CA2122140A1 - Method for freezing engrafting cells - Google Patents

Abstract

The present invention provides a method for preparing engrafting cells for future use, comprising the steps of (a) purifying engrafting cells; (b) concentrating the purified engrafting cells; (c) resuspending the concentrated cells to a concentration of about 10 x 106 to 40 x 106 cells/ml in a solution that substantially maintains cell viability during freezing; and (d) freezing the resuspended cells under a first set of conditions which substantially maintains cell viability.

Description

Wo 93/0774~ Pcr/USs2/09023 ., ~ .
o P~ipti~?n METHOD FOR FREEZING ENGRAFIING CELLS

Cro$s-Referen~e to Relat~d ~j~jQn This application is a continuadon~ part of pending U.S.
Application Serial No. 07/513,543, which was filed April 23, 1990.
T~chnical Fiçld The present invention relates generally ts methods for freezing cells and, more specihcally, to methods for f~eezing engrafting cells.

15 ~. 0~
Bone marrow transplantation (Bl~I~ has emerged as the t}lerapy of choice for patients with certain oncological or hematological diseases (Kamani et ~, "Bone marrow transplantation. Problems and prospects." M~ Cl~ North Amer. 68:657-674, 1984). For cancer therapy, the dos~ge of sonventio~al grtotoxic 20 drugs and radiotherapy is limited because of to~aci~ to bone marrow stem cells.
To overcome this difflculty, patients have been treated with high dose chem~ or radiotherapy followed by allogenic bone marrDw transplantation (BM'I~ as a resclle. Although allogenic BMT bas markedly improved the survival of paffents unth aplastic anemia, acute leukemia, and severe immunodeficiency, many 25 difficulties have limited its clinical application. First, most patients do no~ have a histocompatible sibling donor and the feasl~ility of such transplantations has generally depended on the availability of a suitable donor. Second, the recipients should be treated witb apretr~nsplant immunosuppression. ~hird, allogenic BMT
can be associated with severe complications and morbidi~r caused by graft vs. host 30 disease and infection. .
Compared to allogenic BMl, autologous bone marrow tr~Dsplalltation (ABI~I~ has significant advantages. Ie ha~ been used in combination witb intensive chemotberapy to treat patients wfth various solid tumors wbere there is no involvement with bone marrow, including ~or example 35 malignallt lymp~omas, melanomas, and carcinomas of the lung and breast AB~T itself does not act directly sn tumor cells~ but can facilitate inteDsive ~emothe~py and ra~}iotherapy by -re~oDstitution of immunological and wO 93~Q7745 Pcr/u~92/09023 hematopoietic activities (see Gorin et al. "Chemotherapy and autologous bone marrow transplantation in acute leukemias, malignant lymphomas and solid t~mors," Eur. J. Cancer 17:557-568, 1981). Recently, ABMT has been commonly used to treat patients with leukemia, including acute lymphocytic alld S nonlympho~ytic leukemia, and chronic granulocytic leukemia in the ~lastic phase, as well as patients with lymphoma and breast cancer.
Due to the benefits assoc~ated with chemotherapeutic or irradiation therapies, followed by bone marrow reconstitu~on, preservation of marrow has received considerable scientific attention. However, in order to obtain the 10 benefits of ABMT, the marrow must be stored for a period of time to allow chemotherapy or radiation ~reatment of the paffent. Early work on storing marrow showed ~hat storage at room temperature or 4C led to a rapid loss of theengrafting cells. l'hus a delay in reiDfusion of the ma~row of more than severaldays could lead to failure of the marrow to re-engraft in the patient.
Complications sufficient to delay reinfusion are common amo~g advanced cancer patients, which have made this storage method untenable. Several ~cientists h~vesince shown that whole bone marrow or semi-purified preparations such as buff~
coats can be frozen, thawed, and rei~ed into patients successfully after long storage times.
One major difti~ ulty with current methods, however, is that only relatively low percentages of the ac~ual cells which engraft or recon;titute thebone marrow survive the cryopreservation process. For example, Lemoli et al.
(Haematologica 73:101-104, 1988), collected whole bone marrow and separated out a mononuclear fraction. These cells were f~ozen, but only 50% to 66% of 25 CFU-GM cells were recovered upon th~wing. Figuera et al. (Cryobioiogy 23:47~
475, 1986) utilized a controlled rate freezer to freeze marrow buff~ coats in sto~age bags, but only recovered about 36% to 43% of the CFU-GM cells. Visani et al., Crybiology 20:5~7-590 (1983), froze marrow buf~y coats in a controlled rate ~eezer, a~d only recovered about 70% of cells following cryopreservation.
In addition, present cryopreservation strategies generally utilize large quantities of DMSO iin order to protect the cells dwing ~eezing and thawing. For example, patients receiving semi-p~ied bone marrow typically also receive 10 to 20 ml of DMSO, whi}e patients who are given whole bone marrow may receive as muc~ ~5 150 ml of DMSO.
Administraffon of large qualiffes of DMSO may have a toxic e~ect on patients wbo are already wealcencd by chcmotherapy or ilradiation. Patients wbo haYe been ~en a large qu~tity of DMSO, in addition to experiencing ~ ~/07745 Pcr/US92/09~23 \

3 21221~0 unple~ant breath and odor, ~equently also experience headaches, nausea, chills, dizziness and vomiting. Addit;onally, there is a nsk of more serious problems including heart alThy~as, lesions of the eye, hypertension, renal failure, and even death.
S Finally, cl}rrent med~ods of ~OpIeSe~g bone marrow often lead ~o cell aggregates and lysis of red blood cells These ag~egates aIld the hemoglobin released by the lysed red blood cells are implicated iII pulmonary distress and renal failure, which is occasionally noted in bone marrow reapients.
The present imrention overcomes the toxic effes:ts of DMSQ cell 10 aggregates and lysed red blood cells, and ~e poor cell recoveries of prior ~yopreservation me~hods, and further provides other related ad~antages. ~ ~

Su~aIy of thQInv~ntion ~ .
Briefly stated, it is an ob~ect of ~he present invention to provide --15 methods for prepanng engraf~ng ceLls for future use. More specifically, engraf~g c~lls may be prepared and f~ozen for extended penods of time, and later retrieved for therapeutic or research puIposes. ~.
Within one aspect of the inven~don, a method is provided ~or prep~g en~Fafting cells for future use, comprising the steps of (a) puri~ring 20 engrafting cells from a suitable blood product; (b) concentrating the purif;ed engra~g cells; (c) resuspendiIlg the concentrated cells to a concentration o~
about 10 x 106 to 40 x 106 celLs/ml in a solution that substantially maintains cell tiability during f~ee~ng and tbaw~g; and (d) free~g ~he resuspended cells undera first set of conditions which substan~dally ~t~ cell viabili~r. Wi~hin a related 25 aspect of present ~vention, a method for preparing engrafting cells for future use is provided, compris~ng tbe steps of (a) pu~g engrafting cells from a suitable blood product; (b) concentrating the p~ied engrafting ceL~ c) resuspend~ng the :
concentrated cells L~ a solution that substantially maintains cell ~iabili~ during freeziDg and thawing, the solution contaani~g a total of about 0.00~ ml to about 1 30 ml of DMSO; and ~d) freezing the resu~ended ceLls under a ~rst set of conditions whi~h substa~ maintains cell ~ability. Within one embodiment of the present inYention, the puri~ed engr~ting cells are resuspended to a concentrati~n of about 10 x 106 to about 40 x 106 cells per ml.
W;thin one embodiment, the methods noted above further 35 ~ompnse, subsequcnt to the step of fr~ hawing ~he f~ozen cells under a second set of condiffons wbich subst~tially maintain cell viabili~. Within a Wo 93/0774~ Pcr/us92/0~û23 212~140 pre~erred embodiment, the cells are thawed in a 37DC water bath, and then in a subsequent step, slowly diluted with a physiological buffer.
Within other embodiments of the present invention, the step of purif~g comprises passing ~he blood product over an immunoaffinity column 5 which puri~es the engrafting cells, and the step of concentrating comprises sentrifiugyng the engraft~g ce~s.
In other embodiments, the concentrated cells are resuspended in a solution which contains about 6% HES, or a so}ution that cont~ a to~l of about 0.002 nnl to ab~Dut 1 m1 of DMSO, or preferably, m a solution compr~ng med~a~
10 p~otein, and a penetrating c~yoprotectant. Particularly preferred media include RPMI 1640, TC 199, and Iscoves DMEM.
Within yet another embodiment, the step of freezing is accomplished by ~reezing ~he cells at a controlled rate. Within a presently prefe~ed embodiment, the step of f~eezillg at a con~olled rate is accomplished by 15 (a) cooling the cells down to about 4C; (b) cooling down the 4C cells at a ra~e of ab~ut one degree per minute until the cells reach about 4C; (c) cooling down the ~C sells at a rate of about 05 degrees per minute until the cells reach about -2~C; (d) cooling down the -20C cells at a rate of about 1.0 degree per minute until the cells reach about ~0C; and (e) cooling down the ~0C cells at a rate of 20 about 10.0 degrees per minute until the cells reach about -90C.
Within another aspec~ of the present inven~ion, a composition is pro~ided, comprising (a) a therapeutic dose of engrafting cells produced according to the present invention, and (b) an aqueous solution containing a total of about 0.00~ ml to about 1 ml of DMSO. Within one embodiment, a method is provided 25 for treating immunocompromised patients c~mprising the step of administering to a paffent such a composition. Administraffon may bc performed through use of either a syr~nge or a ddp bag which contains this composition.
These and other aspects of the present invention will become evident upon reference to the following detailed desc~iption and attached 30 drawings.

Brief DescTi~tion of the Drawia~
Figure 1 is a graph which illustrates the freezing of engrafting cells at a controllçd rate.
Figure 2 is a bar graph whicb compares the viability, cell recoveIy, and colony-forming cell (CFC) rccave~y of c~yo~eserved purifed engrafting cells with cryoprese~ved whole bone ma~ow.

wo 93/07745 Pcrluss2/oso23 21221l~0 Figure 3 is a bar graph which compares the viability, cell recoveIy, and C~;C recovery of cryopreseIved purified engraf~ng cells which are frozen at dif~erent cell concentr~tions.
Figure 4 is a bar gr~ph which comI~ares the ~nability, cell recovery, 5 and CFC recove~y of c~yopreserved purified e~graf~ng cells in 75% DMSO, and in lO~o DMSO.

Detailed De~iQn of tbç Ill~ren.~
As noted above, the present ~vention provides methods for 10 preparing engr~ting cells for filture use. Within the context of the preseIlti~lvention, the term "engraf~ng cells" includes to~d~tent hematopoiedc stem cells as well as early progeDitor cells suc~ as colony-folming cells (CFCs~
Representa~ve examples of CFCs include C~U-E, CFU-G, CFU-M~ C~[J-GM, CFU-GEMM, aIld BFU-E cells. Given the fact ~that generally as many as one-15 third to tw~thirds of C~D 34 positive cells may be stem cells or colony-~orming cells, it should be understood that when CD 34 cells are concentrated or p~ ed, engraf~ng celLs are similarly concentrated or~ified.
Utilizing de~ices and methods w~ich are described in m~re detail below, e:llgraf~ng cells may be puri~ed from va~ious blood products, including for 20 example, peripheral blood and whole bone marrow. For purposes of the present inYen~don, engrafting cells are considered to be ~ rified" if at least 20% of the p~ied cells are CD 34 positive cells. Preferably, ~he CD 34 positive cells should be purified to greater than 90% purity. In addition, it is desirable to keep the total numbers of platelets, granulocytes, and red cells as low as possible in order to25 prevent clumpiIIg and the release of degrada~ive enz~mes which decrease engrafting cell reco~ery and viability. More specifically, it is generally desirable that tbe puri~ed engrafffDg ceL1s contain less than about 1% platelets, less than 50% and preferably less than about 25% granulos~es, and less than 10% and pref~rably less than about 1% red cells.
Purification of engra~g cells may be accomplislled throu~ use o~
a ligand w~icb spec;ficaLly reco~s antigens on these cells. For example, antibodies which specifically rcco~e t~e ~ tigeDs may be utilized in the devices and metbods described bclow in order to purif~ engraf~g cells.
Representa~ve e~amples of a~tibodies which specifically reco~ the C~D 34 35 antigen includc MY-10 and HPCA2, (BectoIl-Dic~on, Mou~t~in View, C~lif.), QBEN~10 (~tum Bio~ystem~, Cambridge, U.K) and 12.8 (CellPro, Bothell, Wash.).

WO 93/0774S PCr~US9~/~gO23 21221~o 6 Various methods and de~ices may be utilized to puri~y engrafting cells, including the use of magnetic beads, p~g, and flow cytometI~r (~luorescence Activated Cell Sorting "~ACS") (see, for ~nple7 U.S. Patent Nos 4,714,680 and 4,965,204, hereby in~orporated by reference). Particularly preferred S methods and deYices are immunoaffinity colu~ such as those which are described in U.S. A~plicatio~ SeAal No. 07/513,543, enti~led HImmunoselection Device and Me~hodN (hereby incl~Iporated by reference in its en~re~). Briefly, this applicatio~ describes methods and d~1ices for ~la'dng or separating target particles such as engraf~ng cells, ~om a mixture of non-target and target particles.
10 Included within this application is a di.scussion of devices and methods wherein target particles are sepa~ated in a direct method by passing tbe pa~ticles through a column containing a bed of low nonspecific binding porous material which has a Iigand capable of specifically binding to the target particles. Wi~in one aspect of the application, a device is provided which generally compnses (a) a column 15 hav~ng a proximal end with an inlet port throu~ which ~uid may enter tbe column and a distal end with an outlet port through which fluid may e~t the column, (b) a bed of low nonspecific binding porous material wi~in the column, the porous matenal having a biotin adsorbing group immobilized on the surface thereof, wherein the pores of the porous material are of a size suf~icient to allow the biotin 20 adsorbing group to enter into tbe pores, but nDt so large as to allow collapse of the bed, and wherein the interstitial spaces of the bed a~e of a size sufflcient to allow the particles to flow through the bed. The device may further comprise a means, located within the colurnn, for agitating the porous material upon the application of a~ external force, such that bound target particles are released from the porous 25 material. Within other aspects of this application the target par~dcles are separated by either a one-step or two-step method utilizing a~idin and biotin. It should also be noted, however, that for purposes of the present im~ention other materials may be utilized within the immunoaffinity column, including for example non-porous materials.
A particularly preferred immunoaffinity column is desc~ibed in pending U.5. Application (Ano~s Docket No. 200072.407~ entitled "Improved Apparatus and Method for Cell Separadon" (hereby incorporated by reference in its entire~). Brie~y, witbin one aspect of ~is application, a "cell separa~or" is proYided including a colunm assembh~r for separa~ng target c~lls from a sample 35 flu~d, tbe colum~ asscmbly including a column, a sample fluid supply bag and a fluid collection bag wberein the colu~ is p~aYided for recei~ing the sample fluid from the sample fluid supply bag and for separating the target cells from tbe WO 93~07745 PCr/US92/09023 ~`" 7 21221~0 sample fluid and retaining the target cells, and wherein the fluid collection bag is provided for receiving the ~arget cells after being released from the column, the cell separator comprising an agitation means for agitating the co~tents of the 'column to assist in releasing the sample cells retained in the column, the agita~ion 5 means being responsive to a drive signal for varying amounts ~ agitation of the contents of the column to vaIy ~e rate at which the sample ceLs are released, c~lumn sensor means for providing a column s~gnal indicative of tbe optical dènsity of fluid flowing out of the column and into the nuid collection bag, a column valve means responsive to a column valve control signal for selectively 10 enabling the fh~id coming out of 1he column to flow into the fluid collection bag, and a data processor means for controlling the ol~eration of the cell separator, the data processor means being responsive to the column signal lor providing the drive signal and the column valve control signal to prevent inadequate concentrations of the target cells from being collected. One embodiment of this 15 invention is the OEPRATE LC~ cell separation system w~ich is available from CellPro tBo~ell, Wash.).
The pu~ified engrafting cells are then concentrated. Va}ious methods may be utili~ed to concentrate the purified cells"ncluding for example sedimentation and Bltration, although centrifugat;on is ge~erally preferred.
20 Within a preferred embodiment of the invention, the puri~ed engrafting cells are conce~trated by centrifugation at 150 x g for 10 minutes, and the supernatant isdiscarded.
The concentrated engrafting cells are then resuspended in a solution that substantially m~untains cell viability during freezing and thawmg. Within the 25 con~ext of ~e present invention, cell viabi}ity is "substantially maintained~ if greater than 80%, and pre~erably greater than about 90% of ~he ceLs remain ~iable after freezing and thawmg. A par~icularly prefelled method for determining engrafting cell viability is described in greater detail below in Example 8.
~arious solutions may bc utilized in order to substantially maintain cell viabi1ity during f~eezing and tbal~nng. These solutions maintain cell viability during ~eezing and tbawing by (1)preventing or reduci~g the formation of intracellular ice aystals, (2) increasing osmotic pressure inside cells so tbat the volume reduction upon frcc~ng is rcduccd, and (3) stabilizing the cell membraIle.
35 Tbe basis of the solution may be composed of any physiologically acceptable ~luid, including for examplc simplc sa~ne or buffered saline (e.g., phosp~ate buffered saline or "PBSn), or cell culhlre media Representative examples of cell culture Wo 93/0774~ Pcr/us92/oso23 ~12~1~0 media suitable for use in the present invention include RPMI 1640, T~ 199, or Iscoves DMEM (all available from Gibco BRL Gaithersburg, Md.).
The solution should additioDally contain a penetrating cryoprotectant, a membrane stabili~ng agent, a protein, or preferably, all three.
S Pe~etratillg cryoprotectants such as glyceroL DMSO, and formamide aid in the maintenance of cell viabili~r by iIlcreasiDg the o~moffc pressure insidè dle cells so that the volume reduction upon freezing is reduced. A particularly pre~erred penetra~g cryoprotectant ~ phalmaceutical grade DMSO which is ut~d at a fînal concentra~don of 4~ to 20%, and preferably at a final concentration of 7.5%
10 to 10%. Use of DMSO at these concentrations (given the cell concentrations discussed below) should result in the cells being resuspended in a solution con~g a total of from about 0.00Q ml to about 1 ml of DMSO. As discussed in more detail below, this low total dose of DMSO si~icantly improves the effica~
of therapeutically administering engrafti~g cells.
lS Membrane stabili~g agents are believed to aid in the maintenanceof cell viability by helping to reduce cell damage due to dehydrati~n during ~eezing. Examples of membrane stabili~g agents include hydroxyethyl starch (~S), polyvinyl p~rrolidone (PVP), and glucose. A particularly preferred membrane stabili~ing agen~ is low molecular weight ~IES (Amens~ itical 20 Care, McC~aw Park Ill.), w~ich may be utilized in the solution at a final concentration ranging from 4% ~o 9%, and preferably about 6%.
Proteins, as noted above, are belie7re~ to function by reducing cell damage due to debydration during freezing in a manner similar to membrane ~abilizing agents. Examples of suitable proteins include alb~ (animal or 25 hurnan), hemo~lobin, animal serum (e.g., HYCLONE~, Logan, Utah), a~d human plasma (available from the Puget Sound Blood Center, Seat~e, Wash., or ~om other blood centers, blood banks, or plasma centers). Tbe protein may be utilized at a wide range of concentrations, rallging from about 5% to 90~o in the soludon.
It should be noted, however, tbat if a protein solution is utilized along with a30 media containing Ca+ + or Mg+ + ions, it is preferable to include between S and 20 units of heparin per ml, and preferab~ about 10 units of beparin per ml of the solution in order to prevent clotting or coagulation of the cells by protein. A
particularly preferred protein which is utilized in the present invention is human autologous plasma, because it a~roids thc po6sibility of con~aminating engrafting 35 celJs with foreig~ ses (for ~:xample, HIV), and overcomes compahbili~
difEiculties which might arise if nonc~mpa~ble serum was utilized (for example, fe~al bovine serum) Witbin a prcfcrred embodiment, autologous plasma is w0 93~0774~ Pcr/lJs92/09023 9 21221~D

generated by first centrifuging plasma obtained f~om a donor at 150 x g in order to remove cellular debris. Next, the supematant is removed and centrifuged at tO~OQ0 to 15,000x g in order to remove particulate matter. The supernatant is then passed through a 0.8 ~ filter, and then through a 0 '~ a filter in order to fi~ther 5 clari~r and sterilize the plasma Within a preferred em~diment.of the invention, autologous plasma is ufflized ~t a final concentra~don of about 10% to 20% of the solu~don.
Additionally, defined nutrient supplements which have no protein or only minimal quantities of protein may also be utilized to reduce cell damage due 10 to dehydration during freezing in place of a protein. A particularly preferred d~fined nutrient supplement is Ex Vivo.
As noted above, a preferred solution for use wiehin the present in~en~on comprises media, protein, and a penetrating cIyoprotectant. In one embodiment, ~is solution oomprises TC 199 media containing about 20%
15 autologous plasma, and 75% DMSO.
It is also necessary to res~end the cells in a volume of soluti~n such ~hat dle ~iability and recovery of engrafting cells is substantially maintained upon freezing and thawi~g. In par~cular, if cells are resuspended at a low concentration the number oî viable cells recovered after free~ng greatly decreases. On the other hand, if ~she cells are resuspended at a high concentra~don they may form clumpsJ thereby limiting the number of recoverable cells. Tbus, for purposes of the present invention the en~g cells should be resuspended to a concentration of ab~ut 1 x lo6 to 100 x 106 cells/ml, and preferably to a concentration of about 10 x lo6 to about 40 x 106 cells/ml.
The resuspended cells are ~Ihen frozen under a first set of conditions which substantially maintain: cell viability. Briefly, if cells are frozen too rapidly, ice nucleation begins the formation of ice c~stals which can rupture the cells Alternatively, if cells are frozen too slowly, dehydration of the cell results Therefore, in one embodiment the cells are frozen at a controlled rate. This 30 method is particular~ prcforTed ~or sohltions which do not contain BS. Since it is often difficult to maiiltain a contTolled rate of freezing near -4C (a~ thistemperature ceLls giYe o~ heat tenned tbe ~atent heat of fusionn), a controlled rate freezer such as ~yoMed's Model 1010 (C~yoMed, New Balt~ore, Mich ) wbicb provides 'burst COO]i~lg' near -4C may bc utilized. As sbown in Figure 1, a 35 burst of coolillg is pravided bg ~c free~er at about ~C in order to maintain the constant rate of tem~rature drop in ~e cell suspension wo 93/07745 PCr/U~92/0gO23 2122110 lO

Within a particularly preferred embodiment, freezing comprises the steps of (a) cooling the cells down to about 4C, (b) cooling dowIl the 4C cells at a rate of about 1.0 degree per m~ute until the cells reach about ~C, (c) cooling down the 4C cells at a rate of about 05 degrees per minute un~l the cells reachS about -20~C, (d) cooling down the -20C cells at a rate of about 1.0 degree per mi~ute until the cells reach about 4~C, and (e~ cooling down the ~C cells at a rate of about 10.0 degrees per minute until the cells reach about -9ODC.
It should also be noted, however, that other conditions may be utilized to freeze the engraf~ng cells. ~or example, cells which were resu~ended10 in a solution con~aining about 6% HES may be frozen by placing them directly into an -85C freezer. Subsequently, if desired, they may be placed into liquid Ditrogen.
The resuspended cells may be frozen in any sterile vial sl~itaUe for storage in liquid Ditroge~ Particularly preferred are C~yotubes (Corning Glass 15 Works, Co~g, N.Y.). Alter~atively, if desired, the engrafting cells may also be directly f~ozen in a ~ge. FreeziDg purified engr~ting cells in a ~ge is particularly adYantageolls because d~e same container can be used for both freezing and administration to a patient. This ~its possible con~amination or loss of cells and increases the speed with whicll the recently thawed cells can be 20 given ~o a pa~ent. AlternatiYely, the cells may also be frozen in a freezing bag, commercially available from Fenwac or DeLmed.
As noted above, subsequent to freezing, the cells are thawed under a second set of conditions which substantially maintain cell viabili~. In a preferred embodiment, the cells are ~awed rapidly, preferably in a 3rC water 25 bath.
Once the cells have been thawed, they may be diluted in order to reduce the concentration of a penetra~g c~yoprotectant or other excipients, and also in order to return the cells to tbeir noImal state. For example, cells which were equilibra~ed witb a solution containi~g 10% DMSO are at an osmola~ of 30 1800 mosm, w~ile physiological fluids are at an osmolarity of 300 mosm. lf the thawed cells are placed immediate}y into a pbysiologic solu~on, the cells would quicldy absorb water to equalize tbe osmo~dc p~essure. This rapid absorption of water results in tbe Iysis of ma~y cells. Tberefore, slow dilution of the thawedcells with a physiolo~ uffcr,, such as PBS7 is particularly preferred becau~se it 35 allows time for watcr ~d di,ssolved co~ouIlds to equilibrate across the cell membrane, thcreby limiting the dangerous swelli~g of the cells. A particularly WO 93/07745 PC~/US92/~9023 preferred method for reducing the concentration of DMSO is set forth below in Example 6.

AD~NIsrRAnoN o~ PUR~D ENG~G C~S
S :~
A patient may be immunocompromised for a vanet~ of reasons.
For example, a patient may be immunocompFom~sed d~e to i~herent genetic abnormalities, due to disease, or due to the use of to~cic chemicals or i~adiation in the treatment of cancer. Such a patient may be treated by the administradon ~f a10 composition comprising a therapeutic dose of cngraf~ng cells, and an aqueoL~ssolution cont~g a total o~ about QOO~ ml to about 1 ml of DMSO. Within ~he context of the present invention, a "therapeutic dose" of engxafting cells refers to the number of lCD 34 posidve] cells necessaIy to recons~dtute a pa~ent's immune response. The number of cells required ranges from about Q1 x 106 cells/ml/ldlo :;:
to about 20 x 106 cells/ml/ldlo, although at least Q75 x lo6 cells/ml/lcilo ~of pa~de~t weight) is particularly preferred. Thus, for a patient that weighs 100 l~los, approximately 75 x 106 cells may be a~tered. The cells sbould be tr~sferred iDtravenously ~om a drip bag, or by direct injection from a syriDge. An example of t~is procedure and the resultant benefits is descnbed in more detail below in ~-20 ExamF~le 11.
The following examples are offered by way of illustratioD, and not ~: :
by way of limitation. - :

Preparation Of An Avidinated Biogel .

~L CJUBOXYlA'llONOPAPOLY~RYl~M~E~GE~L ;
Seventeen grams of d~y Biogel P-60~, (50-100 mesh (wet), coa~se beads) (BIORAD, Catalog No. 150, 1630, Richmond, Calif.) are added to 15 l of 05 M NaHCO~/0.5 M Na2C03. Tbe pH is ad~usted to 105 with NaOH and carefully s~irred with a mixcr (RZR1, Carf~mo, Wiarton, Ontario, Canada) so as not ~o damage the beads for appro~amately 20 to 30 minutes. The mixhlre is then 35 placed ~ a 61~C water bath. After the mix~urc reached a tsmperature of 60C, it is i~cubated for al~ additional 2 hs (at 60C~) witb o~casional stir~i~g. The wo ~3/07745 P~r/USs2~9023 21221~10 12 ~ture is then removed from the water bath, and placed in an ice ba~ to bring the mixture temperature down to room temperature.
The beads are washed several ~dmes with distilled or deionized water, followed by several wa~hings with PBS ~g a ooarse glass filter connected 5 to a vacuum. The carbo,~ylated gel may be stored fn PBS at 4C, ~d is stable for up to one year if sterilized or stored wi~h a preserva~ive.

B. AVIDIN CONIUCiAnON OP C~RBOmA~D BIOG~
PBS is first removed from a measured amount of carboxylated 10 Biogel by filtering with a coarse glass filter connected to a Yasuum. The gel is then equilibrated in distilled or deionized water for lS to 30 minutes Equilibration in water causes all e~pansion of the gel to a volume of about 4 times its previou~sly measured amount. The gel is resuspended in 10 ml of distilled ordeionized water for each ml of gel (as originally measured in PBS).
15~y mg of l~thyl-3-(3 dimethylaminopropyl) ca~bodiimide (EDC-HCl) (Sigma Chemical Co., Catalog No. E~750, St. Lol~is, Mo.) is added for each ml of gel as ori~ally measured. The pH is rapidly adjusted to 55 by drspwise ~dditio~ of HCl. Care is taken to maintain the pH at 55; pHs of less than 5.0 or greater than 6.0 result in significantly less astivation of the Biogel. The 20 mixture is stirred for five minutes.
~ qidin (International En~snnes, Inc., Fallbrook~ Calif.) is dissolved at a concentration of between 10 and 100 mg/ml in deionized water. Next, 1 mg of avidin is rapidly added for each ml of gel (as originally measured in PBS). The mixture is stirred for 15 hours. Next, 2 M glycine is added to give a final 25 concentrasion of 0.2 M glycine in the mixture, and s~red for an additional 1 hour.
The gel is washed with several volumes of PBS using a coarse glass filter and vacuum, and stored in PBS at 4C. The gel is stable for appr~ximatelyone year.

~XAMPI~E 2 Isolation Of Engraf~ng Cells A. PR~P~NC ~H~ B~ COAT C~S
A sample of bone maJrow is centrifilged at 240 x g for 15 minutes.
35 The plasma is removed (and is retained ~or latcr use), and the remaining b~
~ cel~ e cc~trifugcd once more at 240 x g for 15 minutes in order to remove red blood ceLls. The buffy coat ~ells are washed Iwice with RPMI by W~ 93/07745 PCr/US~2/09~23 13 21221~0 centrifugation at 1~0 x g ~or 10 minutes. The cells are then resuspended to a final concentration of 1 x 108 white cells/ml in RPMI plus 1% BS~

B, INCUBATION 0~ BU~r COAT Ce~S WnH ANnso~Y
S The suspension of bufljr soat cells is incubated with a final concentration OF 20 I g/ml biotinylated and-CD 34 andbo~y ~CellPro0, Bothell~
Wash.) at room temperature for 25 minutes. The andbody-cell mix~re is then wasbed twice with PBS by celltrifugation at t80 x g for 10 minutes. 'rhe cells are then resuspended at a concentration of 1 x 108 white cells/ml in PBS.

C. COLU~ OP~RAnON AND RBSULrS
A OEP~ CellPro0, Bothell, Wasb.) separadng system was u~ilized essen~ially according to the manufacturer's instructioDs. Bnefly, the instrument was set up, the tubing connected, reagent's were loaded, and the 15 process run was begun ~nth the antlbo~ ~eated cells. lrhe cells were pumped through the colu~, the colu~ was washed with PBS, then the adsorbed cells were released via the magnetically driven impeller. The adsorbed cells were accumulated in a collection bag.

D. RE~
Ten billion bone malrow cells were passed through ~he column; 201) million of the cells were bound to ~Ihe column and were recovered in ~he collection bag. Yiabili~r of the collec~ed cells was 91% as measured by t~pan blue exclusioIL The collected cells were 75% CD 34+ as measured by FACS analysis.

::

Concentration of Engrafting Cells l~e collection bag containi~g ~ied engrafting cells~is gently inYerted in order to mix the cells. The cells are then tra~ erred into two sterile 50ml centrifuge tubes wkirk have been coated with au~ologous plasma. Tbirq millili~ers of TC 199 is used to nnse out ~e ce~ collection bag. The rinse volume is placed into a third 50 ml tube. Tbe bag is then rinsed a second ffme with 20 ml o~ TC 199, w~ich i~ o added to the third 50 ml tube. The t~ree tubes are centri~uged at 150 x g for 10 minutes.

WO ~3~0774~ P~r/US92/0~3 2 1 ~ 0 14 Resuspension of Engra~ng Cells Supernatant is removed from the concentrated engraf~ng cells.
5 One millii~ter of engraf~ng cell media (l'C 199, 40% autologous plasma and 10 U/ml hepar~) is added to each of the ~wo tubes with large pellets, and combined into the small pellet rinse tube. Thfs yields appro~tely 2 25 mls of cell suspension which has a conce~tration of 50 x 106 to 100 x l06 cells/ml.
TC l99 cont~g 10 u/ml of hep~ is added to the cell 10 suspension to bring the final volume to 45 mls. Then, 0.9 ml of autologous plasma and 0.34 ml of DMSO is added.
An equal volume of c~yoprotec~ive~media (TC 199 with 15%
DMSO) is added gradually to the cell su~ensio~ Four and a half ml is ~liquoted into each C~yotube and placed iIi the "precooled ~hamber" of a C~yoMed 15 controlled rate freezer. A "dummy" hlbe witb an identical volume of cryoprotective media is prepared and placed i~ the f~eezer. A thermocouple is placed in the tube in order to record the ~eezing rate in the tube.

EXA~LE S
~reezing Engrafting Cells As shown in Figure 1, the engraf~ng cells are frozen at a controlled rate from room temperature to -90C Briefly, this method is comprised of the foll~wing steps: (a) cooling the cells down to about 4C, (b) cooling down the 4C:
25 cells at a rate of about 1.0 degree per minute until the cells reach about ~C, (c) cooling down the 4C cells at a rate of about 05 degrees per minute until the ceLls reach about -20C, (d) cooling down the -20C cells at a rate of about 1.0degree per minute mltil the cells reacb about 10C, and ~e) cooling down the J,0C cells at a rate of about 10.0 deg~ees per minute until the cells reach about 30 -90C. As sho ~m irl Figure 1, this protocol results in a controlled rate of freezing.

W0 93/07745 PCI`/VS92S09023 ~XAMPlE ~
Thawing Engrafting Cells The cryotube co~ g p~;~ed engra~ing cells is removed from 5 liquid nitrogen, and placed in a sterile ziplock bag. lhe baB containing the tllbe is placed in a 37C water b~th and agita~ed. As ~e iast ice c~ys~ issolves ~he conten~s are tra~erred to a 50 ml centrifuge tube.
Four and one-balf milliliters of warmed engraf~ng Cell Dilution.
Media (TC 199 and 10 u/ml hepari~) is added slowly (one dr~p at a ~me ini~ally) 10 into ~he 50 ml tube. Addition of this media should take approximately 2 minutes Following the first dilution subsequent volumes of 05 mls of engraf~ng Cell Dilu~ion Media may be added followed by gentle mixing of the cells, undl a finalvolume of 30 ml has bee~ attained.

15 ~XA~PLE 7 Comparison of ~yopresen~ed Engrafting Cells and (~y~prese~ved Bone Marrow Engrafting cells were purified and c~yopresen~ed as described above in Examples 2 through 6. Buffj~ coats were also prepared f~om bone marrow and 20 ~eated simi;lar to ~he engrafting cells, except tbat they were not puri~ed. As shoum ill Figure 2, purified engraf~ng cells were significantly more viable9 andsignificalltly more total cells (and CFCs) were recovered than for whole marrow.
EXA~LE~ 8 Determination of CFC Viabili~ and Recovery - .
One ml per 35 mm plate of Iscove's Methylcelllllose (Te~y Fox laboratories, Yancouver, BAtish Cohlmbia, Canada) supplemented with 2 mM
L,glutamine and S0 mg/ml gen~amicin was warmed to 37C. Cells were plated in 30 triplicate at 3-fold dilutions to improve the accura~y of the assay. The highest number of cells plated was 105/plat¢ except for purified cells which were plated at 3 x 103 aIld less. The cells were spread even~ er the surface of each pl~te and then incubated in a humidified incuba~or at 37DC with 5% C02 in air for 10 to 14days. Colonies were countcd if they contained more t~an S0 cells and scored as 35 CFU-GM, B~ E, or other (e,&, C~J~EMM~. The number of various ~pes of colonies weTe summed to give the t~al number of colony-forming cells ~CFC~

wo 93/07745 PC~/US92~090~3 E~LE 9 Effect of Cell Concentration on C~yopreservation Engraft~ng cells were purified and c~yopresenred as described above S in Examples 2 through 6, except that the cells were frozen at different concentrations including: 25, 5, 10, 25, 40, and 50 million cells/ml. As shown in Figure 3, CFC recovery was the greatest when cells were ~ozen at a concentrationranging from 10 x 10~ to 40 x lû6 cells/ml.

, ~ E10 E~ect of DMSO on ~yopreseTvation Engrafting cells were purified and cryopreselved as descnbed above in Examples 2 through 6, except that the cells were frozen at a final concentration 15 of 75% and 10% DMSO. As shown in Figure 4, cell viability~ rec~ve~y, and CFC
recovely were si~ficantly better when only 75% DMSO was utilized.

E~AMPLE 11 Administration of Engraf~ng Cells Four paffents with breast cancer had bone marrow removed by iliac crest aspirations. They were subsequently treated b~f chemotherapy in order to kill the tumor.
Engraf~ing cells were purified essentially as descnbed above from 25 the patent's marrow, and administered to the patients. ~e results of this thesapy is set forth below in Table I.

T~BLE 1 -#CD 34~ Days to Days to# Units Patient Infused Granulo~rte PlateletTransfused Wt.(kg) (Million/kg) Recove~y RecoveryPlatelets . ~ ~
59.8 .98 25 24 13 ~`
35 84.6 1.6~ 17 36 9 57 1.05 32 30 æ

wo 93/07745 PCrJUSs2/09023 17 21221~o ~

Typically, engra~ment (the number of days it takes a patient's granulocyte co~ts to rise above 500 cells/~Ll) talces approximately 20 to 35 days The above patients wbich were treated with purified engrafting cells demonstrated engraftment after only 13 to 32 days. ln addition, as noted above, the total volume 5 of DMSO which was infused into the patients dif~ers markedly from conventionalprocedures. In particular, only about 0.00~ ml to about 1 ml of total DMSO (0.675 ml) was infused into each pa~dent. Cell aggregates and hemoglobin were not detected in the cel}s tD be reinfused. Thus, these patients demonstrated none ofthe toxicities which are typically associated with marrow reinfusion, such as 10 nausea, headache, chills, diz~ness, vo~ting, heart arrhythmia, hypertension, pulmonary distress, or renal failure.
Moreover, patients which receive autologous bone marrow transplallts often experience delayed platelet recovery and require a substantiai number of platelet transfusions (on average about 30). The patients who received15 the above-desclibed therapy, however, required only 12 units of platelets each ~on average3. This reduction in platelet usage decreases ~e potential ~ blood-borne disease transmission, the risk of alloimmuDizatiorl, and the cost (each unit of platelets costs the patient appronmately $300). Thus, it can be seen that the present invention pr~vides substantial advantages over previously available 20 cryopreservation techniques.
From the foregoing, it will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without deviating from the spirit and scope of the invention. Accordingly, the invention is not limited except as by 25 the appended claims.
..

Claims (16)

Claims

1. A method for preparing engrafting cells for future use comprising:
(a) purifying engrafting cells from a suitable blood product;
(b) concentrating the purified engrafting cells;
(c) resuspending the concentrated cells to a concentration of about 10 x 106 to 40 x 106 cells/ml in a solution that substantially maintains cell viability during freezing and thawing; and (d) freezing the resuspended cells under a set of conditions which substantially maintains cell viability.

2. A method for preparing engrafting cells for future use comprising:
(a) purifying engrafting cells from a suitable blood product;
(b) concentrating the purified engrafting cells;
(c) resuspending the concentrated cells in a solution that substantially maintains cell viability during freezing and thawing, said solution containing a total of 4% to 10% DMSO; and (d) freezing the resuspended cells under a set of conditions which substantially maintains cell viability.

3. The method of claims 1 or 2, further comprising, subsequent to the step of freezing, thawing the frozen cells under a set of conditions which substantially maintains cell viability.

4. The method of claim 3 wherein said cells are thawed in a 37°C
water bath.

5. The method of claim 3 further comprising, subsequent to the step of thawing, diluting the thawed cells slowly with a physiological buffer.

6. The method of claim 1 wherein the step of purifying comprises passing the blood product over an immunoaffinity column which purifies the engrafting cells.

7. The method of claim 1 wherein the step of concentrating comprises centrifuging the engrafting cells.

8. The method of claim 2 wherein the concentrated engrafting cells are resuspending to a concentration of about 10 x 106 to about 40 x 106 cells per ml.

9. The method of claims 1 or 2 wherein said solution contains about 6% HES.

10. The method of claim 1 wherein said solution contains a total of 4% to 10% DMSO.

11. The method of claims 1 or 2 wherein said solution comprises media, protein, and a penetrating cryoprotectant.

12. The method of claim 11 wherein the media is selected from the group consisting of RPMI 1640, TC 199, and Iscoves DMEM.

13. The method of claims 1 or 2 wherein the step of freezing comprises freezing the cells at a controlled rate.

14. The method of claim 13 wherein the step of freezing at a controlled rate comprises:
(a) cooling the cells down to about 4°C;
(b) cooling down the 4°C cells at a rate of about 1.0 degree per minute until the cells reach about -4°C;
(c) cooling down the -4°C cells at a rate of about 0.5 degrees per minute until the cells reach about -20°C;
(d) cooling down the -20°C cells at a rate of about 1.0 degree per minute until the cells reach about -40°C; and (e) cooling down the -40°C cells at a rate of about 10.0 degrees per minute until the cells reach about -90°C.

15. A composition comprising:
(a) a therapeutic dose of engrafting cells; and (b) an aqueous solution containing a total of 4% to 10% DMSO.

16. A method for treating immunocompromised patients comprising administering to said patient the composition of claim 15.