HK1021512A - Method for removing tumor cells from tumor cell-contaminated stem cell products - Google Patents

Description

Method for removing tumor cells from tumor cell-contaminated stem cell products

Technical Field

The present invention relates to filtration of blood cells, and more particularly, to a method for selectively removing tumor cells from a tumor cell-contaminated stem cell product. A preferred embodiment of the present invention provides a method of filtering using an in-line filtration device comprising one or more tumor cell filter plates.

Background

Hematopoietic cells are rare, multifunctional cells that have the ability to increase blood cells of all lineages. Self-renewing stem cells are transformed into prophase cells, which are precursors of several different types of blood cells, such as erythroblasts, myeloblasts, and monocytes/macrophages, by a process called commitment. Due to its ability to self-renew, stem cells have a wide range of potential uses in perfusion medicine, particularly in the self-support of cancer patients.

Various methods have been developed to remove stem cells from donors, store them and then transplant them into patients who have been immunosuppressed, for example, by high-dose chemotherapy or total body irradiation. In the past, stem cells were removed from bone marrow by expensive and painful procedures that also required hospitalization and general anesthesia. With the advancement of technology, stem cells and the precursor cells differentiated therefrom have now been obtained from peripheral blood. Thus, stem cell collection can be performed in an outpatient setting without hospitalization, and the term stem cell product (SC product) includes both true stem cells and committed progenitors (i.e., including CD 34)⁺A cell). In addition, stem cell products can also be obtained from peripheral blood in selected surgeries。

SC products, whether from bone marrow or peripheral blood, once collected can be stored for later use, one of the most important applications being transplantation into the body to promote blood recovery from immunosuppressive processes such as chemotherapy.

However, the use of this highly advantageous reinfusion method also has a significant drawback. When the SC product is obtained from a cancer patient, a large number of tumor cells will be collected at the same time, thereby contaminating the SC product. Thereafter, when the SC product is re-infused into the patient, tumor cells are also introduced, thereby increasing the concentration of tumor cells in the patient's blood. Although circulating tumor cells are not directly associated with the recurrence of a particular cancer, as in lymphoma, reinjection of tumor cells has been found at the site of recurrence of the condition. In adenocarcinoma, it has been estimated that about 150,000 tumor cells can be reinjected for one stem cell transplant for an adult having a weight of 50 kg. Furthermore, it has been found that tumor cells present in SC products can survive and can grow by cloning genes in vitro, thus suggesting that they may indeed lead to cancer recurrence after infusion. 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 myelogenous leukemia cells, and acute lymphocytic leukemia cells are all known to be transplanted.

The degree of tumor cell contamination of SC products varied greatly from patient to patient, with the documented data varying from 11 to 78%. Therefore, the harm caused by re-infusion of circulating tumor cells is likely greater than the benefit of stem cell transplantation following invasive chemotherapy, and the development of technologies that can effectively remove tumor cells from SC products would greatly expand the application of this beneficial and valuable clinical treatment.

The isolation of valuable stem cells from products contaminated with harmful tumor cells is today performed using positive selection techniques which identify expression of CD34⁺Stem cells labeled with antigenAnd prophase cells and separating them from the contaminated product. These methods are very labor intensive and require specialized equipment, thereby greatly increasing the cost of patient care and severely limiting the use of SC products in transplantation therapy.

In addition to positive selection, Gudemann et al proposed another method for removing tumor cells from blood, which describes filtration In an intraoperative mechanical automated Infusion (IAT) method to remove urinary tract tumor cells from autologous blood Using a special leukocyte-depleted Membrane filter module that adsorbs charged particles (Gudemann, C^rdConsistency of the ISBT, anchors in Vox Sang.,67(S2), 22.). The drawback of the membrane filtration module employed by Gudemann et al is the inability to selectively retain tumor cells. White blood cells, including stem cells, are also trapped. Therefore, tumor cells were not differentiated from stem cells. Work by Miller et al indicates that the standard blood infusion filter assembly is unable to remove tumor cells from Autologous blood (Miller, G.V., Ramsden, C.W.and Primrose, J.N., autologus transfer: an alternative to transfer with a bound blood staining surgery for cancer, B.J.Surg.1991, Vol.78, June, 713-plus 715).

Because of the valuable effects of transplanting pre-obtained stem cell products, and the resulting increased survival rates, it would be desirable to provide a cost-effective and clinically effective method for selectively removing tumor cells from tumor cell-contaminated stem cell products.

Brief description of the invention

The present invention provides a low cost and clinically effective method for selectively removing tumor cells from tumor cell-contaminated stem cell products (TCCSCs) to maximize recovery of hematopoietic stem cells and committed progenitors.

Accordingly, one aspect of the present invention is a method for removing tumor cells from a tumor cell-contaminated stem cell product, comprising the steps of:

(a) preparing an in-line filtration device comprising:

a housing having an inlet and an outlet therein; and

a tumor cell-depleting filter assembly disposed in said housing between said inlet and said outlet for filtering tumor cell-contaminated stem cell product flowing into said filter device through said inlet, said tumor-depleting filter assembly dividing said housing into a first chamber and a second chamber;

(b) preparing a stem cell product contaminated by tumor cells;

(c) flowing the tumor cell-contaminated stem cell product through the filtration device, wherein tumor cells are retained by a tumor cell-depleting filter element in the filtration device and the stem cell product passes through the tumor cell-depleting filter element and out of the filtration device; then the

(d) Recovering the tumor cell-depleted stem cell product.

Preferably, the tumor cell depleting filter module reduces tumor cells by at least a factor of ten and recovers at least 30% (more preferably 50%) of the stem cells in the tumor cell contaminated stem cell product at a flow rate of at least 50ml per hour through the filter module. For optimal tumor cell retention and stem cell recovery, the filter element has a gas permeability of at least about 750l/min to 1X 10⁴l/min。

Accordingly, one embodiment of the present invention provides a device suitable for use as a tumor cell depleting filter module in an in-line filtration device, wherein the tumor cell depleting filter module reduces tumor cells by at least a factor of ten and recovers at least 30% of the stem cells in the filtered stem cell product. Preferably, the stem cell product is passed through the filter assembly at a flow rate of at least 50ml per hour, and the filter assembly has an air permeability of at least about 750 |/hrmin to 1 × 10⁴l/min, preferably 3.3X 10³l/min。

The tumor cell depletion filter assembly includes various means or mechanically stable matrix for distinguishing between passage or retention of particulate matter in a fluid based on size. In addition, the filter assembly may have a surface chemistry that helps to discern passage or retention. Examples of several types of filter assemblies include, but are not limited to, polymeric membranes having a specified pore size, nonwoven sheets, fiber sheets, aerogels, and the like. Several hydrogels are also contemplated as suitable filter components, particularly hydrogels with mechanical stability mounted on a composite material.

Another embodiment of the present invention is a device suitable for use as a filtration device for removing tumor cells from a tumor cell contaminated stem cell product, comprising:

a housing having an inlet and an outlet therein; and

a tumor cell-depleting filter assembly disposed in said housing between said inlet and said outlet for filtering tumor cell-contaminated stem cell product flowing into said housing through said inlet, said tumor-depleting filter assembly dividing said housing into a first chamber and a second chamber.

According to principles of various aspects and embodiments of the present invention, the tumor cell reduction filter assembly may be one or more Tumor Cell Reduction Filter (TCRF) plates having a shape-retaining three-dimensional network structure of a plurality of fibers and fibrils. Most preferably, the fibers consist of polyester with a titer of about 1.5mm, while the fine fibers consist of cellulose. Since tumor cells are larger than stem cells, the tumor cell depletion filter plate will selectively retain tumor cells, while allowing smaller stem cells to pass through for recovery.

The methods of the invention are useful for removing tumor cells from a stem cell product 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 myelogenous 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 or adenocarcinoma cancer cells.

The present invention thus has therapeutic and diagnostic advantages. By removing tumor cells from the contaminated product, the present method provides not only tumor cell-depleted stem cells for infusion, but also a means for determining the concentration of tumor cells in circulating blood.

Typically, the concentration of tumor cells in circulating blood is very low (every 1.6X 10)⁸4 to 5600 tumor cells in a single monocyte), it is difficult to count them accurately. In accordance with the principles of the present invention, tumor cells will be trapped on the TCRF plate after a known volume of blood product is filtered. These cells can be counted in situ or recovered by methods such as rinsing the TCRF plate with saline in the reverse direction and then counted using, for example, flow cytometry or spectral counting. Since the starting volume of blood product is known, the concentration of tumor cells in this volume can then be calculated from the amount of tumor cells trapped on the TCRF plate.

Depth filtration of cell suspensions is a well-known separation technique for leukocyte depletion of concentrated solutions of red blood cells, the filtration of which is based on two mechanisms, sieving and adhesion. The entrapment of larger cells in the filter plate mesh is a sieving, and the interaction between the blood cell surface and the filter plate material causes adhesion. In the filtration of leukocytes, it has been shown that adhesion traps more leukocytes than sieving. Generally, a depth filtration filter plate is composed of a large number of fibers and fine fibers entangled into a three-dimensional network structure to increase particle adhesion. Depth filtration is therefore also distinguished from surface filtration, where all particle attachment occurs above the surface of the filter plate.

Existing methods for removing tumor cells from contaminated stem cell products are based on positive selection techniques, require very expensive specialized equipment and require long operating times. In contrast, the principles of the present invention employ a low cost, easy to use, in-line depth filtration device that includes a tumor cell depletion filter assembly. It may be gravity or pump driven.

The online filtering device includes: a housing having an inlet and an outlet therein; and a TCRF module disposed in the housing between the inlet and the outlet for filtering TCCSC product flowing through the inlet into the filter apparatus. The TCRF assembly divides the housing into a first chamber that can collect and direct unfiltered fluid flow therein and a second chamber that can collect and direct filtered fluid flow and is in fluid flow communication with the first chamber.

Preferably, the filter device may include an assembly that is within the filter device and that during filtration expels a gas, such as air, out of the filter device through the outlet. The filter assembly is sized such that the distance between the TCRF assembly and the inlet prevents gas from accumulating in the first chamber. Similarly, the filter assembly is sized such that the distance between the TCRF assembly and the outlet prevents gas from accumulating in the second chamber during filtration.

Preferably, the assembly disposed in the apparatus and adapted to allow gas to exit the filtration apparatus through the outlet during filtration includes a flow diverting assembly disposed in the second chamber and between the TCRF assembly and the outlet. The flow direction diverting assembly may comprise a relatively flat member such as a disc including at least one radially extending rib.

The filter assembly may include one or more TCRF plates as the filter assembly and a gasket mountable between the two TCRF plates. The inlet and outlet of the filter device may be oriented coaxially. The housing may include an inlet portion and an outlet portion connected to the inlet portion. The inlet may be provided in the inlet portion and the outlet may be provided in the outlet portion. One or more TCRF plates may be sealed between the inlet portion and the outlet portion or gasket. If the device comprises a plurality of TCRF plates, the plates can be stacked one on top of the other and separated from each other at their circumference by sealing rings.

While this invention is susceptible of embodiment in many different forms, there is shown by way of illustration only preferred embodiments of the invention. It is to be understood that this disclosure is only illustrative of the principles of the invention and is not intended to limit the invention to the embodiments described.

Brief Description of Drawings

Numerous other advantages and features of the present invention will become readily apparent from the following detailed description of the preferred embodiments, the appended claims and the accompanying drawings in which:

FIG. 1 is an isometric view, partially cut away, of a filter apparatus constructed in accordance with the principles of the present invention having a flow direction diverter assembly in its second chamber;

FIG. 2 is a cross-sectional view of the filter apparatus of FIG. 1 illustrating the direction of fluid flow and constructed and used in accordance with the principles of the present invention;

FIG. 3A is a top isometric view of a flow direction diversion assembly used in the filter apparatus of FIGS. 1 and 2; and

FIG. 3B is a bottom isometric view of a flow direction diverting assembly used in the filter apparatus of FIGS. 1 and 2;

figure 4 shows the filter apparatus of figures 1 and 2 in operative association with tubing, a feed bag and a recovery bag.

Detailed description of the preferred embodiments

A preferred embodiment of the present invention is an online gravity-driven filtration device comprising a tumor cell depletion filter (TCRF) assembly, most preferably consisting of one or more TCRF plates. The TCRF plate has a shape-retaining three-dimensional network structure composed of a large number of fibers and fine fibers, and is particularly suitable for selectively removing Tumor Cells (TC), such as lung cancer cells, lymphatic system cancer cells, ovarian cancer cells, testicular cancer cells, ductal cancer cells, breast cancer cells, and adenocarcinoma cancer cells, from a tumor cell-contaminated stem cell (TCCSC) product.

As used herein, the term upstream, top, or upward refers to the location of the TCCSC product prior to filtration through the TCRF components in the filtration apparatus. In contrast, the term downstream, bottom or downward refers to the location where the stem cell (FSC) product has been filtered after filtration through the TCRF components in the filtration device. Further, the terms radially and axially refer to radial and axial directions, respectively, relative to the axis A-A in FIG. 2, passing longitudinally through the middle of the filter apparatus.

While the method of the present invention can be practiced with a variety of embodiments of the filtration device, each embodiment generally includes a housing comprised of an inlet, an outlet, a TCRF module, and a module for allowing gas to exit the filtration device through the outlet. The apparatus preferably includes an assembly for discharging gas from the filtration apparatus in a downstream direction without the need for handling multiple components, using an exhaust gas filtration assembly, or other external components. Preferably, the device is equipped downstream with a flow direction diverting assembly.

Referring now specifically to the drawings, FIGS. 1 and 2 show a filtration apparatus, generally designated 23, which includes an inlet section 1, a TCRF assembly formed by TCRF plates 3, 4, 5 and 6, seal rings 7, 8, 9 and a flow diverting assembly 10. The inlet portion 1 and the outlet portion 2 are sealed together by a joint 32 therebetween. The joint is preferably sealed by ultrasonic welding, heat welding, solvent welding, adhesive bonding or any other method to create a leak-free seal. The TCRF plate 6 is sealed into the outlet portion 2 by pressure, thereby constituting a squeeze seal. The outer circumference of the TCRF plate 6 is squeezed between the shelf 33 of the outlet part 2 and the sealing ring 9. The TCRF plate 5 located on the upper portion of the TCRF plate 6 is sealed in the outlet portion 2 by means of a press seal. The outer circumference of the TCRF plate 5 is compressed between the seal ring 8 and the seal ring 9. The TCRF plate 4 located on the upper portion of the TCRF plate 5 is sealed in the outlet portion 2 by means of a press seal. The outer circumference of the TCRF plate 4 is compressed between the seal ring 7 and the seal ring 8. The TCRF plate 3 located on the upper portion of the TCRF plate 4 is sealed in the outlet portion 2 by means of a press seal. The outer circumference of the TCRF plate 3 is compressed between the sealing ring 7 and a sealing rib 24 projecting axially along the outer circumference of the inlet section 1. The sealing rings 7, 8 and 9 are preferably press fit against the wall 45 of the outlet portion 2. However, ultrasonic welding, heat welding, solvent welding, glue bonding or any other sealing method may be used to seal the sealing rings 7, 8 and 9 in the access portion 2, resulting in a leak-free seal. If the gasket is not press fitted into the outlet portion 2, the gasket 9 may be bonded to the outlet portion 2, the bottom surface of the gasket 8 may be bonded to the lower surface of the gasket 9, and then the bottom surface of the gasket 7 may be bonded to the upper surface of the gasket 8. Although the apparatus shown in fig. 1 and 2 includes 4 TCRF boards 3, 4, 5, and 6, the present invention is not limited thereto, and it may include one or more TCRF boards.

The cavity 21 inside the device 23, which is formed by the inner walls of the inlet part 1 and the outlet part 2, is divided into two chambers by the TCRF plates 3, 4, 5 and 6. The upstream, upper or first chamber 30 is formed by a wall 35 of the inlet section 1, a wall 36 of the inlet section 1 and an upper surface 37 of the TCRF plate 3. The downstream, lower or second chamber is formed by the wall 38 of the outlet section 2, the wall 39 of the outlet section 2 and the lower surface 43 of the TCRF plate 6. In the lower chamber, the lower chamber 29 is divided into two parts by the flow direction diverting assembly 10. A first portion of the lower chamber 29 is enclosed by the bottom surface 43 of the TCRF plate 6 and the upper surface 42 of the flow direction diversion assembly 10. A second portion of the lower chamber 29 is surrounded by a bottom surface 41 of the flow diverting assembly 10 and a surface 39 of the outlet portion 2.

Referring to fig. 3A and 3B, the flow direction diverting assembly is formed of a thin circular disk having radial filter assembly support ribs 12 on a first surface thereof, coaxial protrusions 31 on the outer periphery, and support feet 11 on a second surface thereof. The function of the filter assembly support ribs 12 is to cause the FSC product to flow radially along the flow direction turning assembly first surface. However, instead of support ribs 12, other components that cause radial flow may be used, such as a series of support feet or woven baffles. The function of the support foot 11 is to support the flow direction diverting assembly 10 above the wall 39 of the outlet portion 2. The action of the coaxial protrusion places the flow direction diversion assembly 10 within the lower chamber 29.

In fig. 4, the filter device 23 shown in fig. 1 and 2 is in operative combination with the inlet tube 17, the outlet tube 18, the feed bag 25 and the recovery bag 26. Preferably, the user's resultant assembly is a sterilized assembly, without the feeding bag 5, and the inlet end of the inlet tube 17 is sealed to maintain the sterility of the system. For filtration, an inlet tube 17 (fig. 2) connected to the central tube receptacle 15 of the inlet section 1 was connected to the tail tube of a feed bag 25 containing TCCSC product using a sterile docking device as is well known in the art. Inlet tube 17 is in fluid flow communication with upper chamber 30 through inlet 13. The outlet tube 18 connected to the receiving bag is connected to the outlet tube insertion hole 16 located at the center portion of the outlet portion 2. The outlet tube 18 is in fluid flow communication with the lower chamber 29 through the outlet 14.

The filter device 23 is suspended linearly. The TCCSC product enters the filter 23 through inlet 13 and exits the filter 23 through outlet 14. During filling of the TCCSC product with the filter device 23, all air in between before filtration begins is purged through the outlet pipe 18 into the receiving bag 26 before the FSC product begins to flow out of the filter device 23. This method ensures that little or no air remains in the TCRF boards 3, 4, 5 and 6. Thus, the entire exposed surface of the TCRF plate is used for filtering.

When filtering a TCCSC product, the user first closes the inlet tube near the feed bag 25 connection end with a tube clamp (not shown) and then forms a sterile connection between the inlet end of the inlet tube 17 and the feed bag 25 using sterile docking means well known in the art. A positive sterile connection is made between the inlet tube 17 and the short tube which is part of the feed bag 25. The resulting system is shown in fig. 4. The feeding bag 25 can be suspended by a suitable mechanism, such as a rod 28, by means of hooks 27. The retrieval bag 26 may also be suspended by the mechanism or may be placed on a surface such as a table top or the like.

Referring to fig. 1, 2 and 4, once the clamp (not shown) is opened, the TCCSC product will begin to flow from the feed bag 25, through the inlet tube 17, the inlet 13 and into the upper chamber 30. Air in the inlet tube 17 is forced to flow before the TCCSC product and into the upper chamber 30. The TCCSC product passes from the middle into the upper chamber 30, with the result that the TCCSC product fills the upper chamber 30 from the middle and then radially outward. This radial flow is indicated by arrows in fig. 1 and 2. Since the upper chamber 30 is filled from the middle and radially outwards, the TCRF plates 3, 4, 5 and 6 are also wetted from the middle radially outwards. As the upper chamber 30 is filled, air trapped in the upper chamber 30 will be forced out through the non-wetted parts of the TCRF boards 3, 4, 5 and 6, through the outlet 14, through the outlet tube 18 and into the receiving bag 26. The upper chamber 30 is sized according to the initial TCCSC product flow rate to ensure that all air that was originally in the upper chamber 30 is forced out through the TCRF plates 3, 4, 5 and 6. If the volume of the upper chamber 30 is too large relative to the initial TCCSC product flow rate, some air will remain in the upper chamber 30.

As mentioned above, the TCRF plates 3, 4, 5 and 6 are wetted radially outwards and all the air therein will be forced into the lower chamber 29, through the outlet 14, the outlet tube 18 into the receiving bag 26. Since the TCRF plates 3, 4, 5 and 6 are wetted radially outwards, the FSC product will first flow out from the middle of the TCRF plate 6 and then continue to flow out from the TCRF plate 6 in a radially outward manner. Thus, the first part of the lower chamber 29 is also filled radially outwards from the middle part. As the first part of the lower chamber 29 is filled, all air forced out through the TCRF plates 3, 4, 5 and 6 will be forced radially outwards through the first part of the lower chamber.

Once the first portion of the lower chamber 29 is filled with FSC product, the FSC product will flow radially inward into the second portion of the lower chamber 29, forcing air into the outlet, thereby expelling it downstream. Once the second portion of the lower chamber 29 is filled with FSC product, the outlet 14 and outlet tube 18 will then be filled, and ultimately the recovery bag 26. The flow along the flow diverting assembly is marked by arrows in fig. 2.

Due to the three-dimensional network structure of the TCRF plates 3, 4, 5 and 6, tumor cells will be retained in the filter device 23, while smaller stem cells and committed progenitor cells will flow through them and be recovered into the recovery bag 26. The stem and committed progenitor cells are between about 5 and 15 μm in diameter, whereas the tumor cells are between about 20 and 50 μm in diameter.

Preferably, the TCRF module reduces tumor cells by at least a factor of ten and recovers at least 30% of the stem cells, more preferably 50%. The flow rate of the stem cell product through the TCRF module is at least 50ml per hour. We have found that for good operation of the apparatus, the TCRF module has a gas permeability of at least about 26.8 cubic inches per minute (CFM) (750l/min), and more preferably about 118.5CFM (3.3X 10)³l/min). TCRF components, such as tumor cell depletion filter plates with higher gas permeability, can be made using cellulose or cellulose acetate with higher average surface area.

Since SCP collected is often cryopreserved and stored for later use, it is particularly noted that the TCRF module also traps all granulocytes in the contaminated stem cell product. This is advantageous because granulocytes will not survive and will lyse when filtered SCP is frozen, releasing their cellular contents into the supernatant, which may reduce the survival of the stem cell product after thawing. Therefore, it is preferred to remove the granulocytes prior to cryopreservation.

Examples

Preferred embodiments of the present invention will be described in more detail by the following examples, which are illustrative only and not limiting. Example 1

Model TCCSC products were prepared by 5: 1 mixing of blood mononuclear cells (BMNC) with adenocarcinoma or ductal carcinoma tumor cells (both expressed as tumor cells TC). The above composition was filtered according to the principles of the present invention using a 1.5mm thick cellulose or cellulose acetate-polyester composite TCRF plate with an effective pore size of 10 μm. Due to the three-dimensional network structure of the TCRF plate, the pore size varies between 5-150 μm. The FSC product was analyzed for BMNC and TC content. Wright-Giemsa stained cell centrifugation showed 25 to 60 fold higher recovery of BMNC than TC. Example 2

A model TCCSC product was prepared in which the ratio of BMNC to TC was 50: 1. To aid in accurate reading, TCs were pre-labeled with a fluorescent film dye. The TCCSC product was filtered using the same cellulose acetate TCRF plate as in example 1 and analyzed for FSC product and the TC concentration was not detectable, indicating that the device had at least a 30-fold preferential rejection capacity for TC.

Furthermore, since stem cells are smaller than BMNC, recovery of granulocytes/macrophages and erythroid progenitors after filtration must be greater than recovery of BMNC. Indeed, filtration of BMNC without TC showed that the concentration of hematopoietic stem cells in the recovered product was almost 10 times higher than the unfiltered product; indicating that about 80% of fully active hematopoietic precursors can be recovered at 30-fold lower TC concentrations compared to methods currently used that rely on positive selection principles for TC depletion.

Table 1 shows the results of examples 1 and 2

TABLE 1

Initial ratio BMNC to TC	Log reduction of TC	Log reduction of BMNC
			No TC	NA	1.1
5∶1	2.2,2.8	0.8,1.0
			50∶1	＞1.5	0.6

Example 3

A model TCCSC product was prepared in which the ratio of BMNC to TC was 10: 1. Several filtration experiments of the composition were performed using various TCRF plate media. The percentage of total cell recovery, the ratio of BMNC: TC, the type of cells recovered and the fold increase in the concentration of CFU-GM (granulocyte/macrophage precursor) cells, specific hematopoietic cell colony forming cells, were analyzed after filtration.

Tables 2 and 2A list the results of these filtration experiments ("a" - "V"). As can be seen, the best results were obtained in experiment "U", where two TCRF boards were used, the medium of which was StemShell-3 (SC-3). In this case, 14.9% of total BMNC was recovered and the CFU-GM concentration increased 8.7-fold.

TABLE 2

Experiment of	Breathable CU FT/MIN	TCRF board medium	Number of plates used	% Total cell yield
					A	3-4	LEUKONETTM	2	1.06
B	3-4	LEUKONETTM	4	0.40
					C	3-4	MILLIPORE4528-41	2	1.90
D	3-4	MILLIPORE4528-41	4	1.50
					E	3-4	MILLIPORE4528-41	2	3.00
F	3-4	MILLIPORE4528-41	4	2.45
					G	31	LYDALL LBTM170-54-D	2	20.0
H	31	LYDALL LBTM170-54-D	4	5.20
					I	3-4	BIOCMPTBL 2MG/UL	2	1.20
J	3-4	BIOCMPTBL 2MG/UL	4	1.00
					K	3-4	BIOCMPTBL 5MG/UL	2	2.20
L	3-4	BIOCMPTBL 5MG/UL	4	2.00
					M	3-4	BIOCMPTBL 10MG/UL	2	2.00
N	3-4	BIOCMPTBL 10MG/UL	4	1.00
					O	31	LYDALL LBTM170-64-D	2	4.60
P	31	LYDALL LBTM170-64-D	4	0.40
					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

TABLE 2A

Experiment of	The ratio of BMNC to TC before filtration	The ratio of BMNC to TC after filtration	Cell types after filtration	CFU-GM fold increase after filtration
					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	MRBCLMPHCTMCRPHG	1
H	10∶1	＞100∶1	MRBCLMPHCTMCRPHG	1
					I	10∶1	＞100∶1	MRBC	---
J	NA	NA	MRBC	---
					K	NA	NA	MRBC	---
L	NA	NA	MRBC	---
					M	NA	NA	MRBCLMPHCTMCRPHG	0
N	NA	NA	MRBCLMPHCTMCRPHG	0
					O	10∶1	＞100∶1	MRBCLMPHCTMCRPHG	0
P	10∶1	＞100∶1	MRBCLMPHCTMCRPHG	0
					Q	NA	NA	MRBCLMPHCTMCRPHG	2.4
R	NA	NA	MRBCLMPHCTMCRPHG	1.7
					S	NA	NA	MRBCLMPHCTMCRPHG	4.0
T	NA	NA	MRBCLMPHCTMCRPHG	3.0
					U	NA	NA	MRBCLMPHCTMCRPHG	8.7
V	NA	NA	MRBCLMPHCTMCRPHG	3.7

NA = TC grown too slowly to provide the appropriate number of cells needed for the experiment MRBC = mature red blood cells LMPHCT = lymphocytes MCRPHG = macrophage medium = TCRF plates of various manufacturers. Leukonet^TMAnd the Lydall panel was made of the same filter material from Lydall, Inc (Manchester CT). The material included polyester fiber, cellulosic fine fiber and NW-1845, an acrylic binder (Rohm and Haas). The average titer of the polyester fibers on each filter plate was 0.5mm, with the exception of SC-3, which had an average titer of 1.5 mm. The surface area of the cellulose fine fibers is about 20m²/g。

Varying the percentage of polyester fiber in the material produced a plurality of Lydall panels. Table 2 shows this percentage as a function of the air permeability determined by the Frasier method, using a head pressure of 12.7kg/m²。

Millipore TCRF plate is a Leukonet^TMTCRF plate, wherein,A method of coating a hydrophilic polymer formed by crosslinking hydroxyalkyl acrylate thereon was disclosed in U.S. Pat. No. 4,618,533 issued on 21/10/1986. Biocmptbl TCRF plates were Leukonet coated with different concentrations of phosphorylcholine (Biocompatibles, Ltd., Middlesex, England)^TMA TCRF board.

While the present disclosure is not intended to define a TCRF plate as the TCRF component, it is also not intended to define the number of TCRF plates employed for a single filtration, while optimal tumor cell retention and stem cell recovery have been obtained using two TCRF plates.

The invention has been described with reference to specific embodiments described in detail. It should be recognized, however, that the illustrated embodiments are illustrative only and that the invention is not limited thereto. Modifications and variations within the spirit and scope of the following invention will be readily apparent to those skilled in the art, as they are recognized.

Claims (22)

1. A method of removing tumor cells from a tumor cell contaminated stem cell product comprising the steps of:

(a) preparing an in-line filtration device comprising:

a housing having an inlet and an outlet therein; and

a tumor cell-depleting filter assembly disposed in said housing between said inlet and said outlet for filtering tumor cell-contaminated stem cell product flowing into said filter device through said inlet, said tumor-depleting filter assembly dividing said housing into a first chamber and a second chamber;

(b) preparing a stem cell product contaminated by tumor cells;

(c) passing the tumor cell-contaminated stem cell product through the filter device, wherein tumor cells are retained by a tumor cell-depleting filter assembly in the filter device, and the stem cell product passes through the tumor cell-depleting filter assembly and out of the filter device; then the

(d) Recovering the tumor cell-depleted stem cell product.

2. The method of claim 1, wherein the filter device further comprises a component disposed within the filter device that causes gas to exit the filter device through the outlet.

3. The method of claim 1, wherein the filter element reduces tumor cells by at least a factor of ten and recovers at least 30% of the stem cells in the tumor cell-depleted stem cell product.

4. The method of claim 3, wherein the filter assembly provides a stem cell product flow rate of at least 50ml per hour.

5. The method of claim 3, wherein the filter assembly comprises one or more tumor cell depletion filter plates.

6. The method of claim 3, wherein the filter assembly has an air permeability of at least about 750 l/min.

7. The method of claim 6, wherein the filter assembly has an air permeability of at least about 3.3 x 10³l/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 myelogenous 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 a tumor cell selected from a ductal carcinoma cell or an adenocarcinoma cell.

10. A tumor cell depletion filter module suitable for use in an in-line filtration device, the tumor cell depletion filter module comprising a mechanically stable matrix that reduces tumor cells by at least a factor of ten and recovers at least 30% of the stem cells in a filtered stem cell product.

11. The filter assembly of claim 10, further characterized in that the filter assembly provides a stem cell product flow rate of at least 50ml per hour.

12. The filter assembly of claim 10, further characterized in that the filter assembly comprises one or more tumor cell-depleting filter panels having a three-dimensional network structure comprised of a plurality of fibers and fibrils.

13. The filter assembly of claim 12, wherein the fibers are comprised of polyester having a denier of about 1.5mm and the fine fibers are comprised of cellulose.

14. A filter assembly as recited in claim 10, wherein said filter assembly has an air permeability of at least about 750 l/min.

15. The method of claim 10A filter assembly, further characterized in that said filter assembly has an air permeability of at least about 3.3 x 10³l/min。

16. A filter device suitable for use as a filter for removing tumor cells from a tumor cell contaminated stem cell product, the filter device comprising:

a housing having an inlet and an outlet therein; and

a tumor cell depleting filter assembly disposed in said housing between said inlet and said outlet for filtering tumor cell contaminated stem cell product flowing into said housing through said inlet, said filter assembly dividing said housing into a first chamber and a second chamber.

17. The filter apparatus of claim 16 further comprising a component disposed in said housing for causing gas to exit said housing through said outlet.

18. The filtration device of claim 16, wherein the filtration assembly reduces tumor cells by at least a factor of ten and recovers at least 30% of the stem cells in the filtered stem cell product.

19. The filtration device of claim 18, wherein the filtration assembly provides a stem cell product flow rate of at least 50ml per hour.

20. The filter device of claim 18, wherein the filter assembly comprises one or more tumor cell depletion filter pads.

21. The filter apparatus of claim 18, wherein the filter assembly has an air permeability of at least about 750 l/min.

22. The filter apparatus of claim 21 wherein said filter assembly has an air permeability of at least about 3.3 x 10³l/min。