DEVICES AND METHODS FOR OVERLAYING BLOOD OR CELLULAR SUSPENSIONS
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.
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.
SUMMARYDescribed 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.
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
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 in
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.
Illustrated in
Preferring to
Illustrated in
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
As shown in
An alternative embodiment of a fluid layering device 300 is illustrated in
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. In the illustrative embodiment, 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. 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 the grooves 306 be identical in size, shape, or orientation.
The fluid layering device 300 also includes one or more exhaust vents. In the illustrative embodiment, 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. Preferably, 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. In some embodiments, 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.
Operation of an embodiment of the alternative embodiment of the fluid layering device illustrated in
A first fluid, in this instance a cellular suspension blood, is poured into the open end 332 of the reservoir 30. Once again, 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. 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
As before, careful selection of the dimensions of the one or more exhaust vents can be used to control flow of blood 352 into the container 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, 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.
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
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.
Type: Application
Filed: Aug 10, 2011
Publication Date: Aug 9, 2012
Inventor: Matthew Ralph Marple (Los Angeles, CA)
Application Number: 13/207,323
International Classification: B65B 1/04 (20060101);