Cell concentration devices and methods that include an insert defining a lumen and a cannula assembly

A system and associated method for concentrating and separating components of different densities from fluid containing cells using a centrifuge includes a container defining a cavity for receiving the fluid. The container has a top, a sidewall extending from the top, and a bottom disposed opposite the top and in sealing engagement with the sidewall. An insert is slidably disposed in the cavity of the container and defines a lumen through the insert. The lumen, which includes a hole and a funnel-shaped upper portion in fluid communication with the hole, forms an open fluid path between opposite ends of the insert. The insert has a density such that upon centrifugation a selected component of the fluid resides within the lumen. A container port is disposed in the top of the container to transfer the fluid into the container and to withdraw a fluid component other than the selected component from the container. The system includes a manifold that includes a manifold port, a vent to vent the container, and a connector to couple to the container port. A cannula is receivable in the manifold port and extendable through the container port into the container and into the lumen of the insert to withdraw the selected component from the lumen.

BACKGROUND OF THE INVENTION

In the field of regenerative medicine, access to a broad cross section of sub-dermal tissue is typically required to not only source cells but to also deliver therapy. Fluid tissue that is aspirated or otherwise sourced is often separated into one or more components that are present in the fluid tissue, e.g., plasma, red blood cells, fat cells, stem cells or other nucleated cells. Typically, one or more selected components of the fluid tissue are concentrated into a small volume so that the selected components can be used clinically. For example, there are several commercial devices to separate and concentrate nucleated cells from aspirated bone marrow, fat, or cord blood. Some of these systems employ a floating insert or buoy that is meant to create an interface between the separated fluid components or fractions of interest. The challenge for any apparatus designed to accomplish such a task is the ability to volume reduce the fluid in which the nucleated cells are suspended while recovering as many cells as possible. For example, in marrow aspirate, approximately 1 to 2 percent of the cells suspended in the fluid are the target nucleated cells. Many commercial devices are not able to consistently capture high percentages of nucleated cells while at the same time efficiently volume reduce (i.e., concentrate) the beginning fluid. In other words, many devices are not able to simultaneously obtain a high yield and a high final concentration.

Apparatus and methods for separating components of different densities from a physiological fluid containing cells are described in a previously filed application, International Application No. PCT/US2010/036696, filed on May 28, 2010, published on Dec. 2, 2010 as WO 2010/138895 A2, and incorporated herein by reference in its entirety.

FIG. 1is a diagram illustrating a separation system1700showing different components of a fluid inside the container1702after centrifugation. After centrifugation, the least dense fluid2000will be above the insert1300. The insert can be made of a material of a certain density such that after centrifugation of blood, including blood from marrow, the insert spans the space between the least dense plasma2000and the dense red cells2004, with the intermediate dense material2002, e.g., nucleated cells, residing in the upper funnel-shaped portion1304of the insert. The separation system1700can include a vent1716disposed in the top1706of the container1702and a fluid port1718disposed in or adjacent the top1706. The air vent1716can prevent a vacuum from being created when fluid is withdrawn from the container1702. A cannula assembly1500with a closed end1502is inserted through the injection port1714. Before insertion of the cannula assembly, a clamp1800can be applied to sidewall of container1702to hold the insert1300in place during subsequent fluid extraction. The closed end1502of the cannula assembly butts against the insert and closes the through hole1308of the insert. The closed end of the cannula assembly1500and the insert can form a seal, thus isolating denser fluid component or components beneath the seal from fluid components above the seal. The cannula assembly includes two cannulae, an inner cannula1508and an outer cannula1510, that fit coaxially into each other.

As shown inFIG. 1, the cannula assembly includes a series of two parallel side holes or ports in the two cannulae to line up at different predetermined heights above the closed distal end1502. A first set of side ports1506can be located near the closed distal end1502. A second set of side ports1504can be located above the upper funnel-shaped portion1304of insert1300. Fluid above the distal end1502of the cannula assembly can be removed in at least two fractions or components based on these two different predetermined heights. Fluid can be removed through the cannula assembly1500into connected syringes1802,1804using valve1806. For example, when the top side ports1504are aligned and opened, fluid above the top side ports can be extracted into a first syringe1802through inner cannula1508. By rotating the two cannulae with respect to each other, the top side ports in the cannula assembly1500are misaligned and sealed off, while the bottom side ports are aligned and opened. As shown inFIG. 1, the side ports may be radially offset by 90 degrees, requiring a relative rotation of 90 degrees to change which ports are aligned. When the bottom side ports1506are located just above the seal created by the closed end1502of the cannula assembly, substantially all fluid above the seal, but below the top side ports, can be extracted through inner cannula1508into a second syringe1804.

FIGS. 2A-2Care a series of sequential diagrams illustrating the extraction of fluid components using a separation system2100, the system including a container2102having a movable bottom or plunger2104. Centrifugation separates the fluid in the container by density into separate components or fractions.FIG. 2Aillustrates the position of an insert, such as insert2600, in relation to three components of a fluid in the separation system2100after centrifugation. The components are a low density fraction2000, such as plasma, a medium density fraction2002, such as buffy coat or nucleated cells, and a high density fraction2004, such as red blood cells.

To retrieve the separated layers or fluid components, the user takes the syringe or container2102out of the centrifuge. As shown inFIGS. 2A-2B, the user then uncaps the luer connector of port2118, attaches a plasma extraction syringe2300, and pulls back on the plunger. The calculated combination of 1) the fluid flow of plasma as it is being evacuated from the collection syringe or container2102, which can be lateral to the center injection port, 2) the size of the center hole2608or holes2610in the insert2600, 3) the relative density of the different fluids inside the container2102, and 4) the forces required to extract fluid of different densities under these known parameters, results in substantially only plasma moving into the plasma syringe2300. Because the air vent2116is capped, the collection syringe or container2102is a vacuum. Thus the movable bottom or plunger2104and the insert2600rise in the collection syringe or container2102as the plasma is extracted. The target cells, such as buffy coat, stay in the through hole or holes2610of the insert2600.

As shown inFIGS. 2B-2C, after removal of the plasma2000, the insert2600has risen to the top of the syringe or container2102and effectively seals off port2118connected to the plasma extraction syringe. At this point the user uncaps the air vent2116making the collection syringe or container2102no longer under vacuum pressure. A second target cell extraction syringe2302with a cannula2400attached is then inserted through the center injection port2114. Since 1) the insert2600always ends up at the top of the collection syringe or container2102after removal of the plasma and 2) the height of the insert2600is known, then the distance between the top of the injection port2114and bottom of the through holes2610,2608of the insert2600is always the same after removal of the plasma. The length of the cannula2400is such that it reaches just to the bottom of the center through hole2608in the insert2600after removal of the plasma. Thus, when the user pulls back on the plunger of the target cell extraction syringe2302, after the air vent2116has been uncapped, the target cells residing in the through hole or holes2610,2608are removed.

SUMMARY OF THE INVENTION

A system for separating components of different densities from a physiological fluid containing cells using a centrifuge includes a container having a top, a sidewall extending from the top, and a bottom disposed opposite the top and in sealing engagement with the sidewall. The container defines a cavity for receiving the fluid. The system includes an insert slidably disposed in the cavity of the container. The insert defines a lumen through the insert, the lumen including a hole and a funnel-shaped upper portion in fluid communication with the hole. The lumen forms an open fluid path between opposite ends of the insert. The insert has a density such that upon centrifugation a selected component of the fluid resides within the lumen. A container port is disposed in the top of the container to transfer the fluid into the container and to withdraw a fluid component other than the selected component from the container. The system further includes a manifold that includes a manifold port, a vent to vent the container, and a connector to couple to the container port. A cannula is receivable in the manifold port and extendable through the container port into the container and into the lumen of the insert to withdraw the selected component from the lumen.

The cannula can include a closed end to close the hole in the insert and a side port to withdraw the selected component. The cannula and the insert may form a seal when the closed end of the cannula closes off the hole in the insert. In an embodiment, the cannula a first cannula, and the system further includes a second cannula extendable through the container port to withdraw the component other than the selected component. The second cannula may be receivable in the manifold port. The system may include two manifolds, each including a manifold port, a vent to vent the container, and a connector to couple to the container port, and the first cannula can be receivable in the manifold port of one manifold while the second cannula can be receivable in the manifold port of the other manifold.

The container can be a syringe and the bottom can be movable, i.e., a plunger, which can have a removable handle. In an embodiment, the plunger is a first plunger and the system further includes a second plunger disposed in the syringe below the first plunger to move the first plunger, for example, to transfer the fluid into the container. The system may further include a clamping mechanism to hold the insert in place after centrifugation, the clamping mechanism being configured to press the sidewall of the container inward against the insert.

A method of separating components of different densities from a fluid containing cells using a centrifuge includes receiving the fluid in a separation system such as the separation system described above, and applying centrifugal force to the separation system. The method further includes, after centrifugation, withdrawing a fluid component other than the selected component through the container port; coupling a manifold to the container port, the manifold including a manifold port and a vent to vent the container; extending a cannula through the container port into the container and into the lumen of the insert, the cannula receivable in the manifold port; and withdrawing the selected component with the cannula from the lumen of the insert.

Withdrawing the selected component may include withdrawing the selected component through a side port in the cannula. In an embodiment, the cannula is a first cannula and withdrawing the component other than the selected component includes extending a second cannula through the container port, the second cannula receivable in the manifold port, and withdrawing the component other than the selected component with the second cannula. The manifold can be coupled to the container before the withdrawing of the component other than selected component. The method may further include with a clamping mechanism, holding the insert in place after centrifugation.

A system for separating components of different densities from a physiological fluid containing cells using a centrifuge includes a container, having a bottom, a top disposed opposite the bottom, and a sidewall extending from the top, the container defining a cavity for receiving the fluid. An insert is slidably disposed in the cavity and defines a lumen through the insert, the lumen including a hole and a funnel-shaped upper portion in fluid communication with the hole. The insert has a density such that upon centrifugation a selected component of the fluid resides within the lumen. The lumen forms an open fluid path between opposite ends of the insert. A container port is disposed in the top of the container. An extraction cap is provided to couple to the top of the container, the extraction cap including a cannula assembly receivable in the container port. The cannula assembly is extendable into the cavity of the container to butt against the insert and to withdraw the selected component from the lumen of the insert.

The cannula assembly can include an inner cannula coaxially disposed within an outer cannula. The inner cannula may include a closed end to close the hole in the insert and a side port to withdraw the selected component, the inner cannula and the insert forming a seal when the closed end of the inner cannula closes off the hole in the insert. The outer cannula may include an open end displaced from the distal end of the cannula assembly to withdraw fluid at a predetermined height above the distal end of the cannula assembly.

In an embodiment, the extraction cap includes a first port in fluid communication with the inner cannula and a second port in fluid communication with the outer cannula. The system may further include a first syringe to couple to the first port and a second syringe to couple to the second port. The cap may include an assembly tab adjacent the first and second ports, the assembly tab extending from the cap to prevent the second syringe from coupling to the first port. The system may further include a lock-out element on the second syringe. For example, the lock-out element includes a tab that locks a plunger of the first syringe until second syringe is removed from the cap. In an embodiment, the extraction cap includes an outer part and an inner part, the inner part carrying the needle assembly and being movable relative to the outer part. The cap may include a locking screw coupled to the outer part and positioned at an angle relative to inner part to push the inner part toward the container with rotation of the locking screw.

A method of separating components of different densities from a fluid containing cells using a centrifuge includes receiving the fluid in a separation system, the system including a container having a bottom, a top disposed opposite the bottom, and a sidewall extending from the top, the container defining a cavity for receiving the fluid. A container port is disposed in the top of the container. An insert is slidably disposed in the cavity of the container, the insert including a funnel-shaped upper portion and a hole therethrough, the insert having a density such that upon centrifugation a selected component of the fluid resides within the upper portion of the insert. The method includes applying centrifugal force to the system and inserting a cannula into the container through the container port to butt against the insert, the cannula having one or more side ports displaced from a distal end of the cannula. The method further includes withdrawing the selected component through the side ports in the cannula; ejecting at least a portion of the withdrawn component through the side ports causing one or more fluid jets in the funnel-shape upper portion of the insert to release cells that adhere to the insert; and withdrawing the ejected portion and cells released by the fluid jets through the side ports.

In any of the systems or methods described herein the insert can be rigid. The volume contained in the lumen upper portion of the insert can be between 5% and 20% of the volume of the container cavity. The selected component (also referred to herein as a target fraction) can be buffy coat and the component other than the selected component can be blood plasma.

Embodiments of the current invention overcome the limitations of known devices for concentration of cells sourced from marrow or other tissue. For example, the insert of the separation device does not form a closed recess or a depression or indent to capture cells, but rather allows for the natural sedimentation of the fluid within the container and through the insert. The insert defines a lumen that has at least one relatively large through hole or channel, including a funnel-shaped upper portion, that allows for the free flow of fluid within the container and through the insert and does not interfere with the natural layering of different density components of the fluid. In addition, the insert identifies the location of a layer of interest, including the target cells. The funnel-shaped upper portion and the through hole reduce the cross-sectional area and increase the thickness of the layer of interest. This facilitates extraction of the target cells and contributes to a high yield and high concentration of the target cells.

Embodiments of the current invention overcome limitations of other systems that use inserts or buoys without a through hole and where the fluid path under centrifugation is confined to the distance between the inner wall of a container or tube and the outer walls of the inserts or buoys. In those systems, minor clots, particles, or other inconsistencies in the fluid can lodge between the walls of the tube and the buoys interfering with the natural layering of the different density components of the fluid. The insert(s) described includes a density selected such that after centrifugation the target cells reside within the hole, the funnel-shape upper portion, or lumen defined by the insert. Under gravitational force the insert floats freely within the container with substantially all of the fluid flowing through the hole or lumen of the insert, but not between the outer wall of the insert and the inner wall of the container. The distance between the inner wall of the container and the outer wall of the insert creates enough space to allow the insert to move freely within the container.

Embodiments of apparatus and methods for separating components of a fluid can be combined with devices and methods to access and source, e.g., aspirate, tissue, such as the aspiration needle assemblies described in International Application No. PCT/US2010/036696. Once tissue is sourced, e.g., loaded into a separation system, the system can be centrifuged. Upon centrifugation, the target cells naturally sediment into the through hole or lumen of the floating insert. These cells are then isolated by means of a cannula. The closed end of the cannula can close the hole in the insert. The target cells residing in the hole of lumen of the floating insert may be sealed from fluid below while fluid above the insert is removed through a cannula. The combination of the cell concentration and separation apparatus described herein with an aspiration apparatus allows a clinician the ability to access subcutaneous tissue in a less traumatic manner and then concentrate nucleated cells from that tissue aspirate. The apparatus can be combined, e.g., coupled or connected, by means of tubing and fluid ports, including luer connections, to create a total solution from aspiration to concentration.

DETAILED DESCRIPTION OF THE INVENTION

A prior version of a system for concentrating and separating cells (also referred to herein as a cell concentrator) has been described in International Application No. PCT/US2010/036696, published on Dec. 2, 2010 as WO2010/138895, incorporated herein by reference in its entirety. Here, an improved system is described that has been fabricated and tested. Some key features of the improved system include:

b) a double needle extraction system with jet flushing;

c) an extraction needle lock that forces needle to seat in float; and

d) a syringe lock-out tab to ensure proper order of extraction.

FIG. 3is an exploded view of an improved system10for separating components of difference densities from a physiological fluid according to an example embodiment of the invention. System10includes a base12, a separation vial (container)14, a float (insert)16disposed in the vial14, and an extraction cap18with syringes20,22and a cannula assembly including extraction needles (cannulae)24,25.

To separate buffy coat from red blood cells and plasma, the float16should have a density that matches that of the buffy coat, whose density is roughly 1.06 g/cc. A polystyrene material was selected to manufacture the insert because the material's density is close to that of buffy coat. However, testing indicated that a polystyrene float was lighter than desired. Small slugs56(FIG. 9), e.g., pieces of stainless steel, can be added into the float to adjust its density. The process of testing a device in blood to ensure that the density matches the desired density can be repeated for new batches of plastic, e.g., during manufacturing, or for testing different materials.

The materials of the system are polycarbonate for most of the parts with the vial being PET (Polyethylene teraphthalate plastic) and the float polystyrene, as described above. Other suitable materials for the float may be polyethylene or polypropylene materials which tend to be less sticky to cells than polystyrenes. However, the densities of these materials are lower than that of buffy coat, so a larger metal material may need to be incorporated into the float to achieve the desired density. Another option is to coat the surface of the float and the inside of the vial with a substance that prevents cells form sticking, for example, a coating from the company Hydromer.

FIG. 4is a perspective view of container14including an insert16according to an example embodiment of the invention. Container14is a vial that has a bottom13, a top17disposed opposite the bottom, and a sidewall extending from the top, the top including a lid19to close the container. The vial defines a cavity for receiving fluid. A physiological fluid containing cells, such as blood or marrow, is introduced into the separation vial14via a port28prior to centrifugation. After the fluid is centrifuged, the separation vial14is placed into the base12. As shown inFIG. 5, the base12has a lever26with a cam27that locks the vial to the base by deforming the vial wall15. This deformation locks the float16inside the vial14in place so that it cannot move. In other words, the cam and lever of base12operate as a clamping mechanism that, when engaged, prevents movement of the float. Movement of the float, such as during the extraction procedures described below, can disrupt the target cell layer, e.g., the buffy coat.

Post centrifugation, the serum (plasma) is above the float16, the red blood cells are below the float and the target cells (e.g., buffy coat) are inside the float funnel. This separation of fluid components is analogous to what is shown inFIG. 1. The cap or lid19of the vial14includes a silicone septum30and air vents32. As shown inFIGS. 4 and 5, four air vents32are arranged around the septum30and four protrusions60extend from the lid19. The extraction needles (cannulae)24,25are inserted through the septum30, which functions as a port into the container. The air vents are included so that one does not pull a vacuum when extracting the target cells or the plasma serum.

FIG. 6is a side view of an extraction cap18according to an example embodiment of the invention. Extraction cap18is distinct from the lid19and configured to couple to the top of the vial (container)14. The extraction cap includes the cannula assembly, including needles (cannulae)24and25, which are receivable in container port30.

As illustrated inFIGS. 7 and 8, the extraction cap18includes an outer part31and an inner part33movable with respect to the outer part. The outer part31, which includes apertures62to engage the protrusions60of the lid, snaps onto the top17of the vial14. The inner part33includes two ports36,38for connecting the two syringes20,22to the inner and outer needles (cannulas) of the extraction cannula assembly. An assembly tab42on the inner part33ensures that the larger syringe20can only be connected to the port that is in fluid communication with the outer extraction needle25.

As shown inFIG. 7, a lock-out tab44is mounted on the large syringe20that forces the user to activate the large syringe first. The lock-out tab44blocks the plunger of syringe22. Once syringe20is filled with serum extracted from vial14, syringe20, including lock-out tab44, is removed and the small syringe22may then be activated. This ensures the proper order is followed when extracting fluid components from vial14.

To remove the target cells, the extraction cap18is placed over the vial14, as illustrated inFIGS. 7 and 8. The extraction needles24,25are advanced through the silicone septum (container port)30and into the vial14. The extraction cap18snaps onto the lid of the vial. Once in place, the syringe/needle assembly is pushed down by the operator so that the distal end of the needle assembly bottoms out into the funnel in the float16(FIG. 9). The locking screw34is then tightened. This screw is at an angle. As the screw is tightened, it slightly pushes the needle assembly down to ensure that it fully engages the bottom of the float. In the embodiment shown inFIGS. 7 and 8, the locking screw34is positioned at a small angle within the horizontal plane. In the embodiment ofFIG. 3, the locking screw34is angled out of the horizontal plane to perform the same function.

As shown inFIGS. 8 and 9, the extraction cannula assembly includes an inner needle (inner cannula)24and an outer needle (outer cannula)25. The open distal end46of outer needle25is positioned at a set distance from the distal end48of the inner needle24such that the outer needle sits at or near the top of the float16. The outer needle is used to extract the serum (plasma).FIG. 9illustrates the needle assembly positioned in the float (insert)16. As shown, the inner needle24has a distal taper and multiple holes (ports)40, e.g.,3side holes, at or near its distal end48. The inner needle24sits in the bottom of the float and plugs hole52in the float. The extraction process starts with the outer needle25, which is in fluid communication with large syringe20. The serum is removed until bubbles are seen in the syringe20. Then, the selected component (e.g., stem cells, buffy coat) is removed using the small syringe22and the inner needle24, the selected component being withdrawn through holes40of inner cannula24. As cells of the selected component adhere to the inner wall54of the float (insert)16, the cell solution that has been removed, or portion thereof, is pushed back, e.g., ejected, into the float through the holes40causing one or more jets of fluid in the funnel-shaped portion of the float. This jet flushing releases the cells from the float wall54. The cells are then sucked up during a second withdrawal into the syringe22.

Embodiments shown inFIGS. 3-9and described herein have many advantages. One advantage of the double cannula extraction needle is that the operator only has to push the cannula assembly through the silicone septum of the separation vial once, which reduces the risk of mixing the separated fluid components in the vial. Other systems require needles or cannulae to be inserted serially, which carries a higher risk of infection and of moving the target cells to be extracted. Another advantage is the extraction needle lock, which includes a locking screw that, when tightened, drives the cannula assembly down into the float to ensure that the extraction needle (inner cannula) fully engages the bottom of the float. The locking screw of the needle lock may be positioned at an angle with respect to the extraction needle, such that rotation of the screw drives the extraction needle down. The screw may be at an angle out of the horizontal plane (FIG. 3), or at an angle within the horizontal plane, as illustrated inFIGS. 6-8. Furthermore, the lock-out tab on the larger syringe prevents an operator from using the small syringe first, thereby ensuring proper order of withdrawal of fluid components.

A system for separating components of different densities from a fluid containing cells and for concentrating cells according to another example embodiment of the invention is described below and illustrated inFIGS. 10-17. A system for concentrating bone marrow or blood is described, although the principles of the invention can be applied to other fluids, including other physiological fluids. The system uses a container having a movable bottom, e.g., a syringe with at least one plunger, for both the collection and centrifugation of the fluid specimen.

As illustrated inFIGS. 10A and 10B, a system100for separating components of different densities from a fluid containing cells using a centrifuge includes a collection syringe (container)114having a top117, a sidewall115extending from the top, and a bottom113, e.g., a plunger, disposed opposite the top and in sealing engagement with the sidewall. The container defines a cavity for receiving the fluid. An insert116is slidably disposed in the cavity of the container114. Similar to insert16described above in reference toFIG. 9, insert116defines a lumen through the insert, the lumen including a hole52and a funnel-shaped upper portion50in fluid communication with the hole. The lumen forms an open fluid path between opposite ends of the insert. The insert has a density such that upon centrifugation a selected component of the fluid resides within the lumen. A container port130, e.g., a luer connector, is disposed in the top of the container114to transfer the fluid into the container and to withdraw a fluid component other than the selected component from the container. As illustrated inFIG. 10A, the system further includes at least one manifold160that includes a manifold port162, a vent164to vent the container, and a connector166to couple to the container port130. A cannula170,172(FIG. 14A) is receivable in the manifold port162and extendable through the container port130into the container114and into the lumen of the insert116to withdraw the selected component from the lumen or a component other than the selected component.

As illustrated inFIG. 10A, the system can include a clamping mechanism112that can also double as a syringe holder. The container of the system can be a syringe that includes a plunger having a removable handle (not shown) or a syringe114with two plungers as shown, plunger123having a handle and plunger113being without a handle. A 30 ml plasma extraction syringe120is connected to a cannula170that fits through upper injection port162, manifold160and lower luer connection166. A 5 ml concentrate extraction syringe122is connected to a cannula172that fits through upper injection port162, manifold160and lower luer connection166. The system can include a syringe holder174with washer on bottom and, optionally, O-ring176. For convenience of the user, the system can include two manifolds160, as illustrated inFIG. 10A. One manifold160receives cannula170; the other manifold receives cannula172. A standard 4-way manifold may be used for each manifold160. The standard manifold includes 3 fluid channels and a switch (e.g, valve)168. Using switch168, each of the fluid channels can be selective closed or all channels can be open. Here, all channels are open. Vent164Coupled to the manifold can be a vent that has a micron filter. This way, a sterile environment can be maintained while venting during the extraction of fluid. The injection port162and the connector166can be swabable luer ports, which can be swabbed or wiped, e.g., with alcohol, for good sterile procedure. The swabable luer ports typically include an elastomeric (e.g., rubber) membrane that includes a slit that is normally closed, but parts when a cannula or male luer connector is inserted. Such ports are beneficial when connections are to be air tight.

A step by step overview of the operation of the system will be described. The first step is to use the double plunger syringe114(alternatively, a syringe with a removable handle) to fill the syringe with the blood or marrow specimen to be concentrated.FIGS. 11A-11Cillustrate movement of the plungers of the double plunger syringe114, such as during filling of the syringe with physiological fluid. It should be noted that during filling, the syringe port130is coupled to another container containing the source tissue or to a tissue aspiration needle (not shown). Pulling back on the second plunger123via its handle forces the first plunger113under vacuum pressure to move back and the syringe is thus filled. The second plunger123can be completely removed from the barrel of syringe114leaving behind the first plunger113that is not connected to a handle. The shortened profile of the syringe with the second plunger123removed (or the handle removed, in case of a plunger having a removable handle) fits into commonly used centrifuges.

The second step is to connect to the syringe114containing the specimen (e.g., fluid tissue), after the second plunger has been removed, a small micron vented luer cap178and then place the syringe inside the syringe tube holder174. The holder174has a solid bottom and also has an O-ring176attached to the solid bottom (FIG. 10A). The O-ring lines up with the seal the plunger113makes with the outer wall115of the syringe barrel. Under high g-force, this O-ring serves to prevent any leaking from the syringe into the syringe tube holder174.

Inside of the syringe114is a funnel shaped insert116(also referred to herein as a funnel) with a hole in the center. The density of the funnel is such that after density separation, target cells from blood or marrow will reside inside of the funnel. Consequently, after density separation of blood or marrow, plasma will reside at the top of the syringe114nearest the Luer tip (container port)130, the target cells will reside inside of the funnel116, and red blood cells will reside beneath the funnel nearest the plunger113. Two example funnels116a,116bare shown inFIG. 12.

With respect to the materials used to make the funnel116and the shape of the funnel, it should be noted that various materials and shapes can work. When selecting a material, one consideration is whether the funnel116is to be molded or machined. InFIG. 12, the funnel116ato the left is made of REXOLITE. This material has a density of 1.05 and is easy to machine. Alternatively, a plastic material may be used, such as ABS by Dow Chemical (part #3105 FP EP) that has a density of 1.05 and is ideal for molding applications. A material that has a lower density than desired can have its density increased by adding screws or other material to the body of the funnel, as described above in reference to insert16ofFIG. 9. Thus, various materials and plastics can be combined to fine tune the density of the funnel. For example, the funnel may be made of two parts or two different plastics. With respect to the shape of the funnel, many different shaped funnels will work. For example, a deeper, taller funnel, such as funnel116bpictured to the right inFIG. 12, or a shallow, shorter funnel, such as the clear REXOLITE funnel116aillustrated to the left, can be used in embodiments of the invention. Additionally, a bowl-shaped funnel or a funnel that has an inflection point, such that the angle of the wall is steeper at the bottom compared to the top, are all possible funnel shaped inserts that can be used in embodiments of the invention. Regardless of the shape, the funnel116has an upper funnel shaped portion50and a through hole52at the bottom as illustrated with respect to insert16(FIG. 9).

The third step is to take the syringe114and tube holder174from the centrifuge, place the syringe inside the clamping mechanism112and engage the clamp as illustrated inFIG. 13. The pressure from the clamp will pinch the walls115of the syringe114so that the inner wall of the syringe barrel presses against the funnel116. This pressure will freeze (i.e., lock) the funnel in place. As shown, the clamping mechanism can include a lever26and cam27similar to the clamping mechanism described above in reference toFIGS. 5, 7 and 8.

The fourth step is to remove the vented cap178from the collection syringe114and connect to the collection syringe, via the upper luer connection130, the 30 ml plasma extraction syringe120connected to cannula170via manifold160, as shown inFIG. 14A. Cannula170fits through an upper injection port162connected to manifold160as shown inFIG. 14B. Also connected to manifold162are a side air vent164and a lower luer connection166. Thus, the collection syringe114is not vented during loading of the specimen but is vented, using manifold160, during extraction of the plasma and the target fraction inside the funnel.

After connection the plasma extraction syringe120and cannula170to the collection syringe114, the user pushes the extraction cannula170into the collection syringe. The cannula will advance until it hits the funnel116that has been frozen in place by the clamping mechanism112. As illustrated inFIG. 15, cannula170has a blunt closed end171and a hole180a certain distance above along its shaft. This hole is positioned to be high enough to extract only the plasma above but not the contents of the funnel116. Once the plasma is removed, the plasma extraction cannula170is removed from the collection syringe114and the plasma extraction syringe120is disconnected from the manifold160. Optionally, the syringe120and the manifold160are disconnected from the luer fitting130of the collection syringe and removed.

Once plasma is removed from container114, the same procedure as described above for syringe120and cannula170is repeated with the 5 ml concentrate extraction syringe122and cannula172.FIG. 16illustrates cannula172positioned against funnel (insert)116, the cannula including a side port182displaced from the distal end173of the cannula to withdraw fluid at a predetermined height above the distal end, e.g., within the funnel116, to withdraw substantially all of the target fraction. Extraction cannula172works the same way as the 30 ml extraction cannula except that hole (side port)182positioned near the cannula's blunt distal end173is close to the bottom of the funnel116so that the contents of the funnel are removed through hole182when vacuum pressure is applied via syringe122. Optionally, jet flushing, described above in reference to cannula24ofFIG. 9, may be employed by ejecting fluid through hole182into the funnel116to release cells that adhere to the funnel inner surface.

Thus, in the examples illustrated inFIGS. 15 and 16, each cannula170,172includes a closed end171,173to close the hole52in the insert116and at least one side port180,182to withdraw a component of the fluid, be it the selected component, e.g., buffy coat, or a component other than the selected component, e.g., plasma. Each cannula170,172and the insert116may form a seal when the closed end of the cannula closes off the hole in the insert.

Additional features of the above embodiment, as illustrated inFIGS. 10A-16, are as follows:a) The collection syringe (container)114is centrifuged ‘luer tip up’ and an O-ring or gasket is used to keep the syringe liquid-tight during centrifugation. If a syringe is centrifuged with the luer tip facing up, no cap or a vented cap for the luer tip should be used; otherwise the syringe distorts and leaks.b) The use of luer connectors, injection ports, vented caps and a manifold160as a means to both 1) vent the collection syringe (container) and 2) insert a cannula into the syringe through the luer tip to extract fluid.

The collection syringe114is vented and fluid removed by employing the following novel features (see, e.g.,FIGS. 14A-14B):a) The connection between the extraction cannulas120,122and the extraction syringes170,172is air tight.b) The seal around the upper injection port162and the cannula (170,172) that has pierced it is also air tight.c) The column of the manifold160is air tight with the exception of the air vent164at right angle to the syringes.d) The connection166to the collection syringe114is air tight.

The collection syringe114is vented during the retrieval of the one or more target fractions. A target fraction is removed via a cannula using the negative pressure of an extraction syringe. The air vent164that used to accomplish this is not part of either syringe but is connected to both syringes, e.g., via the manifold160. The luer tip130of the collection syringe114is used as the extraction port.

An alternative extraction process using a syringe PRP (Platelet Rich Plasma) system will be described. In the embodiment described above, e.g., in reference toFIG. 13, the third step in the separation procedure is to take the syringe114and tube holder174from the centrifuge and place it inside of the clamping mechanism112to engage the clamp and freeze the funnel116in place. In the alternative embodiment described below, the clamping mechanism is not used.

An alternative to removing the plasma with a cannula after clamping the funnel116in place is to attach a syringe120to the upper luer130of collection syringe114using a standard fem/fem luer connection and remove the plasma directly, without a cannula, by using the vacuum pressure of the two connected syringes. Since a standard luer connection is contemplated, the plasma can be removed under vacuum pressure by pulling back on the plunger of the plasma extraction syringe120. Thus, as the plasma is removed, the plunger113rises and the funnel116inside the barrel of the collection syringe114also rises. It is contemplated that the user removes the plasma until the funnel116reaches the top of the syringe114. After retrieving the plasma this way, the next step is to remove the plasma extraction syringe120from the collection syringe114and connect to the collection syringe, via the upper luer connection130, a vented 5 ml PRP extraction syringe, e.g., syringe172coupled to manifold160.

The target cells will not re-mix because the walls of the funnel116prevent fluid turbulence or interference from the inner wall of the syringe barrel. In addition, the center hole of the funnel116is sized such that at1G force, surface tension prevents fluid passage from below the funnel into the funnel. The PRP extraction syringe122is connected to a cannula172that fits through an upper injection port162, connected to a manifold116that has a side air vent164and a lower luer connection166. The cannula172can be shorter for this extraction process as the float116is at the top of the collection syringe114. Thus, the collection syringe114is not vented during loading of the specimen but is vented during extraction the target fraction inside the funnel.

After removal of the plasma and after the PRP extraction syringe122and cannula172are connected to the collection syringe114, the user pushes the extraction cannula172into the collection syringe. Since the insert116ends at the top of the syringe in this embodiment, the cannula172can be a set length, such that the cannula will advance the proper distance until it is approximately at the bottom of the funnel116. As described above in reference toFIG. 16, this cannula has a blunt closed end to butt against the funnel116, and a hole (e.g., a port) positioned a certain distance above the blunt end. Typically, the hole is positioned such that it ends up close to the bottom of the funnel when the blunt end of the cannula butts against the funnel. This ensures that the contents of the funnel116can be removed through the cannula172with the PRP extraction syringe122.

Returning toFIGS. 1 and 2, these figures illustrate embodiments described in previously filed application PCT/US2010/036696, which are useful to separate cells of a different fraction of a physiological fluid using centrifugation. The embodiments each include a collection tube or container that contains a funnel (e.g., an insert or float having a funnel-shaped portion) with a through hole in the funnel. The tube is designed to accept fluid, specifically blood or marrow. The funnel has a density such that after centrifugation, cells are captured inside the funnel. Two of the methods described for removing the cells contained inside the funnel after centrifugation are summarized below.

Method 1: The first method involves the following procedures:a) After centrifugation, pinch the funnel in place by applying a clamp on the outside of the tube (e.g., container having a fixed bottom). The pressure on the tube causes the tube to flex. This then causes the inside wall of the tube to pinch against the outside wall of the funnel.b) Once the funnel is secured in place, a double needle apparatus (inner cannula within outer cannula) is inserted through a center port. The blunt tip of the needle mates with the center hole of the funnel blocking off fluid below the funnel from fluid above the funnel.c) The upper access hole (side port) of the double needle extracts all fluid that resides above the top of the funneld) The lower access hole (side port) of the double needle extracts all fluid that resides inside the funnel.

Method 2: The second method involves the following procedure:a) The fluid is loaded into a syringe (e.g., a container having a movable bottom or plunger) containing the funnel; the syringe can have no plunger handle or can have a removable plunger handleb) The syringe has a center luer connection and a side luer connection, both of which are closed.c) In one example, the center luer connection on the underside, inside the barrel of the syringe, has connected to it a blunt needle with side portsd) After centrifugation, the target cells reside inside the funnele) Upper fluid (plasma) is removed via a plasma-syringe through the side luer connection. Because the system is a syringe, it is under vacuum pressure; consequently, as fluid is removed, the funnel and plunger move up.f) Once the majority of the fluid has been removed, the blunt end of the needle meets the center hole of the rising float (funnel) and effectively seals all fluid above the float from fluid below the float. The port retrieving the plasma also mates with the float simultaneously so that no further fluid can be removed from the side port once the blunt needle impales the rising float.g) The plasma syringe is removed and a vented cap added which now makes the system not under vacuum pressure.h) Another syringe is connected to the center port and the target cells are removed

Thus, of the two methods for removing the target cells from the funnel described above, the first involves freezing (e.g., clamping) the float in place and moving the needle assembly into the funnel to get the cells, while the second method involves using the vacuum pressure from the syringe to move the funnel up so that it impales itself into the fixed blunt needle assembly under the cap of the syringe. Please refer to WO 2010/138895 A2, e.g.,FIGS. 13-33and associated text, for a more complete description of the two methods described above.

Described below in reference toFIGS. 18 and 19is yet another apparatus and associated method for extracting cells from the collection funnel of a separation system. The apparatus and method include an upper and a lower funnel that are disposed inside the collection tube (container). As illustrated inFIG. 18, blood is loaded into a tube (container)214that defines a cavity that serves as the collection chamber for fluid. The tube214has an injection port228and an air vent229. Inside the collection tube214and in fluid communication with the injection port228is a flexible, optionally, clear tube205that is attached to a funnel210(referred to as the first or upper funnel) also disposed in the container214. The density of the upper funnel210is less than blood plasma. For example, the density of the upper funnel can be about 1.0.

After centrifugation, the bulk of the upper funnel210is floating above the plasma2000, as illustrated inFIG. 19. The bottom point of the upper funnel210has sunk slightly into the plasma or is in contact with the plasma. As shown, the funnel210is narrower than the diameter of the collection tube214. The funnel210has stabilization fins215at the top that extend from the funnel to approximately the sidewall of the tube214without touching the tube. These fins215are placed in such a manner that the upper funnel210remains oriented vertically with respect to the collection tube. The flexible tube205is attached at one end to the portion of the upper funnel where through hole220is located in the funnel. The other end of the tube205is connected to the port228in the top (e.g., cap) of the collection tube214. Tube205can be a small diameter clear tube that can flex and curl in on itself. Fluid is added to the collection tube214through the port228with a syringe. The fluid then travels through the length of flexible tube205and exits through hole220in the upper funnel210into the hollow chamber of collection tube214. Fluid is removed from the chamber through the same path.

Beneath the upper funnel210is a second funnel216that has a density of about 1.06, which is a higher density than the upper funnel and which allows the lower funnel216to float at the intermediate zone between red cells2004and plasma2000. After centrifugation, most of the platelets and white cells from blood or marrow reside within the lower funnel, as illustrated inFIG. 19. The lower funnel216has a through hole52, similar to other funnel shaped inserts described herein. Because the funnel216is open at the top and the bottom, it results in cleaner fluid flow and cleaner separation of components of the fluid, e.g., red cells, plasma, and target cells in the intermediate layer2002between red cells and plasma.

After centrifugation, the user can attach a syringe to the upper port228and begin to retrieve plasma first. The upper funnel210sinks as fluid in the chamber of tube214is removed but initially remains floating on the top of the fluid. After the desired amount of plasma has been removed, the user can switch syringes and begin to remove the remaining fluid above the lower funnel216and the contents of what is inside the lower funnel.

As additional fluid is removed, the upper funnel210continues to sink until it hits the lower funnel216. The angle of the upper funnel210is steeper than the angle of the lower funnel216so that the upper funnel fits into the lower funnel. The upper funnel210can have bottom stabilization fins225, so that the point of the upper funnel stops a certain distance below the bottom of the lower funnel216. This can also be accomplished by making the upper funnel210a certain height so that the upper stabilization fins215contact the upper surface of the lower funnel216. Additionally, a clamp212can be added after centrifugation that pinches the sidewall of the collection tube, such that the sidewall deforms and pinches against an outer surface of the lower funnel. In this way, the lower funnel216can be clamped in place and does not move as the upper funnel210mates with it during extraction of fluid. This allows the user to only withdraw the contents of the lower funnel216but not any fluid which is contained beneath the lower funnel.

As illustrated inFIG. 19, after centrifugation, red cells2004are below the lower funnel216, target cells of the density desired (e.g., buffy coat)2002are inside the lower funnel, plasma2000is above the target cells, and the upper funnel210is floating on top of the plasma. As described above, the user first removes plasma2000by attaching a syringe (not shown) to the center port228(FIG. 18) of tube214and pulling back on the plunger of the syringe. This causes fluid to flow through the hole (port)220at the bottom of the upper funnel210, through the flex tube205and into the syringe. The user can remove as much plasma as is desired. Once the desired amount of plasma is removed, the user can remove the syringe containing the plasma and attach a second syringe (not shown) and remove the remaining fluid that is contained above and inside the lower funnel216. The stabilization fins215,225and the relative height of the two funnels210,216can be adjusted such that the upper funnel210dead ends against the lower funnel216so that no fluid from beneath the lower funnel is removed during the extraction process.

While this invention has been particularly shown and described with references to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. For example, the double plunger collection syringe described herein may be used to separate fluid components other than those described herein and may be used in applications other than those described herein. Further, the example manifold disclosed herein, including the luer connection ports and micron vent, may be used to transfer fluids in a sterile manner in other applications, and may be used with syringes, cannulas, and containers other than those described herein. Further, inserts other than those illustrated and described herein may be used in combination with containers, cannulas and syringes to separate components of a fluid. For example, inserts need not have a density as described herein.