U.S. patent application number 09/730352 was filed with the patent office on 2001-04-12 for method for removing tumor cells from tumor cell-contaminated stem cell products.
Invention is credited to Castino, Franco, Wickramasinghe, Sumith R..
Application Number | 20010000204 09/730352 |
Document ID | / |
Family ID | 26698567 |
Filed Date | 2001-04-12 |
United States Patent
Application |
20010000204 |
Kind Code |
A1 |
Castino, Franco ; et
al. |
April 12, 2001 |
Method for removing tumor cells from tumor cell-contaminated stem
cell products
Abstract
Methods and apparatus for removing tumor cells from tumor
cell-contaminated stem cells products are disclosed. In one
embodiment, the method relies on an in-line filtration device that
includes a tumor cell reduction filter means, preferably comprised
of one or more tumor cell reduction filter pads. Preferably, the
tumor cell reduction filter means provides at least a ten-fold
reduction in filter cells while allowing for at least a 30%
recovery of stem cells in a filtered stem cell product.
Inventors: |
Castino, Franco; (Sudbury,
MA) ; Wickramasinghe, Sumith R.; (Fort Collins,
CO) |
Correspondence
Address: |
HESLIN & ROTHENBERG, PC
5 COLUMBIA CIRCLE
ALBANY
NY
12203
|
Family ID: |
26698567 |
Appl. No.: |
09/730352 |
Filed: |
December 5, 2000 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09730352 |
Dec 5, 2000 |
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09242857 |
Feb 24, 1999 |
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6177019 |
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09242857 |
Feb 24, 1999 |
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PCT/US99/14774 |
Aug 22, 1997 |
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60034758 |
Jan 6, 1997 |
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60024536 |
Aug 26, 1996 |
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Current U.S.
Class: |
210/640 ;
210/446; 210/508 |
Current CPC
Class: |
B01D 39/1623 20130101;
A61M 1/3633 20130101; B01D 39/18 20130101; A61M 2202/0429
20130101 |
Class at
Publication: |
210/640 ;
210/446; 210/508 |
International
Class: |
B01D 029/13; B01D
027/00; B01D 039/14 |
Claims
We claim:
1. A method for removing tumor cells from a tumor cell-contaminated
stem cell product, comprising the steps of: (a) providing an
in-line filtration device including: a housing having an inlet port
and an outlet port therein; and a tumor cell reduction filter means
disposed within said housing between the inlet port and the outlet
port so as to filter tumor cell-contaminated stem cell products
which flow into the filtration device via the inlet port, said
tumor cell reduction filter means dividing said housing into a
first chamber and a second chamber; (b) providing a tumor
cell-contaminated stem cell product; (c) passing the tumor
cell-contaminated stem cell product through the filtration device
wherein tumor cells are retained by the tumor cell reduction filter
means within the filtration device and the stem cell product passes
through the tumor cell reduction filter means and out of the
filtration device; and (d) recovering a tumor cell-depleted stem
cell product.
2. The method of claim 1 wherein the filtration device further
includes means, disposed within the filtration device, for allowing
gases to vent from the filtration device through the outlet
port.
3. The method of claim 1 wherein the filter means provides at least
a ten-fold reduction in tumor cells and at least a 30% recovery of
stem cells in the tumor cell-depleted stem cell product.
4. The method of claim 3 wherein the filter means provides a stem
cell product flow rate of at least 50 mL per hour.
5. The method of claim 3 wherein the filter means comprises one or
more tumor cell reduction filter pads.
6. The method of claim 3 wherein the filter means has an air
permeability of at least about 750 L/min.
7. The method of claim 6 wherein the filter means has an air
permeability of at least about 3.3.times.10.sup.3 L/min.
8. The method of claim 1 wherein the tumor cell-contaminated stem
cell product comprises a stem cell product contaminated with tumor
cells selected from the group consisting of ovarian cancer cells,
testicular cancer cells, breast cancer cells, multiple myeloma
cells, non-Hodgkin's lymphoma cells, chronic myelogenous leukemia
cells, chronic lymphocytic leukemia cells, acute myeloid leukemia
cells, and acute lymphocytic leukemia cells.
9. The method of claim 1 wherein the tumor cell-contaminated stem
cell product comprises a stem cell product contaminated with tumor
cells selected from the group consisting of ductal carcinoma cells
and adenocarcinoma cells.
10. A tumor cell reduction filter means suitable for use in an
in-line filtration device, the tumor cell reduction filter means
comprising a mechanically stable substrate that provides at least a
ten-fold reduction in tumor cells and at least a 30% recovery of
stem cells in a filtered stem cell product.
11. The filter means of claim 10 further characterized in that the
filter means provides a stem cell product flow rate of at least 50
mL per hour.
12. The filter means of claim 10 further characterized in that the
filter means comprises one or more tumor cell reduction filter pads
having a web matrix construction comprised of a plurality of fibers
and fibrils.
13. The filter means of claim 12 wherein the fibers are formed from
polyester having a denier of about 1.5 mm and the fibrils are
formed of cellulose.
14. The filter means of claim 10 further characterized in that the
filter means has an air permeability of at least about 750
L/min.
15. The filter means of claim 14 further characterized in that the
filter means has an air permeability of at least about
3.3.times.10.sup.3 L/min.
16. An apparatus suitable for use as a filtration device for
removing tumor cells from a tumor cell-contaminated stem cell
product, the filtration device comprising: a housing having an
inlet port and an outlet port therein; and a tumor cell reduction
filter means disposed within the housing between the inlet port and
the outlet port so as to filter tumor cell-contaminated stem cell
products which flow into the housing via the inlet port, the filter
means dividing said housing into a first chamber and a second
chamber.
17. The filtration device of claim 16 further including means,
disposed within the housing, for allowing gases to vent from the
housing through the outlet port.
18. The filtration device of claim 16 wherein the filter means
provides at least a ten-fold reduction in tumor cells and at least
a 30% recovery of stem cells in a filtered stem cell product.
19. The filtration device of claim 18 wherein the filter means
provides a stem cell product flow rate of at least 50 mL per
hour.
20. The filtration device of claim 18 wherein the filter means
comprises one or more tumor cell reduction filter pads.
21. The filtration device of claim 18 wherein the filter means has
an air permeability of at least about 750 L/min.
22. The filtration device of claim 21 wherein the filter means has
an air permeability of at least about 3.3.times.10.sup.3 L/min.
Description
FIELD OF THE INVENTION
1. The invention relates to the filtration of blood cells and more
specifically, to a method for selectively removing tumor cells from
tumor cell-contaminated stem cell products. A preferred embodiment
of the invention provides a method which relies on an in-line
filtration device that includes one or more tumor cell reduction
filter pads.
BACKGROUND OF THE INVENTION
2. Hematopoietic cells are rare, pluripotent cells, having the
capacity to give rise to all lineages of blood cells. Through a
process referred to as commitment, self-renewing stem cells are
transformed into progenitor cells which are the precursors of
several different blood cell types, including erythroblasts,
myeloblasts and monocyte/macrophages. Due to their self-renewing
capacity, stem cells have a wide range of potential applications in
transfusion medicine, and in particular, in the autologous support
of cancer patients.
3. Procedures have been developed whereby stem cells can be
obtained from a donor, stored and later transplanted into a patient
experiencing an immunosuppressive condition, such as following high
dose chemotherapy or total body radiation. In the past, stem cells
were harvested from bone marrow in a costly and painful procedure
which required hospitalization and general anesthesia. New
developments in technology, however, now make it possible to derive
stem cells and committed progenitor cells from peripheral blood.
Collection of stem cell products (SC products), a term which
includes both true stem cells and committed progenitor cells (i.e.,
CD 34.sup.+ cells are included), can thus be done on an outpatient
basis, eliminating the need for hospitalization. In addition, stem
cell products can also be derived from peripheral blood during
elective surgeries.
4. Once collected, the SC products, whether from bone marrow or
peripheral blood, can be stored for future use, one of the most
significant of which is transplantation to enhance hematologic
recovery following an immunosuppressive procedure such as
chemotherapy.
5. There is, however, one significant drawback to the use of this
very beneficial reinfusion procedure. Inevitably, when SC products
are obtained from a cancer patient, a significant number of tumor
cells will also be collected, thereby contaminating the SC product.
Subsequently, when the SC product is reinfused into the patient,
the tumor cells are also reintroduced, increasing the concentration
of tumor cells in the patient's blood stream. While circulating
tumor cells have not been directly linked to the relapse of a
particular cancer, in the case of lymphoma, for example, reinfused
cells have been traced to sites of disease relapse. In cases
involving adenocarcinoma, it has been estimated that for a 50
kilogram adult, approximately 150,000 tumor cells can be reinfused
during a single stem cell transplantation. Moreover, it has been
shown that the tumor cells present in the SC product are viable and
capable of in vitro clonogenic growth, thus suggesting that they
could indeed contribute to post-reinfusion relapse. Ovarian cancer
cells, testicular cancer cells, breast cancer cells, multiple
myeloma cells, non-Hodgkin's lymphoma cells, chronic myelogenous
leukemia cells, chronic lymphocytic leukemia cells, acute myeloid
leukemia cells, and acute lymphocytic leukemia cells are known to
be transplantable.
6. The extent of tumor cell contamination of SC products appears to
vary greatly from patient to patient, and values within the range
of 11 to 78 percent have been recorded. Therefore, as the
reinfusion of circulating tumor cells may well circumvent the
benefits provided by aggressive chemotherapy followed by stem cell
transplantation, the development of techniques that effectively
remove tumor cells from SC products will significantly further the
widespread use of a very beneficial and valuable clinical
procedure.
7. Methods currently used to separate the valuable stem cells from
the undesired tumor cell-contaminated product rely on a positive
selection technique that identifies stem cells and progenitor cells
that express markers for the CD34.sup.+ antigen and remove them
from the contaminated product. These methods are very labor
intensive and require the use of specialized equipment, thus
greatly increasing the cost of patient care and severely limiting
the use of SC products in transplantation procedures.
8. An alternative to positive selection for removal of tumor cells
from blood was provided by Gudemann et al., who described
filtration with special leukocyte depletion membrane filters (which
work by adsorbing charged particles) to remove urologic tumor cells
from autologous blood during an intraoperative mechanical
autotransfusion (IAT) procedure. (Gudemann, C., Wiesel, M. and
Staehler, G., Intraoperative Autotransfusion In Urologic Cancer
Surgery By Using Membrane Filters, XXIII.sup.rd Congress of the
ISBT, abstracts in Vox Sang., 67 (S2), 22.) A disadvantage of the
membrane filters used by Gudemann et al is that they do not
selectively retain tumor cells. White blood cells, including stem
cells, are also retained. Thus, tumor cells are not removed from
stem cells. The work of Miller et al also teaches that standard
blood transfusion filters are ineffective at removing tumor cells
from autologous blood. (Miller, G. V., Ramsden, C. W. and Primrose,
J. N., Autologous transfusion: an alternative to transfusion with
banked blood during surgery for cancer, B. J. Surg. 1991, Vol. 78,
June, 713-715).
9. It is therefore desirable, based upon the valuable benefits
achieved by the transplantation of previously obtained stem cell
products, benefits that ultimately result in increased survival
rates, to provide a low-cost, clinically effective method for the
selective removal of tumor cells from tumor cell-contaminated stem
cell products.
SUMMARY OF THE INVENTION
10. The present invention provides a low-cost, clinically effective
method for selectively removing tumor cells from a tumor
cell-contaminated stem cell (TCCSC) product while allowing for
optimal recovery of hematopoietic stem cells and committed
progenitor cells.
11. Thus, one aspect of the invention is a method for removing
tumor cells from a tumor cell-contaminated stem cell product,
comprising the steps of:
12. (a) providing an in-line filtration device including:
13. a housing having an inlet port and an outlet port therein;
and
14. a tumor cell reduction filter means disposed within said
housing between the inlet port and the outlet port so as to filter
tumor cell-contaminated stem cell products which flow into the
filtration device via the inlet port, said tumor cell reduction
filter means dividing said housing into a first chamber and a
second chamber;
15. (b) providing a tumor cell-contaminated stem cell product;
16. (c) passing the tumor cell-contaminated stem cell product
through the filtration device wherein tumor cells are retained by
the tumor cell reduction filter means within the filtration device
and the stem cell product passes through the tumor cell reduction
filter means and out of the filtration device; and
17. (d) recovering a tumor cell-depleted stem cell product.
18. Preferably, the tumor cell reduction filter means provides at
least a 10-fold unit reduction in tumor cells and at least a 30%
recovery (more preferably a 50% recovery) of stem cells in the
tumor-cell depleted stem cell product, and the flow rate of stem
cell product through the filter means will be at least 50 mL per
hour. To provide optimal tumor cell retention and stem cell
recovery, the filter means has an air permeability of at least
about 750 L/min to about 1.times.10.sup.4 L/min.
19. Thus, one embodiment of the invention provides a device
suitable for use as a tumor cell reduction filter means in an
in-line filtration device wherein the tumor cell reduction filter
means provides at least a ten-fold reduction in tumor cells and at
least a 30% recovery of stem cells in a filtered stem cell product.
Preferably the tumor cell reduction filter means provides a stein
cell product flow rate of at least 50 mL per hour and has an air
permeability of at least about 750 L/min to about 1.times.10.sup.4
L/min, preferably about 3.3.times.10.sup.3 L/min.
20. Tumor cell reduction filter means encompass any sort of device
or mechanically stable substrate that relies on size as the basis
for distinguishing whether particulate matter in a fluid milieu
will be passed or retained. The filter means may additionally have
surface chemistry adapted to facilitate distinguishing between
passage or retention. Examples of the several types of filter means
include, but are not limited to, polymer membranes having defined
pore size, non-woven textile pads, fiber pads, aerogels and the
like. One may also consider as appropriate filter means certain
classes of hydrogels, particularly those attached to a composite
material for mechanical stability.
21. Another embodiment of the invention is an apparatus suitable
for use as a filtration device for removing tumor cells from tumor
cell-contaminated stem cell products comprising:
22. a housing having an inlet port and an outlet port therein;
and
23. a tumor cell reduction filter means disposed within said
housing between the inlet port and outlet port so as to filter
tumor cell-contaminated stem cell products which flow into the
housing via the inlet port, said tumor cell reduction filter means
dividing said housing into a first chamber and a second
chamber.
24. According to the principles of the many aspects and embodiments
of the present invention, the tumor cell reduction filter means may
be one or more tumor cell reduction filter (TCRF) pads having a
shape-sustaining web matrix construction comprised of a plurality
of fibers and fibrils. Optimally, the fibers are comprised of
polyester having a denier of about 1.5 mm and the fibrils are
comprised of cellulose. As tumor cells are larger than stem cells,
the tumor cell reduction filter pads will selectively retain tumor
cells while allowing the smaller stem cells to pass through for
recovery.
25. The method of the invention is suited to removing tumor cells
from tumor cell-contaminated stem cell products contaminated with
tumor cells such as ovarian cancer cells, testicular cancer cells,
breast cancer cells, multiple myeloma cells, non-Hodgkin's lymphoma
cells, chronic myelogenous leukemia cells, chronic lymphocytic
leukemia cells, acute myeloid leukemia cells, and acute lymphocytic
leukemia cells. In particular, the tumor cell-contaminated stem
cell product may be a stem cell product contaminated with ductal
carcinoma cells or adenocarcinoma cells.
26. The present invention thus provides both therapeutic and
diagnostic advantages. By removing tumor cells from the
contaminated product, the method not only provides a supply of
tumor cell-depleted stem cells for transplantation, it also
provides a diagnostic tool for determining the concentration of
tumor cells in circulating blood.
27. Typically, the concentration of tumor cells in circulating
blood is exceedingly low (from 4 to 5600 per 1.6.times.10.sup.8
mononuclear cells), and it is therefore extremely difficult to
obtain an accurate count. According to the principles of the
present invention, following the filtration of a known volume of
blood product, tumor cells will be retained within the TCRF pad.
These cells can then be counted in situ or they can be recovered,
by means such as backwashing the TCRF pad with saline, and counted,
by means such as flow cytometry or spectrometry. As the original
volume of blood product was known, the concentration of tumor cells
in that volume can then be calculated, based upon the volume of
tumor cells retained by the TCRF pad.
28. Depth filtration of cell suspensions is a well-known separation
technique for the leukodepletion of red blood cell concentrates and
is a function of two mechanisms, sieving and adhesion. Sieving is
caused by the mechanical entrapment of larger cells within the
matrix of the filter pad, while adhesion is caused by the
interaction of blood cell surfaces and the filter pad material. In
leukocyte filtration, it is believed that more leukocytes are
retained by the effects of adhesion than by sieving. Generally,
depth filtration filter pads are comprised of a plurality of fibers
and fibrils that are entwined into a web matrix that provides for
increased particle attachment. Depth filtration is thus
distinguished from surface filtration, where the full extent of
particle attachment occurs on the surface of the filter pad.
29. Existing methods of removing tumor cells from contaminated stem
cell products rely on positive selection techniques and require
very expensive, specialized equipment and significant operator
time. In contrast, the principles of the present invention involve
a low-cost, easy-to-use, in-line depth filtration device that
includes a tumor cell reduction filter means. It can be
gravity-driven or pumped.
30. The in-line filtration device comprises a housing having an
inlet port and an outlet port therein, a TCRF means disposed within
the housing between the inlet port and outlet port so as to filter
the TCCSC product that flows into the filtration device via the
inlet port. The TCRF means divides the housing into a first chamber
capable of collecting and directing the flow of unfiltered liquid
therein and a second chamber in fluid flow relationship with the
first chamber capable of collecting and directing the flow of
filtered liquid.
31. Preferably, the filtration device will include means within the
filtration device, for allowing gases such as air to vent from the
filtration device through the outlet port during filtration. The
filtration device may be sized so that the distance between the
TCRF means and the inlet port prevents the accumulation of gases in
the first chamber. Similarly, the filtration device may be sized so
that the distance between the TCRF means and the outlet port forces
gases within the second chamber to enter the outlet port during
filtration.
32. Preferably, the means, disposed within the device, for allowing
gases to vent through the filtration device through the outlet port
during filtration comprises a flow deflector disposed within the
second chamber between the TCRF means and the outlet port. The flow
deflector may comprise a relatively flat member such as a disk, and
the disk may comprise at least one radially extending rib.
33. The filtration device may include one or more TCRF pads as the
filtration means, and a seal ring may be mounted between two of the
TCRF pads. The inlet port and outlet port of the filtration device
may be coaxially oriented. The housing may comprise an inlet
section and an outlet section attached to the inlet section. The
inlet port may be disposed within the inlet section and the outlet
port may be disposed within the outlet section. The one or more
TCRF pads may be sealed between the inlet section and either the
outlet section or a seal ring. If the device contains a plurality
of TCRF pads, the pads may be stacked one on top of the other and
be separated about their periphery by seal rings.
34. Although this invention is susceptible to embodiment in many
different forms, preferred embodiments of the invention are shown.
It should be understood, however, that the present disclosure is to
be considered as a exemplification of the principles of this
invention and is not intended to limit the invention to the
embodiments illustrated.
BRIEF DESCRIPTION OF THE DRAWINGS
35. Numerous other advantages and features of the present invention
will become readily apparent from the following detailed
description of the preferred embodiment, the appended claims and
the accompanying drawings wherein:
36. FIG. 1 depicts an isometric view with portions removed
therefrom of a filtration device having a flow deflector in the
second chamber thereof constructed in accordance with the
principles of the present invention;
37. FIG. 2 depicts a sectional schematic representation of the
filtration device of FIG. 1 depicting the flow of fluid therein and
constructed and usable in accordance with the principles of the
present invention;
38. FIG. 3A depicts a top isometric view of the flow deflector used
within the filtration device of FIGS. 1 and 2;
39. FIG. 3B depicts a bottom isometric view of the flow deflector
used within the filtration device of FIGS. 1 and 2; and
40. FIG. 4 depicts the filtration device of FIGS. 1 and 2 in an
operational assembly with tubing, a supply bag and a recovery
bag.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
41. A preferred embodiment of the present invention relies on an
in-line gravity driven filtration device that includes a tumor cell
reduction filter (TCRF) means that is most preferably comprised of
one or more TCRF pads. A TCRF pad has a shape-sustaining web matrix
construction formed from a plurality of fibers and fibrils and are
especially well suited for the selective removal of tumor cells
(TC), including, for example, lung carcinoma cells, lymphatic
system carcinoma cells, ovarian carcinoma cells, testicular
carcinoma cells, ductal carcinoma cells, mammary carcinoma cells
and adenocarcinoma cells, from a tumor cell-contaminated stem cell
(TCCSC) product.
42. As referred to herein, the terms upstream, top or up refers to
a location of the flow of TCCSC product prior to filtration through
the TCRF means within the filtration device. Conversely, the terms
downstream, bottom or down as used herein refers to a location of
the flow of filtered stem cell (FSC) product, following filtration
through the TCRF means within the filtration device. Moreover, as
used herein, the terms radially and axially refer to the radial and
axial directions, respectively, relative to axis A--A of FIG. 2,
running lengthwise through the center of the filtration device.
43. Although various embodiments of the filtration device may be
used to practice the method of the present invention, each
embodiment comprises a housing typically formed by an inlet
section, an outlet section, a TCRF means, and means for allowing
gases to vent from the filtration device through an outlet port.
The device preferably will include means to enable air within the
filtration device to vent downstream without manipulation of
various components, the use of vent filters or other external
means. Preferably, the device will incorporate a downstream flow
deflector.
44. Referring now specifically to the drawings, FIGS. 1 and 2
depict a filtration device, generally designated as 23, that
comprises an inlet section 1, TCRF means, comprised of TCRF pads 3,
4, 5 and 6, seal rings 7, 8, 9 and flow deflector 10. Inlet section
1 is sealed to outlet section 2 at a joint 32 therebetween.
Preferably the joint is sealed by ultrasonic weld, a heat weld, a
solvent weld, a glue joint or any other means for creating a leak
tight seal. TCRF pad 6 is sealed into the outlet section 2 by
compression thereby forming a compression seal. The outer periphery
of TCRF pad 6 is compressed between shelf 33 of outlet section 2
and a seal ring 9. TCRF pad 5, located on top of TCRF pad 6, is
sealed into outlet section 2 using a compression seal. The outer
periphery of TCRF pad 5 is compressed between seal ring 8 and seal
ring 9. TCRF pad 4, located on top of TCRF pad 5, is sealed into
outlet section 2 also using a compression seal. The outer periphery
of TCRF pad 4 is compressed between seal ring 7 and seal ring 8.
TCRF pad 3, located on top of TCRF pad 4, is also sealed into
outlet half 2 using a compression seal. The outer periphery of TCRF
pad 3 is compressed between seal ring 7 and the seal rib 24
protruding in the axial direction along the outer perimeter of
inlet section 1. Seal rings 7, 8 and 9 are preferably press fit
with wall 45 of outlet section 2. However, seal rings 7, 8 and 9
may be bonded to or into outlet section 2 using an ultrasonic weld,
heat weld, solvent weld, glue or by using any other sealing means
which will create a leak tight seal. If the seal rings are not
press fitted into outlet section 2, then seal ring 9 could be
bonded to outlet section 2 and the bottom surface of seal ring 8
could be bonded to the top surface of seal ring 9 and the bottom
surface of seal ring 7 could be bonded to the top surface of seal
ring 8. Although the device illustrated in FIGS. 1 and 2 includes
four TCRF pads 3, 4, 5 and 6, the invention is not limited thereto
and may include one or more TCRF pads.
45. The cavity 21 formed within the interior of the device 23 by
the inside walls of inlet section I and outlet section 2 is divided
into two chambers by TCRF pads 3, 4, 5 and 6. The upstream, upper
or first chamber 30 is formed by wall 35 of inlet section 1, wall
36 of inlet section 1 and the upper surface 37 of TCRF pad 3. The
downstream, lower or second chamber is formed by wall 38 of outlet
section 2, wall 39 of outlet section 2 and the lower surface 43 of
TCRF pad 6. The lower chamber 29 is divided into two sections by a
flow deflector 10 within the lower chamber. The first section of
lower chamber 29 is bounded by bottom surface 43 of TCRF pad 6 and
top surface 42 of flow deflector 10. The second section of lower
chamber 29 is bounded by bottom surface 41 of flow deflector 10 and
by the surface 39 of outlet section 2.
46. Referring to FIGS. 3A and 3B, the flow deflector is formed of a
thin disk which contains radial filter support ribs 12 on a first
side thereof, alignment tabs 3 1 on the outer periphery, and
support pins 11 on a second side thereof. The filter support ribs
12 function as a means for allowing radial flow of FSC product
along the first side of the flow deflector. However, other means
for allowing such a flow such as a series of support pins or a
woven screen may be used in lieu of support ribs 12. The support
pins 11 function as a means for supporting the flow deflector 10
above wall 39 of outlet section 2. The alignment tabs function as a
means for positioning the flow deflector 10 within the lower
chamber 29.
47. In FIG. 4 the filtration device 23 depicted in FIG. 1 and FIG.
2 is in an operational assembly with inlet tube 17, outlet tube 18,
supply bag 25 and recovery bag 26. Preferably, the user will obtain
the assembly of FIG. 4 sterilized, without supply bag 25, with the
inlet end of inlet tube 17 sealed to maintain system sterility. For
performing filtration, inlet tube 17 (FIG. 2) attached to tube
socket 15 at the center of the inlet section 1 would be bonded to a
pigtail on supply bag 25, which contains a TCCSC product, using a
sterile docking device as is well known in the art. Inlet tube 17
is in fluid flow relationship with upper chamber 30 via inlet port
13. Outlet tube 18, attached to a receiving bag, is bonded to
outlet tube socket 16 located at the center of the outlet section
2. Outlet tube 18 is in fluid flow relationship with bottom chamber
29 via outlet port 14.
48. Filtration device 23 hangs in line. TCCSC product enters
filtration device 23 from its inlet port 13 and FSC product exits
filtration device 23 from its outlet port 14. In the process of
filling filtration device 23 with TCCSC product, all of the air
therein before the filtration process began is purged out of
filtration device 23 through outlet tube 18 into receiving bag 26
before FSC product starts to flow out of filtration device 23. This
process assures that little or no air gets trapped in TCRF pads 3,
4, Sand 6. Therefore, the entire exposed surface area of the TCRF
pads is used for filtration.
49. When filtering TCCSC product, the user would first close inlet
tube 17 near the end to be attached to supply bag 25, with a tube
clamp (not shown) and then make a sterile connection between the
inlet end of inlet tube 17 and supply bag 25 using a sterile
docking device as is well known in the art. The actual sterile
connection is made between inlet tube 17 and a short length of tube
which is a part of supply bag 25. The resulting system is
illustrated in FIG. 4. Supply bag 25 may be suspended from an
appropriate mechanism such as pole 28 with hook 27. Recovery bag 26
may be suspended by the mechanism or may rest on a surface such as
a bench top or the like.
50. Referring to FIGS. 1, 2 and 4, once the tube clamp (not shown)
is opened, TCCSC product will begin to flow from supply bag 25
through inlet tube 17, through inlet port 13, into upper chamber
30. The air that was in inlet tube 17 will be forced ahead of the
TCCSC product and into upper chamber 30. TCCSC product enters upper
chamber 30 at the center, and as a result, upper chamber 30 is
filled with TCCSC product from the center, then radially outward.
This radial flow is illustrated by arrows in FIGS. 1 and 2. Because
upper chamber 30 fills from the center and radially outward, TCRF
pads 3, 4, 5, 6 will also wet from the center and radially outward.
As upper chamber 30 becomes filled, any air remaining in upper
chamber 30 will be forced out through the non-wet portions of TCRF
pads 3, 4, 5 and 6, into lower chamber 29, through outlet port 14,
through outlet tube 18, and into receiving bag 26. Upper chamber 30
should be sized in relation to the initial TCCSC product flow rate
to assure that all of the air initially in upper chamber 30 will be
forced out through TCRF pads 3, 4, 5 and 6. If the volume of upper
chamber 30 in relation to the initial TCCSC product flow rate is
too large, some air will become trapped in upper chamber 30.
51. As indicated, TCRF pads 3, 4, 5 and 6 wet radially outward and
any air that was in them will be forced into lower chamber 29,
through outlet port 14, through outlet tube 18, into receiving bag
26. Due to the radial outward wetting of TCRF pads 3, 4, 5 and 6,
FSC product will first flow out of TCRF pad 6 from its center and
then continue to flow out of TCRF pad 6 in a radial outward
pattern. Thus, the first section of lower chamber 29 will also fill
from its center radially outward. As the first section of lower
chamber 29 fills, any air that had been forced out through TCRF
pads 3, 4, 5 and 6 will also be forced radially outward through the
first section of lower chamber 29.
52. Once the first section of lower chamber 29 is filled with FSC
product, the FSC product will flow into the second section of lower
chamber 29 radially inward, forcing air into the outlet port, and
thereby venting air downstream. Once the second section of lower
chamber 29 is filled with FSC product, outlet port 14 and outlet
tube 18 will then be filled and finally, recovery bag 26. The flow
around the flow deflector is illustrated by arrows in FIG. 2.
53. Due to the web matrix construction of TCRF pads 3, 4, 5 and 6,
tumor cells will be retained within filter device 23 and the
smaller stem cells and committed progenitor cells will pass through
for recovery in recovery bag 26. The diameter of stem cells and
committed progenitor cells ranges between about 5 and about 15
.mu.m, while that of tumor cells ranges between about 20 and about
50 .mu.m.
54. Preferably, the TCRF means provides at least a ten-fold
reduction in tumor cells while also providing at least a 30%
recovery of stem cells and preferably more than 50% recovery. The
flow of stem cell product through the TCRF means should be at least
50 mL per hour. We have found that TCRF means having an air
permeability of at least about 26.8 cubic feet per minute (CFM)
(750 L/min) and more preferably, of about 118.5 CFM
(3.3.times.10.sup.3 L/min) function well in the apparatus of the
invention. TCRF means, such as the tumor cell reduction filter
pads, having a higher air permeability are obtained with the use of
cellulose or cellulose acetates that have a higher average surface
area.
55. As collected SCP are frequently cryopreserved and stored for
later use, it is important to note that the TCRF means will also
retain any granulocytes that may be present in the contaminated
stem cell product. This is advantageous because granulocytes will
not survive the freezing rate of the filtered SCP and will lyse,
releasing their intracellular contents into the superlatant
solution, presumably resulting in the reduced viability of the stem
cell product upon thawing. It is therefore desirable that
granulocytes are removed prior to the cryopreservation process.
EXAMPLES
56. A preferred embodiment of the present invention is hereinafter
described in more detail by means of the following examples which
are provided by way of illustration and not by way of
limitation.
Example 1
57. A model TCCSC product was prepared using blood mononuclear
cells (BMNC) mixed with either adenocarcinoma or ductal carcinoma
tumor cells (collectively, tumor cells, TC) in a 5:1 ratio. The
composition was filtered in accordance with the principles of the
present invention using cellulose or cellulose acetate-polyester
composite TCRF pads 1.5 millimeters in thickness and having an
effective pore size of 10 .mu.m, with a variation from 5-150 .mu.m,
due to the web matrix construction of the TCRF pads. The FSC
product was analyzed for BMNC and TC content. Wright-Giemsa stained
cytospins revealed that the recovery of BMNC was 25-60 fold higher
than that of TC.
Example 2
58. A model TCCSC product was prepared wherein the BMNC to TC ratio
was 50:1. To facilitate an accurate reading of the results, the TC
were pre-labeled with a fluorescent membrane dye. The TCCSC product
was filtered using the same cellulose-acetate TCRF pads as in
Example 1 and upon analysis of the FSC product, the concentration
of TC was undetectable, indicating at least a 30-fold preferential
retention of TC.
59. Furthermore, because stein cells are smaller than BMNC,
post-filtration recovery of granulocytes/macrophages and erythroid
progenitor cells should necessarily be greater than that of BMNC.
In fact, the filtration of TC-free BMNC revealed that the
concentration of hematopoietic stem cells in the recovered product
was almost 10-fold higher than in the unfiltered product;
indicating that approximately 80% of all fully viable hematopoietic
precursors can be recovered under conditions that reduce the
concentration of TC 30-fold, values comparable to those obtained
with currently used TC reduction processes that rely on positive
selection.
60. Table 1 provides the results of Examples 1 and 2.
1TABLE 1 STARTING RATIO LOG REDUCTION LOG REDUCTION BMNC:TC TC BMNC
NO TC NA 1.1 5:1 2.2, 2.8 0.8, 1.0 50:1 >1.5 0.6
Example 3
61. A model TCCSC product was prepared in which the BMNC to TC
ratio was 10:1. Several filtration tests of the composition were
conducted, using various TCRF pad media. Following filtration, the
FSC product was analyzed for the percentage of total cells
recovered, the ratio of BMNC:TC, the type of cells recovered and
the fold increase in concentration of a specific hematopoietic
colony forming cell, CFU-GM (a granulocyte/macrophage
precursor).
62. The results of these filtration tests ("A"-"V") are reflected
in Tables 2 and 2A. As can be seen, optimal results were obtained
in Test "U" wherein two TCRF pads, characterized as TCRF pad media
Stem Cell-3 (SC-3) were utilized. In this case, 14.9% of total BMNC
were recovered, with an 8.7 fold increase in the concentration of
CFU-GM.
2TABLE 2 AIR NO. OF PERMEABILITY TCRF PAD PADS % TOTAL TEST CU
FT/MIN MEDIA USED CELL YIELD A 3-4 LEUKONET .TM. 2 1.06 B 3-4
LEUKONET .TM. 4 0.40 C 3-4 MlLLIPORE 2 1.90 4528-41 D 3-4 MILLIPORE
4 1.50 4528-41 E 3-4 MILLIPORE 2 3.00 4528-41 F 3-4 MILLIPORE 4
2.45 4528-41 G 31 LYDALL LB .TM. 2 20.0 170-54-D H 31 LYDALL LB
.TM. 4 5.20 170-54-D I 3-4 BIOCMPTBL 2 1.20 2 MG/UL J 3-4 BIOCMPTBL
4 1.00 2 MG/UL K 3-4 BIOCMPTBL 2 2.20 5 MG/UL L 3-4 BIOCMPTBL 4
2.00 5 MG/UL M 3-4 BIOCMPTBL 2 2.00 10 MG/UL N 3-4 BIOCMPTBL 4 1.00
10 MG/UL O 31 LYDALL LB .TM. 2 4.60 170-64-D P 31 LYDALL LB .TM. 4
0.40 170-64-D Q 26.8-30.5 SC-1 2 5.00 R 26.8-30.5 SC-1 4 1.30 S
43.6-44.1 SC-2 2 9.04 T 43.6-44.1 SC-2 4 5.00 U 118.5 SC-3 2 14.9 V
118.5 SC-3 4 9.00
63.
3TABLE 2A PRE- POST- FILTER FILTER POST-FILTER RATIO RATIO
POST-FILTER FOLD INCREASE TEST BMNC:TC BMNC:TC CELL TYPES CFU-GM A
10:1 >100:1 MRBC -- B 10:1 >100:1 MRBC -- C 10:1 >100:1
MRBC -- D 10:1 >100:1 MRBC -- E 10:1 >100:1 MRBC -- F 10:1
>100:1 MRBC -- G 10:1 >100:1 MRBC 1 LMPHCT MCRPHG H 10:1
>100:1 MRBC 1 LMPHCT MCRPHG I 10:1 >100:1 MRBC -- J NA NA
MRBC -- K NA NA MRBC -- L NA NA MRBC -- M NA NA MRBC 0 LMPHCT
MCRPHG N NA NA MRBC 0 LMPHCT MCRPHG O 10:1 >100:1 MRBC 0 LMPHCT
MCRPHG P 10:1 >100:1 MRBC 0 LMPHCT MCRPHG Q NA NA MRBC 2.4
LMPHCT MCRPHG R NA NA MRBC 1.7 LMPHCT MCRPHG S NA NA MRBC 4.0
LMPHCT MCRPHG T NA NA MRBC 3.0 LMPHCT MCRPHG U NA NA MRBC 8.7
LMPHCT MCRPHG V NA NA MRBC 3.7 LMPHCT MCRPHG NA = TC growing too
slowly to provide adequate cells for trials MRBC = Mature Red Blood
Cells LMPHCT = Lymphocytes MCRPHG = Macrophages MEDIA = TCRF pads
were made by various manufacturers. Leukonet .TM. and Lydall
64. pads were prepared from the same filter material, available
from Lydall, Inc. (Manchester, Conn.). The material comprised
polyester fibers, cellulose fibrils and NW-1845, an acrylic binder
(Rohm and Haas). The polyester fibers had an average denier of 0.5
mm in each filter pad, except SC-3, where the polyester fibers had
an average denier of 1.5 mm. The cellulose fibrils had a surface
area of about 20 m.sup.2/g.
65. The various Lydall pads were obtained by varying the percentage
of polyester fibers in the material. This is shown in Table 2 as a
function of air permeability, which was determined by the Frasier
method employing a head pressure of 12.7 kg/m.sup.2.
66. The Millipore TCRF pad was a Leukonet.TM. TCRF pad coated with
a hydrophilic polymer formed of a cross-linked hydroxyalkyl
acrylate according to the method of U.S. Pat. No. 4,618,533, issued
Oct. 21, 1986 to Steuck and assigned to Millipore Corp. (Bedford,
Mass.). The Biocmptbl TCRF pad was a Leukonet.TM. TCRF pad coated
with various concentrations of phosphoryl choline. (Biocompatibles,
Ltd., Middlesex, England).
67. While the present invention is not intended to be limited to
the use of TCRF pads as the TCRF means, nor by the specific number
of TCRF pads used during a single filtration, optimal tumor cell
retention and stem cell recovery has been obtained with the
concurrent use of two TCRF pads.
68. This invention has been described in terms of specific
embodiments, set forth in detail. It should, however, be understood
that the embodiments are presented by way of illustration only, and
the invention is not limited thereto. Modifications and variations
within the spirit and scope of the claims that follow will be
readily apparent from this disclosure, as those skilled in the art
will appreciate.
* * * * *