WO1998038940A1

WO1998038940A1 - Method and apparatus for altering the osmotic pressure of cryopreserved white stem cells

Info

Publication number: WO1998038940A1
Application number: PCT/US1998/003936
Authority: WO
Inventors: Philip H. Coelho; Pablo Rubinstein; Cladd E. Stevens
Original assignee: Thermogenesis Corp.; New York Blood Center, Inc.
Priority date: 1997-03-07
Filing date: 1998-03-06
Publication date: 1998-09-11
Also published as: AU6538898A; EP0967928A4; CA2253872A1; JP2001518079A; EP0967928A1

Abstract

A method, apparatus and product formed from cryoprotected white stem cells suitable for transfusion. The osmotic pressure of the white stem cells is reduced by controlled dilution of cryopreservative from the white stem cells.

Description

Method and Apparatus for Altering the Osmotic Pressure of Cryopreserved White Stem Cells

Technical Field The following invention relates generally to instrumentalities which take cryopreserved white stem cells from their frozen condition to a constituency which is compatible with transfusion without compromising the vitality of the white stem cells.

Background Art Transfusions involving white stem cells can provide profound therapeutic benefits in certain situations. Ongoing research by the present inventors has continued to shed light on phenomena which can appreciably alter the vitality of the white stem cells, thereby improving efficacy.

One issue involves the white stem cells themselves, their procurement, concentration and preservation for subsequent use. Details appurtenant thereto are reflected in the present inventors' co-pending application Serial No. 08/349,747, filed December 5, 1994.

The following citations reflect activity by third parties known to applicants and of record in the above-referenced pending application. These citations are included to discharge applicants' acknowledged duty to disclose relevant prior art. It is submitted, however, that none of these citations teach signally nor render obvious when considered in any conceivable combination the next of instant invention set forth hereinafter.

PATENT NO. ISSUE DATE INVENTOR 2,702,034 February 15, 1955 Walter

3,187,750 June 8, 1965 Tenczar, Jr,

4,004,975 January 25, 1977 Lionetti, et al.

4,098,456 July 4, 1978 Bayham

4,332,122 June 1, 1982 Williams 4,343,793 August 10, 1982 Wissler

4,744,907 May 17, 1988 Klimchak

4,887,411 December 19, 1989 Rondeau, et al.

4,902,287 February 20, 1990 Carmen, et al.

4,937,194 June 26, 1990 Pattillo, et al. 4,969,882 November 13, 1990 Carmen, et al.

5,004,681 April 2, 1991 Boyse, et al.

5,023,043 June 11, 1991 Kotzlowski, et al.

5,101,017 March 31, 1992 Rubinstein, et al. 5,104,788 April 14, 1992 Carmen, et al. 5,154,716 October 13, 1992 Bauman, et al. 5,192,553 March 9, 1993 Boyse 5,316,681 May 31, 1994 Serres 5,397,479 March 14, 1995 Kass, et al.

FOREIGN PATENT DOCUMENTS

PATENT NO. ISSUE DATE INVENTOR WO 91/02202 February 21, 1991 Richard WO 92/16800 October 1, 1992 Richard WO 93/03891 March 4, 1993 Knippscheer

OTHER PRIOR ART (Including Author, Title. Date. Pertinent Pages, Etc.)

Korbling, et al, Transfusion "Description of a Closed Plastic Bag System for the Collection and Cryopreservation of Leukaphersis-Derived Blood Mononuclear Leukocytes and CFUc from Human Donors", Volume 20, Number 3, pages 293 - 300, May-June 1960.

Rubinstein, et al., Stored Placental Blood For Unrelated Bone Marrow Reconstitution, May 1993, 27 Pages.

Rubinstein, et al., Processing And Cryopreservation Of Placental /Umbilical Cord Blood For Unrelated Bone Marrow Reconstitution, October 1995, 4 Pages.

The other prior art listed above, catalog the prior art of which the applicants are aware and are tendered to discharge applicants' acknowledged duty to disclose prior art. These references diverge even starkly from the instant invention specifically distinguished hereafter.

Disclosure Of Invention

The instant invention takes the cryoprotected white blood cells of the previous pending application and conditions the unfrozen white stem cells for subsequent transfusion. In the above-referenced patent application, the white stem cells will have been modified with a starch, such as HES, and cryopreservatives including DMSO and Dextran. The DMSO is understood to pass through the walls of the white stem cells and displace water therein, raising the osmotic pressure of the white stem cells. Concurrently, the Dextran further insulates the white stem cells by their affinity to the outer periphery of the cell, surrounding the cell and further displacing the water. It is the sequestration of the water from the white stem cells which protect the cells from the sharp crystalline nature of the water as it freezes and protects the white stem cells by minimizing the crystalline water's affinity to puncture the cells. However, prior to transfusion, the osmotic pressure within the white stem cells must be returned to a lower pressure compatible with ambient conditions within the recipient of the white stem cells in order to enhance the vitality of the cells. Otherwise, the white stem cell's vitality would be compromised by the pressure differential upon transfusion to the recipient. In addition the DMSO should be diluted and a majority of the DMSO removed prior to cell transfusion. In order to achieve pressure normalization, the white stem cells are first thawed and then transferred to an aseptic transfusion bag having air and a volumetric capacity approximately eight times greater than the capacity of the storage container originally housing the frozen white stem cells. Assume that the white stem cell freezing bag initially held 25 milliliters of product. Once transferred to the transfusion bag, a volume of sterile saline preferably six (6) times greater (e.g., in the present case, 150 milliliters of sterile saline) is admitted into the bag from a diluent bag. Preferably, this dose of sterile saline is admitted at a slow rate, i.e. a drop at a time, via a drip reducer preferably over a four to ten minute span. This slow drip rate allows the osmotic pressure to be reduced, gently, asymptotically approaching the recipient's osmotic pressure. Thus, after the volume of sterile saline has been mixed into the transfusion bag, the white stem cells will have been pressure normalized. Further, the DMSO is caused to go into solution with excess saline. The white stem cells are thereafter sequestered from the DMSO/saline solution. The transfusion bag can now administer the white stem cells by a transfusion

IV coupling. The effect of the dilution by using sterile saline involves the gentle reduction of osmotic pressure in the white stem cells from an elevated pressure (compared to ambient osmotic pressure of a transfusion recipient) to one which is compatible with the recipient's osmotic pressure. This assures that the transfusion will not initiate white stem cell fracture upon transfusion which would alter the treatment's efficacy. Also dilution of the DMSO, suspension of the DMSO and removal of the DMSO from the white stem cells reduces the likelihood of DMSO induced side effects.

Industrial Applicability The industrial applicabillity of this invention shall be demonstrated through discussion of the following objects of the invention.

Accordingly, it is the primary object of the present invention to provide a new and novel method and apparatus for preserving the vitality of cryopreserved white stem cells. It is a further object of the present invention to provide an instrumentality which delivers the white stem cell with an osmotic pressure compatible with the osmotic pressure of an intended recipient while simultaneously diluting the cryopreservative with a diluent, such as saline. The DMSO is sent into solution with the saline.

It is a further object of the present invention to provide an instrumentality as set forth above which can achieve reliability and repeatability. It is a further object of the present invention to provide an instrumentality as set forth above which is extremely safe to use.

Viewed from a first vantage point it is an object of the present invention to provide a method for normalizing the osmotic pressure of frozen white stem cells having a cryopreservative for administration to a patient, the steps including: thawing the white stem cells, diluting the cryopreservative, and reducing the osmotic pressure of the white stem cells prior to administration to the patient.

Viewed from a second vantage point it is an object of the present invention to provide an aseptic apparatus for taking a white stem cell bag having frozen cyroprotected white stem cells and conditioning the white stem cells prior to therapeutic administration to a patient, comprising, in combination: a first aseptic bag having means for accessing an interior of the white stem cell bag to receive white stem cells therefrom, means for reducing the osmotic pressure of the cryoprotected white stem cells, and means for transfusing the pressure corrected white stem cells. Viewed from a third vantage point it is an object of the present invention to provide a previously frozen white stem cell product having a viable white stem cell concentration of at least 80% and an osmotic pressure compatible with a recipient.

These and other objects will be made manifest when considering the following detailed specification when taken in conjunction with the appended drawing figures.

Brief Description Of Drawings

Figure 1 is a perspective view of the apparatus of the present invention. Figure 2 is a flow chart of the methodology associated with the bags of figure 1. Figure 3 is a schematic depiction of the white stem cell being diluted.

Best Mode(s) For Carrying Out The Invention

Referring to the drawings, wherein like reference numerals denote like parts throughout the various drawing figures, reference numeral 10 is directed to the bag set according to the present invention. In its essence, the bag set 10 includes a transfusion bag 20 which receives the white stem cells including cryopreservative from a white stem cell bag B via spikes 30. The transfusion bag 20 receives dilution, preferably in the form of sterile saline from a diluent bag 40. After the white stem cells have been diluted with saline, the solution containing the cryopreservative is moved from the transfusion bag 20 to the diluent bag 40 and the white stem cells remaining in the transfusion bag 20 are transfused via transfusion tube 50.

More specifically, the white stem cell bag B includes a pair of spaced parallel portals P. These portals P are accessed by a pair of spaced parallel spikes 2 spaced from each other a distance comparable to the portals P, once the caps C from the portals P have been removed. Similarly, these spikes 2 are provided with covers 4 which must be removed in order to access the spikes which are protected therewithin. Slightly upstream from the spikes 2 are spacers 6, which provide a positive stop abutment to limit the degree of incursion of the spikes 2 within the bag B. Each of the spikes has a hollow interior allowing the white stem cells and cryoprotectant, when thawed, to pass within tubes 8 that in turn communicate via a manifold 12 to a conduit 14 leading into a channel 16 and thence into the transfusion bag 20. Because the bag B is substantially full, it is preferred that the transfusion bag 20 include aseptic air A therewithin to provide ease of transfer either by squeezing air from the transfusion bag 20 into bag B and /or squeezing the contents from bag B to thwart vapor lock. In other words, air can be allowed into the bag B whereupon the cryoprotected white stem cells can be released from the bag

B into the transfusion bag 20.

Once the cryoprotected white stem cells have been introduced into the transfusion bag 20, the conduit 14 is occluded with a clamp 18a. The conduit 14 communicates with the channel 16 via a Y-adapter 22a. The Y-adapter 22a also allows access to the interior of the transfusion bag 20 via a passageway 24. The passageway 24 is protected by another clamp 18b. The passageway 24 communicates with a T-adapter 26 which is protected on a left side by a clamp 18c and on the right side by clamp 18d. Clamp 18d controls access between the transfusion bag 20 and the diluent bag 40 via an access 28. Interposed along access 28 is a drip reducer 32. This drip reducer 32 controls the rate at which fluid proceeds through access 28 from the diluent bag 40 to the transfusion bag 20 once the appropriate clamps 18 have been manipulated.

Preferably, the diluent bag 40 is provided with gradations 34 indicating volume. The diluent bag 40 includes a holder 36 as does the transfusion bag 20 to allow each bag to be supported in an elevated position. The diluent bag 40 also is preferably provided with a spike 2, spike cover 4 and spacer 6 as shown. In addition, the diluent bag 40 is similarly provided with a port P comparable to the ports on the white stem cell bag B. Saline is initially transferred from the diluent bag 40 to the transfusion bag 20 at a controlled rate via the drip reducer 32 by opening a clamp 18g (between reducer 32 and diluent bag 40), opening clamps 18d and 18b and closing all other clamps. Typically, the process can take four to ten minutes. Preferably, the dilution is equal to the six times the volume of the contents of the white stem cell bag B and proceeds at a slow rate. For example, assume 25 milliliters of cryoprotected white stem cells have been moved into the transfusion bag 20. 150 milliliters of saline are administered from the diluent bag 40 at a slow rate. The saline and the cryoprotected white stem cells are mixed. This is done at a slow rate because this reaction is exothermic and achieves the object to preserve the vitality of the white stem cells.

After gentle mixing in the transfusion bag 20, the osmotic pressure of the white stem cells has been normalized to approximately a pressure which the white stem cells will experience when transfused into a patient. Further, the cryoprotectant will have passed into solution with the excess diluent, saline. Next, the cryoprotectant saline solution in the transfusion bag 20 is delivered to the diluent bag 40 through a pipeline 60. Pipeline 60 is accessed via another "Y" adapter 22b located on channel 16 and merges into access 28 between the drip reducer 32 and diluent bag 40 via another Y-adapter 22c. A low micron filter 62, interposed in pipeline 60, allows only the cryoprotectant saline solution therebeyond. Filter 62 is optional and instead (or in combination therewith) the cryoprotectant solution can be decanted or expressed off the white stem cells. Centrifuging the transfusion bag 20 can assist in driving the larger, heavier white stem cells to the bottom of bag 20. The pipeline 60 is protected at both ends by clamps 18, one clamp 18e nearer transfusion bag 20 and clamp 18f nearer diluent bag 40. The drip reducer 32 is also cut off from pipeline 60 via a clamp 18g located on access 28 between the pipeline 60 and the reducer 32. Clamps 18a and 18b are also closed when removing fluid from transfusion bag 20 to diluent bag 40 through filter 62.

A saline flush from source 80 may next wash out bag B and then be introduced into transfusion bag 20 as was done with the initial contents of bag B (as described above). Alternatively, the saline flush of bag B can be performed before removing the cryoprotectant /saline solution from transfusion bag 20 to diluent bag 40 should it appear desireable to "wash" the residual cryoprotectant from the bag B flush. Next, the clamp 18b (controlling access to the passageway 24) is opened and all other clamps (18a, 18e) along passageway 24 are closed. The clamp 18d (to the right of T 26) is closed; the clamp 18c to the left of T 26 is opened (i.e. along transfusion tube 50). The transfusion bag 20 is hung on an IV pole via its hook 36 and a transfusion connection 52 of transfusion tube 50 is connected to a conventional IV for administration to the recipient.

Moreover, having thus described the invention, it should be apparent that numerous structural modifications and adaptations may be resorted to without departing from the scope and fair meaning of the instant invention as set forth hereinabove and as described hereinbelow by the claims.

Claims

I Claim:

Claim 1 - A method of normalizing the osmotic pressure of frozen white stem cells having a cryopreservative for administration to a patient, the steps including: thawing the white stem cells, diluting the cryopreservative, and reducing the osmotic pressure of the white stem cells prior to administration to the patient. Claim 2 - An aseptic apparatus for taking a white cell bag having frozen cyroprotected white stem cells and conditioning the white stem cells prior to therapeutic administration to a patient, comprising, in combination: a first aseptic bag having means for accessing an interior of the white cell bag to receive white stem cells therefrom, means for reducing the osmotic pressure of the cryoprotected white stem cells, and means for transfusing the pressure corrected white stem cells.

Claim 3 - A previously frozen white stem cell product having a viable white stem cell concentration of at least 80% and an osmotic pressure compatible with a recipient. Claim 4 - An aseptic cryopreserved white stem cell transfer and separation apparatus, comprising, in combination: an aseptic bag; means for transferring cryopreserved white stem cells from a remote source to an interior of said bag; and means for conditioning said stem cells for subsequent transfusion into a recipient.

Claim 5 - The aseptic apparatus of claim 4 wherein said transfer means includes a pressure source operatively coupled to said remote source for pressurably forcing the white stem cells from said remote source to said aseptic bag. Claim 6 - The aseptic apparatus of claim 5 wherein said pressure source includes air within said interior of said aseptic bag.

Claim 7 - The aseptic apparatus of claim 6 wherein said transfer means further includes a plurality of conduits and channels.

Claim 8 - The aseptic apparatus of claim 7 wherein said conditioning means includes a stem cell osmotic pressure normalization means. Claim 9 - The aseptic apparatus of claim 8 wherein said pressure normalization means includes a dilutent source operatively coupled to said aseptic bag via said plurality of conduits and channels.

Claim 10 - The aseptic apparatus of claim 9 wherein said pressure normalization means further includes flow limiting means operatively coupled between said dilutent source and said aseptic bag.

Claim 11 - The aseptic apparatus of claim 10 wherein said flow limiting means includes a drip reducer.

Claim 12 - The aseptic apparatus of claim 11 wherein said dilutent source includes saline contained within an aseptic container.

Claim 13 - The aseptic apparatus of claim 12 wherein said conditioning means further includes cryopreserve segregation means for segregating the cryopreserve form the white stem cells.

Claim 14 - The aseptic apparatus of claim 13 wherein said cryopreserve segregation means includes a filter said filter coupled between said dilutent source and said aseptic bag.

Claim 15 - The aseptic apparatus of claim 14 wherein said cryopreserve segregation means further includes centrifuging means operatively coupleable to said aseptic bag for driving the larger heavier white stem cells to the bottom of said aseptic bag.

Claim 16 - The aseptic apparatus of claim 15 wherein said plurality of conduits and channels includes a network comprising, in combination: at least one conduit coupled between said remote source and said aseptic bag; a drip conduit removably coupled between said dilutent source and said aseptic bag; a segregation conduit removably coupled between said aseptic bag and said filter, and further between said filter and said dilutent source; and a transfusion conduit removably coupled between said aseptic bag and the recipient.

Claim 17 - The aseptic apparatus of claim 16 wherein said transfer, drip, segregation, and transfusion conduits include valves at extremities thereof to control fluid flow individually therein.

Claim 18 - The aseptic apparatus of claim 2 wherein said accessing means includes at least one conduit removably coupled between said aseptic bag and the white cell bag, and pressure means for forcing the white cells from the white cell bag to said aseptic bag. Claim 19 - The aseptic apparatus of claim 18 wherein said pressure means includes air within said aseptic bag.

Claim 20 - The aseptic apparatus of claim 19 wherein osmotic pressure reduction means includes a dilutent source operatively coupled to said aseptic bag via a fluid flow reducer coupled between said aseptic bag and dilutent source.

Claim 21 - The aseptic apparatus of claim 20 wherein said transfusion means includes a conduit coupled to said aseptic bag at one end and coupleable to a recipient at another end.

Claim 22 - The aseptic apparatus of claim 21 further comprising means for segregating cryoprotectant from white stem cells.

Claim 23 - The aseptic apparatus of claim 22 wherein said segregation means includes a filter transposed between said dilutent source and said aseptic bag.

Claim 24 - The aseptic apparatus of claim 23 further comprising means for flushing white stem cells from the white cell bag and into said aseptic bag. Claim 25 -The aseptic apparatus of claim 24 wherein said flushing means includes a saline source coupleable to the white cell bag.

Claim 26 - The aseptic apparatus of claim 25 wherein said dilutent is saline.

Claim 27 - The aseptic apparatus of claim 26 wherein the cryoprotectant includes DMSO. Claim 28 - The aseptic apparatus of claim 26 wherein the cryoprotectant further includes a starch and dextran.