Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
1998-07-17
2001-03-06
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C210S781000, C210S782000, C435S002000, C435S243000, C435S308100
Reexamination Certificate
active
06197579
ABSTRACT:
FIELD OF THE INVENTION
The invention is concerned with centrifugable devices that are suitable for washing isolated cells. The invention is also concerned with a method of washing certain rare cell populations using such containers.
BACKGROUND OF THE INVENTION
Advances in cell separation technology have spawned therapeutic methods in which a subject's cells can be removed from the bloodstream or bone marrow and fractionated to provide specific cell types for re-introduction into the subject or of a recipient patient. For example, U.S. Pat. No. 5,840,502, filed Aug. 31, 1994, describes methods for enriching cell fractions and indications for use of such fractions.
In enriching a cell fraction from a cell suspension, it is many times desirable to add reagents, such as cell-specific antibodies or buffering agents, to the cell suspension as part of the fractionation procedure. Such reagents must be removed before re-introduction of the cells into a patient. Alternatively or in addition, it is many times desirable to change the ionic conditions of the cells prior to use of the cells, such as in the therapeutic indications referred to above.
As such cell fractionation procedures become routine in the clinical setting, it is desirable that handling and manipulation of the cells be minimized, so that cells can be processed in a minimum amount of time and with a minimum amount of exposure to potential contamination.
Typically, isolated cells are washed by either resuspending the cells in the same centrifuge tube or bag in which they were originally centrifuged or by transferring the cells to a different centrifugable container. Cells are resuspended in the wash buffer and re-centrifuged at relatively low centrifugal forces (approx. 1000×g). The cells form a soft pellet from which the wash supernatant must be removed prior to subsequent washings or resuspension in the final buffer.
Removal of supernatant can be effected either by decantation or by gentle aspiration. Decantation, while a relatively fast operation, often results in cell loss, differentially depleting those cells having relatively low specific gravities and which sediment on the top of the pellet. For this reason, decantation has generally not been considered to be a reliable means for removing supernatants from cells, particularly when such cells have relatively low specific gravities.
Aspiration, on the other hand, is labor-intensive, and unless careful attention is given to each individual tube, may also selectively result in loss of “lighter” cells from the pellet into the discarded washing solution. In addition, aspiration requires introduction of a probe into the cell container. This may also introduce contamination into the container.
One attempt to solve the foregoing problems is found in U.S. Pat. No. 5,047,004 (Wells) which describes an automatic decanting centrifuge in which swinging buckets are locked in an extended angle following centrifugation, to effect gravity decantation of the fluid therein. This system, while providing relative ease and automaticity to the decantation process, necessarily exposes the cells to contamination, by its open top design. Moreover, there is no provision for ensuring that the more slowly sedimenting “light” cells are retained in the pellet.
U.S. Pat. No. 5,474,687 describes a method and specialized tube for enriching an exemplified fraction of rare cells, CD34
+
hematopoietic progenitor cells, in a single-step density gradient, by selectively collecting the “light” cells that migrate to the cell solution-density gradient interface. However, these cells must be pelleted and washed prior to use. Such pelleting and washing is typically carried out at relatively low speeds (500-1000×g) in preparative centrifuge tubes that are available commercially. Using such conventional washing methods, it has been found that the CD34
+
cells are differentially lost during the washing procedure, since they are “light” cells that tend to sediment to the top of the pellet formed during the washing process.
The present invention provides a cell washing device that overcomes the problems just described. The device includes a tube with a sealable cap or lid that provides for sterile transfer and handling of cells. Specifically, cells or liquid medium can be added to the tube by means of a sterile port which traverses the cap. In addition, according to an important feature of the invention, wash supernatant can be decanted from the cell pellet through an upper port without disturbing the pellet, obviating the need for careful supernatant removal procedures. Moreover, the tube design is such that even “lighter” cells present in the upper portion of the cell pellet are retained during the decantation process.
This tube and process provide the advantages of (i) a closed system for sterile manipulation of transfer of materials into and out of the tube, (ii) a design that allows for thorough decantation of the cell supernatant by inversion of the tube, without appreciable or differential loss of the cells at the top of the pellet. This latter feature of the invention facilitates high yield recovery of rare cells that might otherwise be lost or at least severely depleted during the washing process.
These and other features of the invention are described in the sections which follow.
SUMMARY OF THE INVENTION
In one aspect, the invention includes a cell washing device. According to an important feature of the invention, device is designed and constructed to allow for decantation by inversion of the supernatant from a cell pellet, without appreciable loss of cells from the pellet, and, importantly, without selective loss of the cells present in the upper portion of the cell pellet.
In one embodiment, the cell washing device includes an elongate centrifuge tube having generally cylindrical side walls and a conical bottom. According to an important feature of the invention, the conical bottom forms an apical angle of between about 50 and about 90°. The device also includes a lid sealed to the top of the tube, and the lid includes at least two access ports positioned and constructed for flow of liquid or gas therethrough.
In a particular embodiment of the invention, the access ports include (i) a liquid passage port adapted for sterile passage of liquid into and out of the tube, and (ii) an air vent capable of providing filtered air to the interior of the tube. In one embodiment, the device includes a ridge encircling the inlet port and said air vent.
In another embodiment, the lid of the cell washing device includes a concave lower portion adapted to funnel liquid from the tube into the liquid passage port when the tube is held in an inverted position. The liquid passage port may include a “LUER-LOK” connector. According to a related embodiment, the liquid passage port may communicate with a conduit tube which extends into the tube. A third port may be added to the lid of the device. In still another embodiment, this additional port is adapted to serve as an air vent for supporting culture of cells in the device, where the device also includes cell culture medium. The device may also include a support adapted to maintain the tube in an upright position.
In further related embodiments, the lower inner portion of the tube of the device is designed and adapted to promote retention of pellet in the lower portion of the tube. Various examples of retention means are illustrated. Such means include, but are not limited to, texturing on the inner surface walls of the lower portion of the tube, the presence of ridges on or grooves in the lower portion, the presence of fins projecting from the sides toward the center of the tube, the presence of longitudinal dividers in the lower portion of the tube, combinations of the foregoing, and the like.
In a related aspect, the invention includes a method of removing unwanted media from an isolated cell fraction. Typically, the cell fraction is a rare cell fraction, that is a fraction of cells that constitutes less than about 1% of the initial cell sus
Hasan Shirin W.
Van Vlasselaer Peter
Dendreon Corporation
Frazier Jeffery D.
Iota Pi Law Group
Lankford , Jr. Leon B.
LandOfFree
Cell washing device and method does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Cell washing device and method, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Cell washing device and method will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2517721