U.S. patent application number 16/835053 was filed with the patent office on 2020-10-15 for cell concentration devices and methods that include an insert defining a lumen and a cannula assembly.
The applicant listed for this patent is EndoCellutions, Inc.. Invention is credited to Jeffrey R. Chabot, Neil F. Duffy, JR., Andy H. Levine, Andrew McGillicuddy, John Meade, Neil S. Tischler.
Application Number | 20200324285 16/835053 |
Document ID | / |
Family ID | 1000004925907 |
Filed Date | 2020-10-15 |
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United States Patent
Application |
20200324285 |
Kind Code |
A1 |
Levine; Andy H. ; et
al. |
October 15, 2020 |
CELL CONCENTRATION DEVICES AND METHODS THAT INCLUDE AN INSERT
DEFINING A LUMEN AND A CANNULA ASSEMBLY
Abstract
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.
Inventors: |
Levine; Andy H.; (Newton
Highlands, MA) ; Meade; John; (Mendon, MA) ;
Tischler; Neil S.; (Acton, MA) ; Chabot; Jeffrey
R.; (Medford, MA) ; McGillicuddy; Andrew;
(Hanover, MA) ; Duffy, JR.; Neil F.; (Brighton,
MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EndoCellutions, Inc. |
Marshfield |
MA |
US |
|
|
Family ID: |
1000004925907 |
Appl. No.: |
16/835053 |
Filed: |
March 30, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
14764115 |
Jul 28, 2015 |
10603665 |
|
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PCT/US2014/013636 |
Jan 29, 2014 |
|
|
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16835053 |
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61897587 |
Oct 30, 2013 |
|
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61757993 |
Jan 29, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 3/50215 20130101;
B01L 2200/026 20130101; A61M 1/029 20130101; B01L 2300/0858
20130101; B01L 3/0282 20130101; B01L 2200/0689 20130101; B01L
3/5082 20130101; B01D 21/262 20130101; B01L 2200/0684 20130101;
B01L 2400/0478 20130101; B01L 2400/0409 20130101; A61M 1/3693
20130101; B01L 2300/046 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; A61M 1/02 20060101 A61M001/02; A61M 1/36 20060101
A61M001/36; B01D 21/26 20060101 B01D021/26; B01L 3/02 20060101
B01L003/02 |
Claims
1. A system for separating components of different densities from a
fluid containing cells using a centrifuge, the system comprising: 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 defining a cavity for receiving the fluid;
an insert slidably disposed in the cavity and defining a lumen
through the insert, the lumen including a hole and a funnel-shaped
upper portion in fluid communication with the hole, the insert
having a density such that upon centrifugation a selected component
of the fluid resides within the lumen, the lumen forming an open
fluid path between opposite ends of the insert; a container port
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; a manifold including a manifold port, a vent to vent the
container, and a connector to couple to the container port; and a
cannula 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.
2. The system of claim 1, wherein the cannula includes a closed end
to close the hole in the insert and a side port to withdraw the
selected component, the cannula and the insert forming a seal when
the closed end of the cannula closes off the hole in the
insert.
3. The system of claim 1, wherein the cannula is a first cannula,
and further including a second cannula extendable through the
container port to withdraw the component other than the selected
component.
4. The system of claim 3, wherein the second cannula is receivable
in the manifold port.
5. The system of claim 1, wherein the container is a syringe and
the bottom is a plunger.
6. The system of claim 5, wherein the plunger has a removable
handle.
7. The system of claim 5, wherein the plunger is a first plunger
and further including a second plunger disposed in the syringe
below the first plunger to move the first plunger.
8. The system of claim 1, further including a clamping mechanism to
hold the insert in place after centrifugation, the clamping
mechanism configured to press the sidewall inward against the
insert.
9. The system of claim 1, wherein the selected component is buffy
coat and the component other than the selected component is blood
plasma.
10. The system of claim 1, wherein the insert is rigid.
11. The system of claim 1, wherein the volume contained in the
lumen upper portion of the insert is between 5% and 20% of the
volume of the container cavity.
12. A method of separating components of different densities from a
fluid containing cells using a centrifuge, the method comprising:
receiving the fluid in a separation system, the system comprising 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 defining a cavity for receiving the fluid,
an insert slidably disposed in the cavity and defining a lumen
through the insert, the lumen including a hole and a funnel-shaped
upper portion in communication with the hole, the insert having a
density such that upon centrifugation a selected component of the
fluid resides within the lumen of the insert, the lumen forming an
open fluid path between opposite ends of the insert, and a
container port disposed in the top of the container; applying
centrifugal force to the separation system; 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; and 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.
13. The method of claim 12, further comprising closing the hole in
the insert with a closed end of the cannula.
14. The method of claim 13, wherein closing the hole in the insert
comprises forming a seal between the cannula and the insert.
15. The method of claim 12, wherein the container is a syringe and
the bottom is a plunger.
16. The method of claim 15, wherein the plunger is a first plunger,
and further including moving the first plunger with a second
plunger disposed in the syringe below the first plunger to transfer
the fluid into the container.
17. The method of claim 12, wherein withdrawing the selected
component includes withdrawing the selected component through a
side port in the cannula.
18. The method of claim 12, wherein the cannula is a first cannula
and wherein 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.
19. The method of claim 12, wherein the manifold is coupled to the
container before the withdrawing of the fluid component other than
the selected component.
20. The method of claim 12, further comprising, with a clamping
mechanism, holding the insert in place after centrifugation.
21. A method of separating components of different densities from a
fluid containing cells using a centrifuge, the method comprising:
receiving the fluid in a separation system, the system comprising 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 slidably disposed in the cavity,
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, and a container port disposed in the
top of the container; applying centrifugal force to the system;
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; 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.
22. A method of separating components of different densities from a
fluid containing cells using a centrifuge, the method comprising:
receiving the fluid in 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 defining a
cavity for receiving the fluid; applying centrifugal force to the
container; after centrifugation, inserting a cannula through an
elastomeric membrane and into the container; withdrawing a selected
component of the fluid through the cannula; and with a vent,
allowing air through the elastomeric membrane and into the
container to displace fluid withdrawn through the cannula.
23. The method of claim 22, wherein the container includes an
insert slidably disposed in the cavity, the insert, upon
centrifugation, allowing fluid components of a density lower than a
certain density to rise above the insert and fluid components of a
density higher than the certain density to sink below the
insert.
24. The method of claim 23, wherein the insert includes a
funnel-shaped upper portion and a hole therethrough, the insert
having a density such that upon centrifugation the selected
component of the fluid resides within the upper portion of the
insert.
25. The method of claim 22, further comprising coupling a manifold
to the container, the manifold including the elastomeric membrane
and the vent.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 14/764,115, filed Jul. 28, 2015, which is the U.S. National
Stage of International Application No. PCT/US2014/013636, filed on
Jan. 29, 2014, published in English, which claims the benefit of
U.S. Provisional Application No. 61/757,993, filed on Jan. 29, 2013
and U.S. Provisional Application No. 61/897,587, filed on Oct. 30,
2013. The entire teachings of the above applications are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] 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.
[0004] FIG. 1 is a diagram illustrating a separation system 1700
showing different components of a fluid inside the container 1702
after centrifugation. After centrifugation, the least dense fluid
2000 will be above the insert 1300. 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 plasma 2000 and the dense red cells 2004,
with the intermediate dense material 2002, e.g., nucleated cells,
residing in the upper funnel-shaped portion 1304 of the insert. The
separation system 1700 can include a vent 1716 disposed in the top
1706 of the container 1702 and a fluid port 1718 disposed in or
adjacent the top 1706. The air vent 1716 can prevent a vacuum from
being created when fluid is withdrawn from the container 1702. A
cannula assembly 1500 with a closed end 1502 is inserted through
the injection port 1714. Before insertion of the cannula assembly,
a clamp 1800 can be applied to sidewall of container 1702 to hold
the insert 1300 in place during subsequent fluid extraction. The
closed end 1502 of the cannula assembly butts against the insert
and closes the through hole 1308 of the insert. The closed end of
the cannula assembly 1500 and 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 cannula 1508 and an outer cannula 1510, that
fit coaxially into each other.
[0005] As shown in FIG. 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 end
1502. A first set of side ports 1506 can be located near the closed
distal end 1502. A second set of side ports 1504 can be located
above the upper funnel-shaped portion 1304 of insert 1300. Fluid
above the distal end 1502 of 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
assembly 1500 into connected syringes 1802, 1804 using valve 1806.
For example, when the top side ports 1504 are aligned and opened,
fluid above the top side ports can be extracted into a first
syringe 1802 through inner cannula 1508. By rotating the two
cannulae with respect to each other, the top side ports in the
cannula assembly 1500 are misaligned and sealed off, while the
bottom side ports are aligned and opened. As shown in FIG. 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 ports 1506 are located just above the seal
created by the closed end 1502 of the cannula assembly,
substantially all fluid above the seal, but below the top side
ports, can be extracted through inner cannula 1508 into a second
syringe 1804.
[0006] FIGS. 2A-2C are a series of sequential diagrams illustrating
the extraction of fluid components using a separation system 2100,
the system including a container 2102 having a movable bottom or
plunger 2104. Centrifugation separates the fluid in the container
by density into separate components or fractions. FIG. 2A
illustrates the position of an insert, such as insert 2600, in
relation to three components of a fluid in the separation system
2100 after centrifugation. The components are a low density
fraction 2000, such as plasma, a medium density fraction 2002, such
as buffy coat or nucleated cells, and a high density fraction 2004,
such as red blood cells.
[0007] To retrieve the separated layers or fluid components, the
user takes the syringe or container 2102 out of the centrifuge. As
shown in FIGS. 2A-2B, the user then uncaps the luer connector of
port 2118, attaches a plasma extraction syringe 2300, 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 container 2102, which can be lateral to the center injection
port, 2) the size of the center hole 2608 or holes 2610 in the
insert 2600, 3) the relative density of the different fluids inside
the container 2102, and 4) the forces required to extract fluid of
different densities under these known parameters, results in
substantially only plasma moving into the plasma syringe 2300.
Because the air vent 2116 is capped, the collection syringe or
container 2102 is a vacuum. Thus the movable bottom or plunger 2104
and the insert 2600 rise in the collection syringe or container
2102 as the plasma is extracted. The target cells, such as buffy
coat, stay in the through hole or holes 2610 of the insert
2600.
[0008] As shown in FIGS. 2B-2C, after removal of the plasma 2000,
the insert 2600 has risen to the top of the syringe or container
2102 and effectively seals off port 2118 connected to the plasma
extraction syringe. At this point the user uncaps the air vent 2116
making the collection syringe or container 2102 no longer under
vacuum pressure. A second target cell extraction syringe 2302 with
a cannula 2400 attached is then inserted through the center
injection port 2114. Since 1) the insert 2600 always ends up at the
top of the collection syringe or container 2102 after removal of
the plasma and 2) the height of the insert 2600 is known, then the
distance between the top of the injection port 2114 and bottom of
the through holes 2610, 2608 of the insert 2600 is always the same
after removal of the plasma. The length of the cannula 2400 is such
that it reaches just to the bottom of the center through hole 2608
in the insert 2600 after removal of the plasma. Thus, when the user
pulls back on the plunger of the target cell extraction syringe
2302, after the air vent 2116 has been uncapped, the target cells
residing in the through hole or holes 2610, 2608 are removed.
SUMMARY OF THE INVENTION
[0009] 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.
[0010] 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.
[0011] 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.
[0012] 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.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] 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.
[0021] 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The foregoing will be apparent from the following more
particular description of example 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 embodiments of the present invention.
[0023] FIG. 1 illustrates a separation system showing different
components of a fluid after centrifugation.
[0024] FIGS. 2A-2C are a series of sequential diagrams illustrating
the extraction of fluid components using a separation system having
a movable bottom.
[0025] FIG. 3 is an exploded view of a system for separating
components of difference densities from a physiological fluid
according to an example embodiment of the invention.
[0026] FIG. 4 is a perspective view of a container including an
insert according to an example embodiment of the invention.
[0027] FIG. 5 is a perspective view of the container of FIG. 3
positioned in a base including a clamping mechanism.
[0028] FIG. 6 is a side view of an extraction cap according to an
example embodiment of the invention.
[0029] FIG. 7 is a side view of a system including the container
and base of FIG. 5 and the extraction cap of FIG. 5.
[0030] FIG. 8 is a sectional view of the system of FIG. 7.
[0031] FIG. 9 illustrates a needle assembly positioned in an
insert.
[0032] FIG. 10A illustrates a system for concentrating cell
according to another example embodiment of the invention.
[0033] FIG. 10B is a perspective view of a double plunger syringe
including an insert.
[0034] FIGS. 11A-11C illustrate movement of the plungers of the
double plunger syringe of FIG. 10A.
[0035] FIG. 12 illustrates inserts for use with a system for
concentrating cells according to example embodiments of the
invention.
[0036] FIG. 13 illustrates the collection syringe of FIG. 11
positioned in a holder including a clamping mechanism.
[0037] FIG. 14A illustrates elements of the system of FIG. 10A
arranged for extraction of fluid components from the container.
[0038] FIG. 14B is a detailed view of the cannula and manifold of
FIG. 14A.
[0039] FIG. 15 illustrates a cannula positioned against an insert,
the cannula including a side port displaced from the distal end of
the cannula to withdraw fluid at a predetermined height above the
distal end, e.g., above the insert, to withdraw substantially only
plasma.
[0040] FIG. 16 illustrates a cannula positioned against an insert,
the cannula including a side port displaced from the distal end of
the cannula to withdraw fluid at a predetermined height above the
distal end, e.g., within the insert, to withdraw substantially all
of the target fraction.
[0041] FIG. 17 illustrates an alternative embodiment including an
extraction syringe coupled to a collection syringe for withdrawing
a fluid component other than a selected component, e.g. for
withdrawing plasma.
[0042] FIG. 18 illustrates a separation system according to another
example embodiment of the invention.
[0043] FIG. 19 illustrates the separation system of FIG. 18 showing
different components of a fluid after centrifugation.
DETAILED DESCRIPTION OF THE INVENTION
[0044] A description of example embodiments of the invention
follows.
[0045] 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: [0046] a) a locking base; [0047] b) a
double needle extraction system with jet flushing; [0048] c) an
extraction needle lock that forces needle to seat in float; and
[0049] d) a syringe lock-out tab to ensure proper order of
extraction.
[0050] FIG. 3 is an exploded view of an improved system 10 for
separating components of difference densities from a physiological
fluid according to an example embodiment of the invention. System
10 includes a base 12, a separation vial (container) 14, a float
(insert) 16 disposed in the vial 14, and an extraction cap 18 with
syringes 20, 22 and a cannula assembly including extraction needles
(cannulae) 24, 25.
[0051] To separate buffy coat from red blood cells and plasma, the
float 16 should 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 slugs 56 (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.
[0052] 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.
[0053] FIG. 4 is a perspective view of container 14 including an
insert 16 according to an example embodiment of the invention.
Container 14 is a vial that has a bottom 13, a top 17 disposed
opposite the bottom, and a sidewall extending from the top, the top
including a lid 19 to 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 vial 14
via a port 28 prior to centrifugation. After the fluid is
centrifuged, the separation vial 14 is placed into the base 12. As
shown in FIG. 5, the base 12 has a lever 26 with a cam 27 that
locks the vial to the base by deforming the vial wall 15. This
deformation locks the float 16 inside the vial 14 in place so that
it cannot move. In other words, the cam and lever of base 12
operate 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.
[0054] Post centrifugation, the serum (plasma) is above the float
16, 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 in FIG. 1. The cap
or lid 19 of the vial 14 includes a silicone septum 30 and air
vents 32. As shown in FIGS. 4 and 5, four air vents 32 are arranged
around the septum 30 and four protrusions 60 extend from the lid
19. The extraction needles (cannulae) 24, 25 are inserted through
the septum 30, 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.
[0055] FIG. 6 is a side view of an extraction cap 18 according to
an example embodiment of the invention. Extraction cap 18 is
distinct from the lid 19 configured to couple to the top of the
vial (container) 14. The extraction cap includes the cannula
assembly, including needles (cannulae) 24 and 25, which are
receivable in container port 30.
[0056] As illustrated in FIGS. 7 and 8, the extraction cap 18
includes an outer part 31 and an inner part 33 movable with respect
to the outer part. The outer part 31, which includes apertures 62
to engage the protrusions 60 of the lid, snaps onto the top 17 of
the vial 14. The inner part 33 includes two ports 36, 38 for
connecting the two syringes 20, 22 to the inner and outer needles
(cannulas) of the extraction cannula assembly. An assembly tab 42
on the inner part 33 ensures that the larger syringe 20 can only be
connected to the port that is in fluid communication with the outer
extraction needle 25.
[0057] As shown in FIG. 7, a lock-out tab 44 is mounted on the
large syringe 20 that forces the user to activate the large syringe
first. The lock-out tab 44 blocks the plunger of syringe 22. Once
syringe 20 is filled with serum extracted from vial 14, syringe 20,
including lock-out tab 44, is removed and the small syringe 22 may
then be activated. This ensures the proper order is followed when
extracting fluid components from vial 14.
[0058] To remove the target cells, the extraction cap 18 is placed
over the vial 14, as illustrated in FIGS. 7 and 8. The extraction
needles 24, 25 are advanced through the silicone septum (container
port) 30 and into the vial 14. The extraction cap 18 snaps 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 float 16 (FIG. 9). The
locking screw 34 is 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 in FIGS. 7 and 8, the locking screw 34 is
positioned at a small angle within the horizontal plane. In the
embodiment of FIG. 3, the locking screw 34 is angled out of the
horizontal plane to perform the same function.
[0059] As shown in FIGS. 8 and 9, the extraction cannula assembly
includes an inner needle (inner cannula) 24 and an outer needle
(outer cannula) 25. The open distal end 46 of outer needle 25 is
positioned at a set distance from the distal end 48 of the inner
needle 24 such that the outer needle sits at or near the top of the
float 16. The outer needle is used to extract the serum (plasma).
FIG. 9 illustrates the needle assembly positioned in the float
(insert) 16. As shown, the inner needle 24 has a distal taper and
multiple holes (ports) 40, e.g., 3 side holes, at or near its
distal end 48. The inner needle 24 sits in the bottom of the float
and plugs hole 52 in the float. The extraction process starts with
the outer needle 25, which is in fluid communication with large
syringe 20. The serum is removed until bubbles are seen in the
syringe 20. Then, the selected component (e.g., stem cells, buffy
coat) is removed using the small syringe 22 and the inner needle
24, the selected component being withdrawn through holes 40 of
inner cannula 24. As cells of the selected component adhere to the
inner wall 54 of 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 holes 40 causing one or more jets of
fluid in the funnel-shaped portion of the float. This jet flushing
releases the cells from the float wall 54. The cells are then
sucked up during a second withdrawal into the syringe 22.
[0060] Embodiments shown in FIGS. 3-9 and 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 in FIGS.
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.
[0061] 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 in FIGS. 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.
[0062] As illustrated in FIGS. 10A and 10B, a system 100 for
separating components of different densities from a fluid
containing cells using a centrifuge includes a collection syringe
(container) 114 having a top 117, a sidewall 115 extending from the
top, and a bottom 113, 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 insert 116 is slidably
disposed in the cavity of the container 114. Similar to insert 16
described above in reference to FIG. 9, insert 116 defines a lumen
through the insert, the lumen including a hole 52 and a
funnel-shaped upper portion 50 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 130, e.g., a luer connector, is disposed in the top
of the container 114 to transfer the fluid into the container and
to withdraw a fluid component other than the selected component
from the container. As illustrated in FIG. 10A, the system further
includes at least one manifold 160 that includes a manifold port
162, a vent 164 to vent the container, and a connector 166 to
couple to the container port 130. A cannula 170, 172 (FIG. 14A) is
receivable in the manifold port 162 and extendable through the
container port 130 into the container 114 and into the lumen of the
insert 116 to withdraw the selected component from the lumen or a
component other than the selected component.
[0063] As illustrated in FIG. 10A, the system can include a
clamping mechanism 112 that 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 syringe 114 with
two plungers as shown, plunger 123 having a handle and plunger 113
being without a handle. A 30 ml plasma extraction syringe 120 is
connected to a cannula 170 that fits through upper injection port
162, manifold 160 and lower luer connection 166. A 5 ml concentrate
extraction syringe 122 is connected to a cannula 172 that fits
through upper injection port 162, manifold 160 and lower luer
connection 166. The system can include a syringe holder 174 with
washer on bottom and, optionally, O-ring 176. For convenience of
the user, the system can include two manifolds 160, as illustrated
in FIG. 10A. One manifold 160 receives cannula 170; the other
manifold receives cannula 172. A standard 4-way manifold may be
used for each manifold 160. The standard manifold includes 3 fluid
channels and a switch (e.g, valve) 168. Using switch 168, each of
the fluid channels can be selective closed or all channels can be
open. Here, all channels are open. Vent 164 Coupled 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 port 162 and the connector 166 can 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.
[0064] A step by step overview of the operation of the system will
be described. The first step is to use the double plunger syringe
114 (alternatively, a syringe with a removable handle) to fill the
syringe with the blood or marrow specimen to be concentrated. FIGS.
11A-11C illustrate movement of the plungers of the double plunger
syringe 114, such as during filling of the syringe with
physiological fluid. It should be noted that during filling, the
syringe port 130 is coupled to another container containing the
source tissue or to a tissue aspiration needle (not shown). Pulling
back on the second plunger 123 via its handle forces the first
plunger 113 under vacuum pressure to move back and the syringe is
thus filled. The second plunger 123 can be completely removed from
the barrel of syringe 114 leaving behind the first plunger 113 that
is not connected to a handle. The shortened profile of the syringe
with the second plunger 123 removed (or the handle removed, in case
of a plunger having a removable handle) fits into commonly used
centrifuges.
[0065] The second step is to connect to the syringe 114 containing
the specimen (e.g., fluid tissue), after the second plunger has
been removed, a small micron vented luer cap 178 and then place the
syringe inside the syringe tube holder 174. The holder 174 has a
solid bottom and also has an O-ring 176 attached to the solid
bottom (FIG. 10A). The O-ring lines up with the seal the plunger
113 makes with the outer wall 115 of the syringe barrel. Under high
g-force, this O-ring serves to prevent any leaking from the syringe
into the syringe tube holder 174.
[0066] Inside of the syringe 114 is a funnel shaped insert 116
(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 syringe 114 nearest the luer tip
(container port) 130, the target cells will reside inside of the
funnel 116, and red blood cells will reside beneath the funnel
nearest the plunger 113. Two example funnels 116a, 116b are shown
in FIG. 12.
[0067] With respect to the materials used to make the funnel 116
and 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 funnel 116 is to be molded or
machined. In FIG. 12, the funnel 116a to 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 insert 16 of FIG. 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 funnel 116b pictured to the right in FIG. 12, or a
shallow, shorter funnel, such as the clear REXOLITE funnel 116a
illustrated 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 funnel 116 has an upper funnel shaped
portion 50 and a through hole 52 at the bottom as illustrated with
respect to insert 16 (FIG. 9).
[0068] The third step is to take the syringe 114 and tube holder
174 from the centrifuge, place the syringe inside the clamping
mechanism 112 and engage the clamp as illustrated in FIG. 13. The
pressure from the clamp will pinch the walls 115 of the syringe 114
so that the inner wall of the syringe barrel presses against the
funnel 116. This pressure will freeze (i.e., lock) the funnel in
place. As shown, the clamping mechanism can include a lever 26 and
cam 27 similar to the clamping mechanism described above in
reference to FIGS. 5, 7 and 8.
[0069] The fourth step is to remove the vented cap 178 from the
collection syringe 114 and connect to the collection syringe, via
the upper luer connection 130, the 30 ml plasma extraction syringe
120 connected to cannula 170 via manifold 160, as shown in FIG.
14A. Cannula 170 fits through an upper injection port 162 connected
to manifold 160 as shown in FIG. 14B. Also connected to manifold
162 are a side air vent 164 and a lower luer connection 166. Thus,
the collection syringe 114 is not vented during loading of the
specimen but is vented, using manifold 160, during extraction of
the plasma and the target fraction inside the funnel.
[0070] After connection the plasma extraction syringe 120 and
cannula 170 to the collection syringe 114, the user pushes the
extraction cannula 170 into the collection syringe. The cannula
will advance until it hits the funnel 116 that has been frozen in
place by the clamping mechanism 112. As illustrated in FIG. 15,
cannula 170 has a blunt closed end 171 and a hole 180 a 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
funnel 116. Once the plasma is removed, the plasma extraction
cannula 170 is removed from the collection syringe 114 and the
plasma extraction syringe 120 is disconnected from the manifold
160. Optionally, the syringe 120 and the manifold 160 are
disconnected from the luer fitting 130 of the collection syringe
and removed.
[0071] Once plasma is removed from container 114, the same
procedure as described above for syringe 120 and cannula 170 is
repeated with the 5 ml concentrate extraction syringe 122 and
cannula 172. FIG. 16 illustrates cannula 172 positioned against
funnel (insert) 116, the cannula including a side port 182
displaced from the distal end 173 of the cannula to withdraw fluid
at a predetermined height above the distal end, e.g., within the
funnel 116, to withdraw substantially all of the target fraction.
Extraction cannula 172 works the same way as the 30 ml extraction
cannula except that hole (side port) 182 positioned near the
cannula's blunt distal end 173 is close to the bottom of the funnel
116 so that the contents of the funnel are removed through hole 182
when vacuum pressure is applied via syringe 122. Optionally, jet
flushing, described above in reference to cannula 24 of FIG. 9, may
be employed by ejecting fluid through hole 182 into the funnel 116
to release cells that adhere to the funnel inner surface.
[0072] Thus, in the examples illustrated in FIGS. 15 and 16, each
cannula 170, 172 includes a closed end 171, 173 to close the hole
52 in the insert 116 and at least one side port 180, 182 to
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 cannula 170, 172 and the insert 116 may form a
seal when the closed end of the cannula closes off the hole in the
insert.
[0073] Additional features of the above embodiment, as illustrated
in FIGS. 10A-16, are as follows: [0074] a) The collection syringe
(container) 114 is 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. [0075] b) The use of luer
connectors, injection ports, vented caps and a manifold 160 as 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.
[0076] The collection syringe 114 is vented and fluid removed by
employing the following novel features (see, e.g., FIGS. 14A-14B):
[0077] a) The connection between the extraction cannulas 120, 122
and the extraction syringes 170, 172 is air tight. [0078] b) The
seal around the upper injection port 162 and the cannula (170, 172)
that has pierced it is also air tight. [0079] c) The column of the
manifold 160 is air tight with the exception of the air vent 164 at
right angle to the syringes. [0080] d) The connection 166 to the
collection syringe 114 is air tight.
[0081] The collection syringe 114 is 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 vent 164 that used to accomplish this is not part of either
syringe but is connected to both syringes, e.g., via the manifold
160. The luer tip 130 of the collection syringe 114 is used as the
extraction port.
[0082] An alternative extraction process using a syringe PRP
(Platelet Rich Plasma) system will be described. In the embodiment
described above, e.g., in reference to FIG. 13, the third step in
the separation procedure is to take the syringe 114 and tube holder
174 from the centrifuge and place it inside of the clamping
mechanism 112 to engage the clamp and freeze the funnel 116 in
place. In the alternative embodiment described below, the clamping
mechanism is not used.
[0083] An alternative to removing the plasma with a cannula after
clamping the funnel 116 in place is to attach a syringe 120 to the
upper luer 130 of collection syringe 114 using 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 syringe 120. Thus, as the plasma is removed, the plunger
113 rises and the funnel 116 inside the barrel of the collection
syringe 114 also rises. It is contemplated that the user removes
the plasma until the funnel 116 reaches the top of the syringe 114.
After retrieving the plasma this way, the next step is to remove
the plasma extraction syringe 120 from the collection syringe 114
and connect to the collection syringe, via the upper luer
connection 130, a vented 5 ml PRP extraction syringe, e.g., syringe
172 coupled to manifold 160.
[0084] The target cells will not re-mix because the walls of the
funnel 116 prevent fluid turbulance or interference from the inner
wall of the syringe barrel. In addition, the center hole of the
funnel 116 is sized such that at 1 G force, surface tension
prevents fluid passage from below the funnel into the funnel. The
PRP extraction syringe 122 is connected to a cannula 172 that fits
through an upper injection port 162, connected to a manifold 116
that has a side air vent 164 and a lower luer connection 166. The
cannula 172 can be shorter for this extraction process as the float
116 is at the top of the collection syringe 114. Thus, the
collection syringe 114 is not vented during loading of the specimen
but is vented during extraction the target fraction inside the
funnel.
[0085] After removal of the plasma and after the PRP extraction
syringe 122 and cannula 172 are connected to the collection syringe
114, the user pushes the extraction cannula 172 into the collection
syringe. Since the insert 116 ends at the top of the syringe in
this embodiment, the cannula 172 can be a set length, such that the
cannula will advance the proper distance until it is approximately
at the bottom of the funnel 116. As described above in reference to
FIG. 16, this cannula has a blunt closed end to butt against the
funnel 116, 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 funnel 116 can be removed through the cannula 172 with the PRP
extraction syringe 122.
[0086] Returning to FIGS. 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.
[0087] Method 1: The first method involves the following
procedures: [0088] 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. [0089] 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. [0090] c) The upper access hole (side port) of the double
needle extracts all fluid that resides above the top of the funnel
[0091] d) The lower access hole (side port) of the double needle
extracts all fluid that resides inside the funnel.
[0092] Method 2: The second method involves the following
procedure: [0093] 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 handle [0094] b) The syringe has a center luer
connection and a side luer connection, both of which are closed.
[0095] 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 ports [0096] d) After centrifugation, the
target cells reside inside the funnel [0097] e) 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. [0098] 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. [0099] g) The plasma syringe is removed and a vented cap
added which now makes the system not under vacuum pressure. [0100]
h) Another syringe is connected to the center port and the target
cells are removed
[0101] 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.
13-33 and associated text, for a more complete description of the
two methods described above.
[0102] Described below in reference to FIGS. 18 and 19 is 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 in FIG. 18, blood
is loaded into a tube (container) 214 that defines a cavity that
serves as the collection chamber for fluid. The tube 214 has an
injection port 228 and an air vent 229. Inside the collection tube
214 and in fluid communication with the injection port 228 is a
flexible, optionally, clear tube 205 that is attached to a funnel
210 (referred to as the first or upper funnel) also disposed in the
container 214. The density of the upper funnel 210 is less than
blood plasma. For example, the density of the upper funnel can be
about 1.0.
[0103] After centrifugation, the bulk of the upper funnel 210 is
floating above the plasma 2000, as illustrated in FIG. 19. The
bottom point of the upper funnel 210 has sunk slightly into the
plasma or is in contact with the plasma. As shown, the funnel 210
is narrower than the diameter of the collection tube 214. The
funnel 210 has stabilization fins 215 at the top that extend from
the funnel to approximately the sidewall of the tube 214 without
touching the tube. These fins 215 are placed in such a manner that
the upper funnel 210 remains oriented vertically with respect to
the collection tube. The flexible tube 205 is attached at one end
to the portion of the upper funnel where through hole 220 is
located in the funnel. The other end of the tube 205 is connected
to the port 228 in the top (e.g., cap) of the collection tube 214.
Tube 205 can be a small diameter clear tube that can flex and curl
in on itself. Fluid is added to the collection tube 214 through the
port 228 with a syringe. The fluid then travels through the length
of flexible tube 205 and exits through hole 220 in the upper funnel
210 into the hollow chamber of collection tube 214. Fluid is
removed from the chamber through the same path.
[0104] Beneath the upper funnel 210 is a second funnel 216 that has
a density of about 1.06, which is a higher density than the upper
funnel and which allows the lower funnel 216 to float at the
intermediate zone between red cells 2004 and plasma 2000. After
centrifugation, most of the platelets and white cells from blood or
marrow reside within the lower funnel, as illustrated in FIG. 19.
The lower funnel 216 has a through hole 52, similar to other funnel
shaped inserts described herein. Because the funnel 216 is 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 layer 2002 between red
cells and plasma.
[0105] After centrifugation, the user can attach a syringe to the
upper port 228 and begin to retrieve plasma first. The upper funnel
210 sinks as fluid in the chamber of tube 214 is 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
funnel 216 and the contents of what is inside the lower funnel.
[0106] As additional fluid is removed, the upper funnel 210
continues to sink until it hits the lower funnel 216. The angle of
the upper funnel 210 is steeper than the angle of the lower funnel
216 so that the upper funnel fits into the lower funnel. The upper
funnel 210 can have bottom stabilization fins 225, so that the
point of the upper funnel stops a certain distance below the bottom
of the lower funnel 216. This can also be accomplished by making
the upper funnel 210 a certain height so that the upper
stabilization fins 215 contact the upper surface of the lower
funnel 216. Additionally, a clamp 212 can 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 funnel 216 can be
clamped in place and does not move as the upper funnel 210 mates
with it during extraction of fluid. This allows the user to only
withdraw the contents of the lower funnel 216 but not any fluid
which is contained beneath the lower funnel.
[0107] As illustrated in FIG. 19, after centrifugation, red cells
2004 are below the lower funnel 216, target cells of the density
desired (e.g., buffy coat) 2002 are inside the lower funnel, plasma
2000 is above the target cells, and the upper funnel 210 is
floating on top of the plasma. As described above, the user first
removes plasma 2000 by attaching a syringe (not shown) to the
center port 228 (FIG. 18) of tube 214 and pulling back on the
plunger of the syringe. This causes fluid to flow through the hole
(port) 220 at the bottom of the upper funnel 210, through the flex
tube 205 and 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 funnel 216. The stabilization
fins 215, 225 and the relative height of the two funnels 210, 216
can be adjusted such that the upper funnel 210 dead ends against
the lower funnel 216 so that no fluid from beneath the lower funnel
is removed during the extraction process.
[0108] The teachings of all patents, published applications and
references cited herein are incorporated by reference in their
entirety.
[0109] 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.
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