Dellê et al., The Use of Iodixanol for the Purification of Rat Pancreatic Islets, T
RANSPLANTATION PROCEEDINGS, Vol. 39, No. 2, pages 467-469); neural cells from brain tissue (e.g., Sims & Anderson, Isolation of mitochondria from rat brain using Percoll density gradient centrifugation, NATURE PROTOCOLS, Vol. 3, 2008, pages 1228-1239); ovarian follicles (e.g., Martinez-Madrid et al., Ficoll density gradient method for recovery of isolated human ovarian primordial follicles, FERTILITY AND STERILITY, Vol. 82, No. 6, 2004, pages 1648-1653); spermatozoa from epididymis (e.g., Haldar et al., Ficoll Gradient Isolation of Immature Sperm of High Purity and Intactness From Goat Epididymis, SYSTEMS BIOLOGY IN REPRODUCTIVE MEDICINE, Vol. 24, No. 2, 1990, pages 125-128); and plant cells (e.g., Attree & Sheffield, An evaluation of Ficoll density gradient centrifugation as a method for eliminating microbial contamination and purifying plant protoplasts, JOURNAL PLANT CELL REPORTS, Vol. 5, No. 4, 1986, pages 288-291; Liang et al., Isolation of Spinach Leaf Peroxisomes in 0.25 Molar Sucrose Solution by Percoll Density Gradient Centrifugation, PLANT PHYSIOL., Vol. 70, 1982, pages 1210-1212). - In general, the fluid layering device can be coupled through a coupling extension to an open end of a container, such as a conical centrifuge tube, already including the base material. Once attached, the device regulates flow of the first fluid from the reservoir into the conical tube so that a suitable overlay layer is formed without substantially disturbing a surface of the base material, regardless of the skill and care of the user.
- A side view of an embodiment a
fluid layering device 100 is illustrated inFIG. 1 . Thedevice 100 includes an elongatedcylindrical housing 102 defining an open endedreservoir 104 at one end and aninsertable coupling portion 106 at an opposite end. One or morehelical channels 108 are defined along aperipheral side wall 144 of thecoupling portion 106. Thehelical channels 108 extend from a top or proximal end of thecoupling portion 106 to a bottom or distal end of thecoupling portion 106. There are also a number of triangular openings 110 that connect thecylindrical reservoir 104 tohelical channels 108 that wrap around the coupling portion orbase 106 of the device 100 (the base is fully inserted into the conical centrifuge tube). The number of triangle openings 110 andhelical channels 108 depend on the size/model of the invention. There is acircular exhaust vent 118, which allows gas to be vented from the inside of the conical centrifuge tube to the environment. - In at least some embodiments, the
entire device 100 can be made of a common material. In this embodiment, the device is made from a moldable material, such as a hard, translucent, non-flexible plastic such as polystyrene (PS), acrylonitrile butadiene styrene (ABS), or polycarbonate (PC). The reason for translucency is so the user can view the progress of the blood/cellular suspension in the device in order to determine whether or not the process has completed. The engineering grade plastics (PS, ABS, PC) are required so that the device will retain structurally integrity when inserted to and removed from its conical centrifuge tube. -
FIG. 2 details the cross-section of thedevice 100 and illustrates theexhaust vent 112 and an interior barrier wall 114. In the illustrative embodiment, the barrier wall is a conical divider 114. The exhaust vent is composed of arectangular tunnel 116 that leads from the conical centrifuge volume to acircular tunnel 118 that exits a proximal side wall of the device. The cross-section of thistunnel helical channels 108. The conical divider physically separates the contents of the cylindrical reservoir from the conical centrifuge tube. Its conical shape assists in the complete evacuation of the reservoirs contents into the centrifuge tube. -
FIG. 3A illustrates a proximal end view of the fluid flow device illustrated inFIG. 1 andFIG. 2 . A proximal end or rim of the reservoir wall 120 defines an open end of thereservoir 104. Aproximal surface 122 of the conical divider 114 defines a bottom of thereservoir 104. Theproximal surface 122 follows the contour of the conical divider 114 terminating in aconical peak 124 centrally located along a base surface of thereservoir 104. Anannular trough 126 extends between a base portion of theproximal surface 122 and the base of the interior surface of the reservoir walls 120. One ormore openings annular trough 126. In the illustrative embodiment threetriangular openings 128 are disposed about the annular trough. Also visible is a proximal portion of an exhaust vent housing 130. The exhaustvent exit tunnel 118 is illustrated in phantom, exiting the reservoir wall 120 at the exhaustvent outlet port 132. A fluid placed in thereservoir 104 will be directed by theconical peak 124 toward theannular trough 126. The fluid is able to exit thereservoir 104 through thetriangular openings 128. - Illustrated in
FIG. 3B is a distal end view of thefluid layering device 100. Aproximal extension 106 defines along its outer peripheral surface a peripheral sealing wall 140. Anannular shoulder 142 is defined along a base portion of the reservoir wall 120 extending in radial direction beyond theperipheral sealing wall 144. One or more fluid ports are defined along a proximal end of theproximal extension 312. In the illustrative embodiment threefluid ports proximal extension 106. A distal surface 148 of the conical divider 114 extends across an opening of theproximal extension 106. An innerconical peak 150 is shown centrally to the distal surface 148. The distal surface 148 includes an exhaustvent inlet port 152. The exhaustvent inlet port 152 is in fluid communication with the exhaustvent exit tunnel 118 allowing gas to exit through the exhaustvent outlet port 132. - Preferring to
FIG. 4A , thefluid layering device 100 is positioned in axial alignment with an elongated open endconical container 200. Thefluid layering device 100 is positioned such that a distal end of theinsertable portion 106 faces anopen end 202 of the open endedcontainer 200. Coupling of the two devices is accomplished by advancing thefluid layering device 100 towards theopen end 200 advancing theinsertable portion 106 within theopen end 202 of thecontainer 200. Thefluid layering device 100 is advanced axially until theannular shoulder 142 abuts anannular rim 204 of theopen end 202. An illustration of thefluid layering device 100 fully inserted within the open endedcontainer 200 is shown inFIG. 4B . Also visible is the exhaustvent outlet port 132 positioned proximally to theannular shoulder 142 such that theexhaust outlet port 132 remains unobstructed after insertion. - Illustrated in
FIG. 5A a is a lateral cross-section of the open endedcontainer 200 with thefluid layering device 100 fully inserted into itsopen end 202.FIG. 5B illustrates in more detail arrangement of theperipheral sealing wall 144, thehelical groove 108 and aninner wall surface 210. In particular afluid channel 220 is formed along thehelical groove 108 being defined by thehelical groove 108 and an adjacent portion of theinner wall surface 210. Also shown in more detail is the abutting arrangement of theshoulder 142 against theannular rim 204. - Operation of an embodiment of a fluid layering device in controlling flow of a fluid into an open ended conical container is illustrated in the series of
FIG. 6A throughFIG. 6D . Initially, a base material, such as Ficoll is inserted into an open end of theupright container 200. Thebase material 230 pools along the conical bottom end, such that a surface of the base material is spaced apart from theopen end 202 by a distance h1. Referring toFIG. 6A , thefluid layering device 100 is coupled to thecontainer 200 as described above and illustrated inFIG. 4B andFIG. 5A . When fully inserted, a distal end of theinsertable portion 106 resides at a height h2 above the surface of the base material. Thereservoir 104 extends proximally from theopen end 202 to a height h3. As illustrated inFIG. 6B , a first fluid, in this instance a cellular suspension—blood, is poured into anopen end 105 of thereservoir 104. Preferably, the container-fluid layering device arrangement is positioned in upright or vertically, such that gravity will drive the flow of blood into thecontainer 200. In some embodiments, the arrangement can be positioned at an angle, but preferably not much more than about 30 degrees measured from vertical. - As shown in
FIG. 6C , gravity induces a downward flow, forcingblood 240 from thereservoir 104 into the channels formed betweenhelical grooves 108 and the inner surface of thecontainer 210. The fluid 240 exits thegrooves 108 along theinterior walls 210 of the substantiallyupright container 200. Gravity continues to driveblood flow 242 downward towards the exposed surface of thebase material 230. Surface tension keeps theblood flow 242 substantially directed along theinner side walls 210. As the volume of blood increases in a layer above thebase material 230, air pressure within thechamber 244 increases. Theexhaust vent 115 allows air to bleed off from thechamber 244, thereby reducing the pressure and allowing for continuedblood flow 242. Careful selection of the dimensions of theexhaust vent 115 can be used to control flow ofblood 242 into thecontainer 200. For example, a substantiallynarrow exhaust vent 115 restricts the flow of gas similarly restricting flow ofblood 240 into thechamber 244. When thereservoir 104 contents have been substantially transferred, thefluid layering device 100 can be removed carefully as shown inFIG. 6C . Care must be exercised when separating thedevice 100 from the container so as not to disturb the layered arrangement offluid 240 and base material 230 (e.g., blood and Ficoll). In most applications and particularly when working withblood 240, thefluid layering device 100 is preferably sterilized. Although it may be possible to re-sterilize (e.g., autoclave) thedevice 100, it is generally anticipated that it will be disposable based on the relative simplicity of design and minimum costs. - An alternative embodiment of a
fluid layering device 300 is illustrated inFIG. 7A-7D . Thedevice 300 includes abarrier wall 302 defining a perimeter conforming to a lateral cross section of the open endedcontainer 330. In the illustrative embodiment, the perimeter is substantially circular. Thebarrier wall 302 defines aproximal surface 303 facing the fluid reservoir and adistal surface 305 facing the base material. Aperipheral sealing wall 304 extends between theproximal surface 303 and thedistal surface 305 and along the outer perimeter of thebarrier wall 302. When inserted into an open end of thecontainer 330, the barrier wall prevents fluid flow from the reservoir toward the base material. - The
fluid layering device 300 also includes one ormore grooves 306 formed in theperipheral sealing wall 304, configured to allow a controlled flow of fluid from the reservoir toward the base material. In the illustrative embodiment, sixsuch grooves 306 are evenly distributed around the circular perimeter of thebarrier wall 302. Each groove is formed asrectangular groove 306. The rectangular shape of thegroove 306 is not meant to be limiting in any way. Other shaped grooves are contemplated, such as triangular, elliptical, circular, polygons, random shapes, and combinations of any such shaped. In the illustrative embodiment, the grooves are directed parallel to a longitudinal axis of the device. In some embodiments, the grooves may be angled, and/or curved, for example, in a helix arrangement. It is not necessary that all of thegrooves 306 be identical in size, shape, or orientation. - The
fluid layering device 300 also includes one or more exhaust vents. In the illustrative embodiment, thedevice 300 includes two such vents. Eachexhaust vent 307′, 307″ (generally 307). Eachvent 307 includes an elongatedexhaust vent lumen 314 defined by aproximal handle 310 and extending between an exhaustvent inlet port 308 and an exhaustvent exit port 316. Thehandle 310 extends proximally away from the proximal surface of thebarrier wall 302. Preferably, thehandle 310 extends axially for a length sufficiently longer than any intended insertion depth, such that at least a proximal portion of thehandle 310 extends beyond an open end of thecontainer 330. In some embodiments, the handle includes an open area to facilitate removal of thefluid layering device 300 from the container by providing a surface upon which a finger, fingers, or suitable instrument may apply an axial removing force to remove thedevice 300 from thecontainer 330. - Operation of an embodiment of the alternative embodiment of the fluid layering device illustrated in
FIG. 7A-7D and described above is illustrated in the series ofFIG. 8A-8C . Once again, a base material, such as Ficoll is inserted into an open end of theupright container 330. The base material pools along the conicalbottom end 331, such that asurface 333 of thebase material 319 is spaced apart from theopen end 332 by a distance h1. Referring toFIG. 8A , a distal portion of thefluid layering device 300 including thebarrier wall 302 is inserted into the open end of thecontainer 330. When fully inserted, a distal surface of thebarrier wall 302 resides at a height h2 above thesurface 333 of thebase material 319 as illustrated inFIG. 8B . Areservoir 332 extends proximally from a proximal surface of thebarrier wall 332 to theopen end 332 to a height h3. A proximal portion of the handle remains exposed, extending to a height h5 measured from theopen end 332. Preferably, a sufficient portion of theopen area 312 remains exposed to allow for insertion of a finger or suitable instrument during removal process. - A first fluid, in this instance a cellular suspension blood, is poured into the
open end 332 of thereservoir 30. Once again, the container-fluid layering device arrangement is preferably positioned upright or vertically, such that gravity will drive the flow ofblood 350 into thecontainer 330. In some embodiments, the arrangement can be positioned at an angle, but preferably not much more than about 30 degrees measured from vertical. - As shown in
FIG. 8C , gravity induces a downward flow, forcingblood 350 from thereservoir 332 into the channels formed between grooves 306 (FIG. 7C andFIG. 7D ) and the inner surface of thecontainer 330. Theblood 350 exits thegrooves 306 along the interior walls of the substantiallyupright container 330. Gravity continues to driveblood flow 352 downward towards the exposedsurface 333 of the base material 319 (e.g., Ficoll). Surface tension keeps theblood flow 352 substantially directed along the inner side walls. As the volume of blood increases in a layer above the base material, air pressure within the chamber increases. The exhaust vent allows exhaustedair 354 to bleed off from the chamber, thereby reducing the pressure and allowing for continued blood flow. - As before, careful selection of the dimensions of the one or more exhaust vents can be used to control flow of
blood 352 into thecontainer 330. For example, a substantially narrow exhaust vent restricts the flow of gas similarly restricting flow of blood into the chamber. When the reservoir contents have been substantially transferred, thefluid layering device 300 can be removed carefully by puling the exposed portion of thehandle 310. Care must be exercised when separating the device from the container so as not to disturb the layered arrangement ofblood 352 andFicoll 319. -
FIG. 9 illustrates yet another embodiment of afluid layering device 380. Thedevice 380 includes aproximal reservoir 382 and aninsertable base portion 394. Thereservoir 382 includes an open endedfluid chamber 384 and anelongated side wall 385 extending along a cord dissecting a cylindrical wall of thereservoir 382. Fluid poured into theopen end portion 384 flows toward abarrier wall 392 forming a base portion of thereservoir 382. One ormore drain apertures 390 are disposed along an outer periphery of thebarrier wall 392, allowing fluid stored within the container to enter one or more longitudinally extendingchannels 398 defined along aperipheral sealing wall 396. Fluid flows along thelongitudinal channels 398 between thechannels 398 and aninterior wall 210 of the open-endedcontainer 200. Gas displaced by the inflowing fluid exits thecontainer 200 through anexhaust vent 388. The exhaust vent directs expelled gas (e.g., air) into avent portion 386 defined between an opposite side of the elongated separatingwall 385 and an opposing portion of the cylindrical side wall, such that exiting exhaust air is not interfered with by fluid stored within thereservoir 382. -
FIG. 10A illustrates an open endedcontainer 400 comprising a layered arrangement ofblood 402 over Ficoll-paque PLUS 404. Thelayered arrangement layered arrangement FIG. 10B illustrates contents of the container illustrated inFIG. 10A after applied to a centrifuge process. Abottom layer 406 of granulocytes and erythrocytes is formed along the very bottom of thecontainer 400. Disposed above thislayer 406, is a firstintermediate layer 408 of Ficoll-paque PLUS. Above the firstintermediate layer 408 is a secondintermediate layer 410 of lymphocytes, and above that is atop layer 412 of plasma and platelets. In some embodiments, thetop layer 412 can be removed, for example by simply pouring it out of the container as illustrated inFIG. 10C . -
FIG. 11 illustrates an embodiment of aprocess 500 for directing fluid flow into an open ended container. In an initialization step not shown, an open-ended container is partially filled with a base material. This is generally accomplished as part of protocols known to those skilled in the art, such as a protocol for PBMC isolation. The fluid layering device is then coupled to the container from its open end at 502. The coupled arrangement defines a fluid reservoir for temporarily storing a first fluid during the layering process. Also defined are one or more fluid channels providing fluid communication between the reservoir and an interior region of the container in a space or chamber formed between a barrier wall and a surface of the base material. - Next, a fluid is added to the reservoir at 504. The fluid flows under the influence of gravity through the one or more fluid channels into the chamber above the surface of the base material at 506. In particular, the fluid flow is directed along an inner surface of the container wall, avoiding any free-falling droplets that would otherwise disturb the surface tension of the base material. The side-wall flow continues under the influence of gravity until it reaches the surface of the base material. The fluid then begins to pool along the surface. As the volume of fluid increases in the chamber, pressure of any gas within the chamber, such as air is raised. Gas (air) is exhausted from the chamber at 508 by pressure induced by the fluid flow. Exhausted air reduces the pressure and allows for continued fluid flow. Ultimately an equilibrium can be reached between the inflow of fluid and outflow of exhaust gas. The rate of flow can be controlled by at least one of the dimension of the fluid channels (e.g., length, shape, direction, diameter) and dimensions of one or more exhaust vents.
- The fluid layering device is removed from the chamber at 510. Generally, the device is removed after all of the fluid has flowed from the reservoir into the chamber. Removal of the device allows for further processing, such as centrifuging at 510 (optional).
- Other embodiments will be evident to those of skill in the art. It should be understood that the foregoing detailed description is provided for clarity only and is merely exemplary. The spirit and scope of the present invention are not limited to the above examples, but are encompassed by the following claims.
Claims (25)
1. A fluid layering device configured to control a flow of fluid into an open-ended container, comprising:
a fluid barrier configured to prevent passage of fluid from a proximal reservoir toward a distal end of the open-ended container;
a peripheral seal extending along an outer perimeter of the fluid barrier, the peripheral seal configured for sealing engagement along an interior surface of the open-ended container;
at least one groove formed in the peripheral seal, configured to provide a controlled flow of fluid across the fluid barrier and along the interior surface of the open-ended container; and
an exhaust vent configured to vent from the open-ended container gas displaced by the controlled flow of fluid.
2. The fluid layering device of claim 1 , further comprising a proximal reservoir defined at least partially by a proximal surface of the fluid barrier.
3. The fluid layering device of claim 2 , wherein the reservoir is open-ended, defined by an elongated cylindrical wall extending proximally from the fluid barrier.
4. The fluid layering device of claim 3 , further comprising a coupling arrangement adjacent to the open end of the reservoir.
5. The fluid layering device of claim 4 , wherein the coupling arrangement comprises a thread.
6. The fluid layering device of claim 1 , wherein the fluid barrier comprises at least one drain port in fluid communication with a proximal end of each of the at least one grooves.
7. The fluid layering device of claim 1 , further comprising a proximal handle allowing for insertion and removal of the fluid layering device with respect to the open-ended container.
8. The fluid layering device of claim 1 , wherein the exhaust vent terminates in an exhaust port disposed along an outer surface of the fluid layering device.
9. The fluid layering device of claim 1 , wherein each of the at least one grooves extends helically along a cylindrical surface defining the peripheral seal.
10. The fluid layering device of claim 1 , further comprising a shoulder positioned to abut at least a portion of a rim of the open-ended container when the fluid layering device is inserted therein, the shoulder position with respect to a proximal end of the fluid layering device to control height of the fluid barrier along a longitudinal axis of the open-ended container.
11. The fluid layering device of any of the above claims, wherein the fluid layering device is sterilized.
12. The fluid layering device of any of the above claims, wherein the fluid layering device is formed from material selected from the group consisting of: plastics; polymers; resins; glass; ceramics; metals; and combinations thereof.
13. The fluid layering device of any of the above claims, wherein fluid stored within the fluid layering device is observable through a sidewall of the device.
14. The fluid layering device of claim 13 , wherein at least a portion of the fluid layering device is translucent or transparent.
15. A method for controlling a flow of fluid into an open-ended container, comprising:
positioning a fluid barrier above a surface of a base material disposed in a distal end of the open-ended container;
adding a first fluid into a reservoir positioned proximal to the fluid barrier;
directing a flow of fluid from the reservoir across the fluid barrier and into the distal end of the open-ended container, fluid passing the fluid barrier flowing toward the base material along an interior surface of the open-ended container; and
exhausting from the distal end of the open-ended container gas displaced by the controlled flow of fluid,
wherein a layer of the first fluid is deposited over the base material without substantially disturbing a surface of the base material.
16. The method of claim 15 , wherein positioning the fluid barrier comprises inserting at least a proximal portion of a fluid layering device into an open end of the open-ended container.
17. The method of any of claims 15 -16, further comprising removing the fluid barrier from the open-ended container.
18. The method of claim 17 , further comprising centrifuging the layered material.
19. The method of any of claims 15 -17, wherein the base material is Ficoll.
20. The method of any of claims 15 -18, wherein the fluid is a cellular suspension.
21. The method of claim 20 , wherein the cellular suspension is blood.
22. A fluid layering device configured to control a flow of fluid into an open-ended container, comprising:
means for positioning a fluid barrier above a surface of a base material disposed in a distal end of the open-ended container;
means for adding a first fluid into a reservoir positioned proximal to the fluid barrier;
means for directing a flow of fluid from the reservoir across the fluid barrier and into the distal end of the open-ended container, fluid passing the fluid barrier flowing toward the base material along an interior surface of the open-ended container; and
means for exhausting from the distal end of the open-ended container gas displaced by the controlled flow of fluid.
23. A fluid layering device configured to control a flow of fluid into an open-ended container containing a base material, comprising:
a longitudinally extending cylindrical side wall open at its proximal end;
a fluid barrier disposed across a distal end of the cylindrical side wall, a proximal surface of the fluid barrier and an interior surface of the cylindrical side wall forming an open-ended reservoir;
an insertable portion extending distally from the fluid barrier; the insertable portion comprising:
a longitudinally extending sealing wall positioned to form a fluid-tight seal along a peripheral interior surface of a proximal portion of the open-ended container;
at least one groove extending along the sealing wall and terminating in a peripheral fluid port configured to ensure fluid flowing into the open-ended container flows along an interior surface of the open-ended container;
at least one drain in fluid communication between the reservoir and a proximal end of each of the at least one grooves; and
an exhaust vent in fluid communication with the open-ended container, wherein the vent regulates the flow of gas displaced by the controlled flow of fluid, thereby contributing to a rate of fluid flow.
24. The fluid layering device of claim 23 , wherein at least one groove extends helically along the sealing wall.
25. The fluid layering device of claim 23 , further comprising a threaded coupling engagement along a distal portion of the cylindrical wall.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13/207,323 US20120199241A1 (en) | 2009-02-11 | 2011-08-10 | Devices and methods for overlaying blood or cellular suspensions |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US15178709P | 2009-02-11 | 2009-02-11 | |
PCT/US2010/023971 WO2010093840A1 (en) | 2009-02-11 | 2010-02-11 | Devices and methods for overlaying blood or cellular suspensions |
US13/207,323 US20120199241A1 (en) | 2009-02-11 | 2011-08-10 | Devices and methods for overlaying blood or cellular suspensions |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2010/023971 Continuation WO2010093840A1 (en) | 2009-02-11 | 2010-02-11 | Devices and methods for overlaying blood or cellular suspensions |
Publications (1)
Publication Number | Publication Date |
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US20120199241A1 true US20120199241A1 (en) | 2012-08-09 |
Family
ID=42562066
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/207,323 Abandoned US20120199241A1 (en) | 2009-02-11 | 2011-08-10 | Devices and methods for overlaying blood or cellular suspensions |
Country Status (2)
Country | Link |
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US (1) | US20120199241A1 (en) |
WO (1) | WO2010093840A1 (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10220384B2 (en) * | 2011-08-09 | 2019-03-05 | Jae Go Kwon | Devices and methods for overlaying blood or cellular suspensions |
US10371609B2 (en) * | 2011-08-09 | 2019-08-06 | Jae Go Kwon | Devices and methods for overlaying blood or cellular suspensions |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4469151A (en) * | 1982-06-07 | 1984-09-04 | Wilson Billie J | Fluid layering device |
US7176034B2 (en) * | 2002-07-03 | 2007-02-13 | St. Joseph's Healthcare | Apparatus and method for filtering biological samples |
-
2010
- 2010-02-11 WO PCT/US2010/023971 patent/WO2010093840A1/en active Application Filing
-
2011
- 2011-08-10 US US13/207,323 patent/US20120199241A1/en not_active Abandoned
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Publication number | Publication date |
---|---|
WO2010093840A1 (en) | 2010-08-19 |
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STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- INCOMPLETE APPLICATION (PRE-EXAMINATION) |