CA2259878A1 - System and methods for handling fluids - Google Patents

Abstract

Systems and methods for transferring a fluid (e.g., to process stem cells) in a controlled manner are disclosed.

Description

CA 022~9878 1999-01-22 SYSTEM AND METHOD FOR HANDLING FLUIDS

This application claims the priority of U.S. provisional patent application 60/072,468, filed January 26, 1998, which is incorporated by reference.

Technical Field S The present invention relates to systems and methods for h~m11ing fluids such as biological fluids, particularly with respect to preserving blood components, such as stem cells.

Back~round of the Invention Blood from a newborn's umbilical cord and placenta (hereinafter referred to as "cord blood") includes hematopoietic stem cells. Stem cells (also known as progenitor cells) are capable of forming different types of mature, terminally dirr~lenliated blood cells. Due to their undirÇelel,~ia~ed nature, stem cells have a variety of th~r~eulic uses. For example, transfused stem cells can replace blood cells in the bone lllallOW
which may have been de~lroyed by disease or me-lic~l treatment (e.g., radiation).
It would be desirable to create a bank for preserving and storing cord blood.
An i,-le".~lional bank of cord blood would permit researchel~ and patients nPe~ling stem cells to have quick access to a wide variety of cord blood samples. More importantly, such a bank would permit easy access to a large base of potential m~tchPs.
However, the creation of an hlLellLalional bank of cord blood requires the collection 2 0 and storage of vast amounts of cord blood. The collected cord blood would have to be preserved and stored quickly and efficiently.
In conventional processes for collecting, preserving and storing cord blood, placental blood is collected from an umbilical cord and typically processed as follows.
The cord blood is collected into a conventional blood bag cont~ining an anti-coagulant, 2 5 and a se~limPntation agent such as hydroxyethyl starch is added to the bag. The bag is then centrifuged to form a white cell-rich supernatant fraction and a red cell-rich sediment fraction, wherein the starch improves the efficiency of red cell se~limPntation and/or aggregation. The white cell-rich ~upelnaL~ fraction, which comprises white cells (including the stem cells) and plasma, is Llal~r~ d to another blood bag, and is further centrifuged to form an enriched white cell sediment fraction (also cont~ining some plasma) and a plasma-rich ~lpelllatant fraction. The supelllatant plasma fraction 5 is removed from the bag, leaving an enriched white cell sediment fraction including the stem cells. This enriched white cell fraction is then combined with a freshly plepaled cryoprotectant solution.
The cryoprotectant solution, that is added to the bag cont~ining the enriched white cell fraction, is formed by withdrawing dimethylsulfoxide (DMSO), dextran, and 10 possibly other components, from sepalate, sterile treatment solution containers to form a mixture. The mixture is subsequently drawn into a syringe, and added to the enriched white cell fraction (through an injection port or site) to the bag.
A stem cell freezing bag is then spike-conn~cted to the enriched white cell bag,and the fluid cont~ining the white cells and cryoproteclalll mixture is passed to the stem 15 cell freezing bag. The bag and its colllellL~ are then frozen. The stem cells are subsequently thawed and washed (to remove the cryoprotectant) for subsequent uses.
For example, the stem cells can be analyzed and/or transfused.
Conventional systems and processes for cryoprotecting cord blood products suffer from a number of disadvantages. For example, cord blood products can be 20 co~ .,.in~ed by microorg~ni~m~ (particularly bacteria) ellL~ g the system during the cryoprotection process, since the formulation of the cryoplole-;L~lL llli~lule and the allsrel of the mixture to the enriched white cells is carried out in a com~rolllised or "open" system. The process includes inserting syringes into various injection ports or into sterile containers, each having a different llli~-lu~ component therein, withdrawing 2 5 a volume of the component, and adding the components to a mixing container to form the cryoprotectant lni~Lule. The cryopLo~ecL~ Lule is then withdrawn from the mixing container and is added to the white cell bag via a syringe. The use of spikes or syringes creates punctures or openings in the various containers and/or injection ports, and these openil~s expose the contents of the containers (e.g., the cryoprotecLallt 30 mixture and the stem cells) to potential bacterial co."~ on from the outside CA 022~9878 1999-01-22 ellvi~ ell~.
Additionally, the various containers in the cord blood h~n-lling system are typically conn~cte~ via spike connectors, which also create ~ull~;Lules or openings that expose the container contents to potential bacterial co~ ion.
There are other drawbacks to the conventional systems and methods. For example, the processes for form~ ting the cryoprotectant lni~lUle, and for adding the solution to the white cell bag, are each time and labor intensive, and each aspect relies on the skill of individual opelatol~ carrying out these procedures. Additionally, the syringes and pumps used to prepare and add the mixture to the white cell bag may not be calibrated with great accuracy. Since each cryoprotectant solution mixture isp,epaled and added to the white cell bag individually, the concentration of ingredients in the cryoprotectant mixture, as well as the rate of addition and volume of mixture added to the bag, can vary from ope,dtor to operator, from day to day, and from laboratory to laboratory. As a result, it is difficult, if not impossible, to standardize the overall process and system. This variability can adversely affect the quality and/or the yield of the stem cells, and make it difficult to prepare the cells in a ullirollll, repeatable manner.
Moreover, typical cord blood h~ntlling systems, e.g., including conventional blood bags, can be bulky, incompatible and/or inefficient for use in h~ntlling stem 2 0 cells. For example, the volume of cord blood collected is substantially less than the volume of conventional blood bags, making it difficult to centrifuge the bag and/or express the contents of the bag efficiently.
The present invention provides for ameliorating at least some of the disadvantages of the prior art. These and other advantages of the present invention will 2 5 be apl)aren~ from the description as set forth below.

Sull~ al ~ of the Invention The present invention provides a treatment fluid metering system dimensioned for controllably l~al~r~lillg a treatment solution to a desired location, such as a container, wherein the L~ealll~lll solution contacts a fluid and/or a substance to be CA 022~9878 1999-01-22 treated. In a plerelled embodiment, the system provides for transferring a controlled predetermined volume of a treatment solution such as a cryoprotectant mixture at a controlled predetellllil~ed rate to a container having stem cells disposed therein.
The present invention also relates to systems and methods for h~nflling a fluid,e.g., collecting, processing and freezing at least one desired component of the fluid, and provides for reducing the potential for co~ .in~tion of the desired component(s) of the fluid during h~nrlling. For example, in one embodiment wherein the desired component comprises stem cells, the invention provides for combining the cells with a preform~ te~l sterile treatment solution, e.g., a storage solution such as a cryoprote-;lanl mixture.
In a plerell~d embodiment, the present invention provides for combining stem cells with a preform~ te~ sterile cryoprotectant mixture, wherein the mixture iscontrollably transferred to a container having the cells disposed therein. In one embo-lim~.nt, the system provides for transferring a cryoprotectant mixture to acontainer having stem cells therein while m~int~inin~ a closed system.

Brief Description of the Drawin~s Figure 1 illustrates a sch~m~tic view of an embodiment of a fluid h~n~ling system according to the invention, including a collection set, a processing set, and a post thaw set.
2 0 Figure 2 illustrates an embodiment of the collection set shown in Figure 1.
Figure 3 illustrates an embodiment of the proces~ing set shown in Figure 1, showing a treatment solution lll~le~ g system for connection to a container of tre~tm~nt solution.
Figures 4 A-E illustrate, in pe,spec~i~e views, providing fluid co~ ion 2 5 between the treatment solution metering system and the container of treatment solution shown in Figure 3.
Figure 5 illustrates an embodiment of the post thaw set shown in Figure 1.
Figure 6 illustrates a sch~m~tic view of another embodiment of a fluid h~n-llingsystem according to the invention, including a collection set, a processing set, and a CA 022~9878 1999-01-22 post thaw set.
Figure 7 illustrates an embodiment of the post thaw set shown in Figure 6.
Figures 8 A-B illustrate an embodiment of a device for centrifuging the collection set.
Figures 9 A-C illustrate embodilne.l~ of a sampling arrangement for taking a sample in accordance with the invention.

Specific Description of the Invention In an embodiment of the invention, a fluid metering system is provided 10 co~ lisillg a collllector suitable for providing fluid co~."",~ir~tion with a first container, the first container comprising a treatment solution container having a volume of treatment solution disposed therein, and a conduit in fluid co~--...ln-ic~tion with the connector, wheleill the system is dimensioned for tl~l~rellillg from the first container into a second container dowl~lle~ll of the conduit a controlled predetermined volume 15 of the treatment solution that is less than the volume of the treatm~.nt solution in the first container.
In another embodiment, a fluid lll~lelhlg system comprises a connector suitable for providing fluid commllnication with a first container, the first container comprising a tre~tm~nt solution container and having a defined internal volume, and a conduit in 2 o fluid co,-",.~ tion with the connector, wherein the system is dimensioned for llal~rellillg from the first container into a second container dowl~L,e~n of the conduit a controlled predete~ in~d volume of the treatment solution that is less than the internal volume of the tre~tm~nt solution container.
An embodiment of a fluid metering system according to the invention comprises 25 a connector suitable for providing fluid col.~ ation with a first container, the first container colllplising a tre~tm~nt solution container having a volume of treatment solution disposed therein, and a conduit in fluid col...,.~ tion with the connector, wherein the system is dimensioned for tl~l~r~llillg from the first container into a second container dowlLsll._~n of the conduit a controlled predelelmilled volume of the 3 0 treatment solution at a controlled predete. ..li~.~d rate, the controlled predetermined CA 022~9878 1999-01-22 ~. .

volume being less than the volume of the treatment solution in the first container.
Other embo~limP-nts of the fluid llltlerhlg system are dimensioned for transferring a controlled predetermined volume of treatment solution wherein thecontrolled predete~ Pd volume substantially comprises the volume of treatment 5 solution in the first container.
In accordance with another embodiment, a fluid metering system comprises a connector suitable for providing fluid co~.. ~nir~tion with a first container, the first container comprising a treatment solution container having a volume of treatmentsolution disposed therein, and a conduit in fluid co.. ~-ir~tion with the connector, 10 wherein the system is dimensioned for Llal~r~lling from the first container into a second container dowl~l~alll of the conduit the treatment solution at a controlled predetermined rate.
A biological fluid tre~tmPnt system according to an embodiment of the invention comprises a sealed tellnillally sterilized container, and a treatment fluid 15 mixture including a cryoprotectant, wherein the sealed terminally sterilized container has the fluid lllL~lUl~ disposed therein.
An embodiment of a method according to the invention provides a method for Lla~r~ g a treatment solution colll~lisillg passing a controlled predeLelmilled volume of a ll~aLn~lll solution (e.g., from a first container) through a fluid metering system.
2 o The controlled pred~lelll~il~ed volume can be less than, or subst~nti~lly equal to, the volume of treatment solution in the first container In another embodiment, a method for llal~relling a treatment solution comprising passing a treatment solution through a fluid nlelelillg system at a controlled predetc~ ed rate. In a pler~lled embodiment, the method provides for passing a 25 controlled predele....il-Pd volume of the treatment solution through the metering system at a controlled predetell~ ed rate.
In accordance with an embodiment of the invention, a set for processing a white blood cell-cont~ining fluid comprises a first container plasticized with triethylhexyltrimellitate, and a second container suitable for freezing white blood cells, 30 wherein said second container is in fluid co-n-----~-iration with the first container.

CA 022~9878 1999-01-22 The following definitions are used in accordance with the invention.
(A) Fluid. Fluid includes any liquid, or gas, or mi~lules thereof. In a prefelled embodiment, the fluid is a biological fluid, as defined below. Other suitable fluids include various suspensions or solutions. In an illustrative embodiment, the fluid comprises a tissue culture fluid, which may include, for example, one or more proteills such as hormones, enzymes, cell expression products, and/or one or more cell types.
(B) Biological Fluid. A biological fluid includes any treated or untreated fluidassociated with living org~ni~m.~, particularly blood, including whole blood, blood from the placenta, blood from the umbilical vessels, cord blood (i.e., blood from the placenta and the umbilical cord), warm or cold blood, and stored or fresh blood;treated blood, such as blood diluted with at least one physiological and/or storage solution, including but not limited to saline, nutrient, anticoagulant and/or cryo~rotecli~e solutions; blood components, such as platelets, platelet collce~ ate (PC), platelet-rich plasma (PRP), plasma, components obtained from plasma, packed red cells (PRC), buffy coat (BC); blood products or blood components derived from blood (including peripheral blood and cord blood) and/or bone marrow; blood components such as white cells, platelets and/or red blood cells sepalated from plasma and resuspended in a physiological fluid or a cryoprotective fluid. The biological fluid may have been treated to remove some of the components before being processed 2 0 according to the invention. As used herein, blood product or biological fluid refers to the components described above, and to similar blood products or biological fluids obtained by other means and with similar ~lope,lies. Preferably, the biological fluid includes cells, especially stem cells.
(C) Unit. Typically, a unit of cord blood is the quantity collected or drawn 2 5 from a single placenta via the umbilical cord. A unit can also refer to the quantity of biological fluid drawn from a donor or derived from one unit of whole blood. Thevolume of a unit typically varies, differing from collection to collection. Multiple units of some blood components, particularly platelets and buffy coat, may be pooled or combined, typically by combining four or more units.
3 o Each of the components of the invention will now be described in more detail CA 022~9878 1999-01-22 below, wherein like components have like lefelellce numbers. Figures 1 and 6 illustrate embo~ i of system 1000 for h~n(lling biological fluid, including a collection set 100, a processing set 200 including a treatment solution metering system 250, and a post thaw set 300A (Figure 1) or 300B (Figure 6). Embodiments of the collection set 100, the processing set 200 including the treatment solution metering system 250, and post thaw set 300A and 300B are shown in more detail in Figures 2-5 and 7, respectively.
In the following general description, the embodiments of the system 1000 as illustrated in Figures 1 and 6 can be utilized similarly, unless noted otherwise, e.g., 1 o with respect to the embodiments of post thaw set 300A and 300B.
Using the illustrated embodiments of Figures 1 and 2 for refelel1ce, biological fluid, e.g., cord blood, is collected in collection container 123 in the collection set 100 (Figure 2), and processed to form a red cell-rich sediment fraction and a white cell-rich supernatant fraction. Collection set 100 (having the cord blood therein) is conn~ cted to the rest of the system 1000 (Figure 1).
Turning now to Figures 1 and 3, the white cell-rich ~upell~lll fraction is passed from collection set 100 into the receiving container 232 in processing set 200 including keallnelll solution metering system 250. The white cell-rich fluid in receiving container 232 is further processed to provide a white cell-enriched, plasma 2 0 volume-reduced fluid colll~,ising stem cells, and the majority of the plasma is subsequently passed into plasma container 233. The white cell-enriched plasma volume-reduced fluid remains in receiving container 232, and a treatment solution (e.g., a plefo....~ te~ sterile cryoploleclanl mi~lule contained in tre~tm~nt solution container 211) is added to the white cell-enriched fluid in receiving container 232 2 5 through treatment solution lllelelh~g system 250.
The treatment solution Ill~le.illg system 250 provides for mel~lillg the flow oftreatment solution from the container 211 into the receiving container 232. Figures 4 A-E illustrate placing treatment solution container 211 in fluid c~.. "ication with treatment solution metering system 250 via connector 215.
3 o Turning back again to Figure 3, the white cell-enriched fluid, now mixed with, CA 022~9878 1999-01-22 and suspended in, treatment solution, is passed into storage container 234, and the fluid is frozen in the container.
Subsequently, and now using Figures 1 and 5 for lere~ence, the frozen treatment solution/white cell-enriched fluid is subsequently thawed and passed from the freezing bag 234 (from processing set 200) into transplant container 344A in post thaw set 300A where the stem cells are washed (e.g., to deplete the tre~tm~.nt solution from the cells) before further use. Alternatively, using another embodiment shown in Figures 6 and 7 for reference, the thawed fluid is passed from freezing bag 234 into post thaw set 300 B (Figures 6 and 7). Typically, further use of the cells includes analysis, manipulation (e.g., cloning and gene therapy) and/or transfusion.
The components of the fluid h~n(lling systems are described in more detail below, initially focussing on the processing set.

PROCESSING SET and TREATMENT SOLUTION METERING SYSTEM
Figures 1 (and 6), 3 and 4 illustrate embo-lim~nt.c of a processing set 200 1 5 including a treatment solution l,lete~ g system 250 as part of a biological fluid h~n-lling system 1000.
For convenience, the set and system will be described below for preparing stem cells for storage, and for controllably adding a cryoprotectant Il~i~L~ule to a container having the cells disposed therein. However, the set and metering system can also be 2 0 utilized in a variety of other applications and protocols, that can involve biological fluid or non-biological fluid. Exemplary applications and protocols include, but are not limited to, prepa~il g th~,ap~u~ic agents such as drugs and/or nutrients, adding an anticoagulant to a blood collection bag, preparing eggs and/or sperm, and h~n~1ling other fluids such as tissue culture fluids. Other applications and protocols can include, 2 5 for example, tleatillg cells and/or preparing cell-free solutions or suspensions.
In these illustrated embo-lim~nt~, the processing set 200 includes a treatment solution metering system 250, a plurality of conduits 201-205, and a plurality of containers 232-234. For convenience and clarity, the containers will be referred to below as the receiving container (or the white cell container) 232, the plasma container CA 022~9878 1999-01-22 233, and the freezing bag 234. The illustrated set 200 also includes access ports 224-229 (such as injection ports and/or connector ports), and flow control devices 31.
Exemplary flow control devices, that can be adjustable flow devices, include, but are not limited to, clamps (including adjustable clamps such as screw clamps), valves, and 5 the like.
Additionally, as shown in Figures 3, 4B, and 4E, an embodiment of the set 200 also includes a treatment solution container 211, having a treatment solution (e.g., a cryoprotectant ~ ule) disposed therein.
Figure 4E illustrates an embodiment of the treatment solution metering system 1 0 250 in processing set 200 in more detail, wherein the system 250 is disposed at a predete....il~ head height 218 during use. The illustrated embodiment of the treatment solution metering system 250 comprises connector 215 including a base 213, at least one conduit 203 in fluid co.. ~ic~tion with the connector 215, and a vent 214. The connector 215 provides fluid co..~ -ir~tion between the rest of the system 250 and the treatment solution container 211.
In the illustrated embodiment, connector 215 colll~lises a pene~ lg connector, such as a spike or needle having a prede~ellllilled length, and conduit 203 comprises tubing (e.g., microbore tubing) having a pred~lellllhled length and inner tel . In another embodiment (not shown), the connector comprises a length of 2 0 tubing (such as microbore tubing) wherein placing the connector in fluid co... ,-ication with the treatment solution container includes sterile docking, e.g., as disclosed in U.S. Patent No. 4,610,670. Illustratively, a conduit similar to conduit 203 can be "sterile docked" to ano~er conduit that was previously ~ ch~ to the treatment solution container.
2 5 Turning again to Figure 4E, the connector 215 can be attached to, or integrally formed with, the base 213. The base 213 is typically wider than the width of the rest of the connector. In those embodiments wherein connector 215 comprises a pen~llatillg connector having a pell~llalillg end 215(a) (e.g., as shown in Figures 4B
and 4E), the system 250 typically also includes a fitting or jig 17, e.g., to provide an interface between the connector 215 and the container 211. If desired, the jig can CA 022~9878 1999-01-22 engage with the connector 215 (for example, by eng~ging with base 213) and/or the container 211 (for example, by eng~ging with shoulder 216).
In those embodiments wherein the system includes a vent 214, e.g., wherein the treatment solution container 211 comprises a substantially non-collapsible container, the vent preferably comprises a filter that allows air or gas (but not bacteria) to pass into the container 211, and resists the passage of liquid thelellll-)ugh.
If desired, the set can also include at least one additional component such as but not limited to, at least one filter such as a bacterial filter (not shown), for example, interposed btlween the treatment solution container 211 and receiving container 232 1 0 for filtering the treatment solution. Other illustrative additional components include, but are not limited to, at least one of a vent, a container, a conduit, a transfer leg closure, and a sampling arrangement.
The llea~ t solution metering system 250 shown in Figure 3 and 4E is dimensioned for tlal~r~,lhlg the treatment solution from container 211 into receiving 1 5 container 232 (that contains white cells including stem cells disposed therein) in a controlled manner. For example, as will be described in more detail below, the metering system 250 provides for passing into the receiving container a controlled predetermined volume of cryoprotectant mixture at a controlled predetermined rate.
Typically, the stem cells are processed (e.g., concentrated) before LI~L~rellhlg2 0 the cryoplole~ l mixture into the receiving container 232. For example, processing set 200 (shown in Figure 3), that includes metering system 250, is first placed in fluid co,-"~ ir~tion with a collection set 100 (shown in Figures 1 and 2), so that the white cells (including the stem cells) suspended in plasma can be passed from the collection set 100 into the processing set 200 via conduits 201 and 202. If desired, the set 200 2 5 can be placed in fluid connection with set 100 via spike 237 (Figure 3). Alternatively, the sets can be conn~cte~ while m~int~inin~ a closed system, e.g., via sterile docking.
Referring now to Figure 3, the white cells suspended in plasma in receiving contaillel 232 are celllliruged to form a supelllatant fraction coln~lisillg plasma, and a sediment fraction colllplisillg white cells and some plasma, wherein the white cells 3 0 include the stem cells. The supernatant fraction is passed from receiving container 232 CA 022~9878 1999-01-22 into plasma container 233 via conduits 202 and 205, leaving a white cell-enriched, plasma volume-reduced fluid in the leceiving container (or white cell container) 232.
The ~ solution met~ g system 250 is placed in fluid colllll,.ll-ie~tion with the lle~ lelll solution container 211 via connector 215. In accordance with the invention, the treatment solution metering system 250 is dimensioned for transferring the treatment solution from container 211 into the white cell container 232 (that contains the stem cells therein) in a controlled manner.
For example, in one embodiment, the metering system 250 provides for lL~l~r~,lling a controlled predele.lllilled volume of treatment solution from the treatment solution container 211 into the white cell container 232. Typically, the transferred controlled predetermined volume is less than the volume of treatmentsolution in the container 211. Illustratively, and using the embodiment illustrated in Figure 4E for rererellce, the flow of cryoprotectant mixture from the container 211 through conduit 203 into white cell container 232 will stop once the level of fluid no longer covers the tip 215(a) of the connector 215, even though fluid remains in the container 211. Thus, while the container still contains fluid, the flow stops predictably, and autom~tic~lly, after delivering a predelellllilled volume of fluid, without m~nl-~lly clamping (e.g., col~lliclillg the inner diameter of) the conduit 203 to control the volume of fluid delivered.
In one embodiment wherein lllel~ling system 250 does not include a penetrating connector, e.g., wheieill container 211 (cont~ining a predetermined volume of fluid) can be conn~ctçd to the system 250 via sterile docking, the flow stops autom~tic~lly with treatment solution rem~ining in the container. For example, the container 211 can comprise a non-vented, non-collapsible container wherein a predelelll~illed residual 2 5 volume will remain in the container. Alternatively, for example, in some of those embodiments wherein container 211 is vented and/or is collapsible, the flow will stop when the container is empty, e.g., wherein essentially no treatment solution remains in the container.
The treatment solution metering system 250 also provides for llal~rel~ g the 3 o treatment solution from the treatment solution container 211 into the receiving CA 022~9878 1999-01-22 container 232 at a controlled predetelll,il~d rate. Illustratively, in an embodiment, the system includes at least one conduit with a selected inner di~mPter (e.g., to provide resistance) and the set is disposed at a selected head height during use to provide any desired controlled predete. ~ d flow rate. Accordingly, embo~im~nt~ of the metering 5 system provide a desired controlled predete, . "i~-~cl flow rate with and without penetrating connectors, and without m~m-~lly clamping a conduit to control the rate.
Figure 4E illustrates one embodiment of the system arranged at a predelellllhled head height 218.
As noted earlier, the tle~ llelll solution metering system according to the 1 0 invention can be utilized in a variety of applications and protocols. For example, since the viscosity of a desired tre~tm~nt solution is known or can be det~ in~d, and the flow rate is dependent on the viscosity and re~i~t~n~e, the system can be dimensioned for tra~ hlg the desired treatment solution in a controlled manner. In accordance with the invention, an exemplary controlled predetermin~d flow rate is in the range of 15 about 0.1 ml/min to about 1 ml/min, or more. In some embodimelll~, e.g., someembodhl~llts wherein the lleatlllellt solution comprises a cryoprotectant mixture, the predele,ll~illed flow rate is in the range of about 0.2 ml/min to about 0.5 ml/rnin.
If desired, as shown in Figures 4 A-E for example, a jig 17 can be interposed betweell the comle~;lor 215 and the container 211, e.g., to guide (more preferably, to 2 0 more precisely define) the cormection between the connector 215 and the container 211. The jig 17 shown in these illustrated embodiments includes an extended portion 17(a) having a controlled thir~n~ss, and an aperture 17(b). The jig also includes side walls 17(c), a first open area 17(d) belweell extensions 17(e), and a second open area 17(f) between extensions 17(g) and slots 17(h). Embo-lim~-nt~ of the jig can include 2 5 moveable elements. For example, as shown in Figures 4C and 4D, one section of the jig, that includes 17(a) and 17(e), moves relative to another section, that includes 17(c) and 17 (g).
In an illustrative embodiment, the jig 17 can reproducibly control the depth of needle insertion, and the alignment of the needle with the stopper of the container 211.
3 0 If desired, the jig can "lock" or "fix" the connector into place, e.g., by eng~gin~ with CA 022~9878 1999-01-22 the connector base 213, and with a shoulder 216 or groove in the container 211. After the fluid has been ~lal~felled from the container 211, the jig is typically disengaged from the container and the connector, and the jig can be used with another treatment solution l,lelelil1g system. The jig can be used with a plurality of treatment solution 5 ~ eling systems with accurate, reproducible, and predictable results (e.g., ll~l~rer of a controlled predetermin~d volume of fluid).
Since the treatment solution is added to the white cell container cont~ining thestem cells in a controlled pred~lellllined manner, the present invention provides a "standard" that allows different laboratories and operators to treat cells in a uniform, 10 repeatable manner so as to m~int~in the viability of the cells.
If desired, systems and methods according to the invention provide for further standardizing a protocol, particularly a stem cell treatment protocol, while further ,~illi,-.i~i,-g the potential for co~ .in~tion, especially bacterial co~ lion, of the treated material. For example, in accordance with another embodiment of the 15 invention, the lleallllenl solution, e.g., a cryoprotecldlll n~L~lule~ is supplied as a standardized plero. .~ ted sterile solution. Preferably, the solution is preform~ te(l with respect to component concentration and total volume. Since the solution can be supplied as a standardized sterile plefol...lll~ted solution, this avoids both the variability of individually prepared solutions and the labor illlensive effort of such 2 0 operations. Additionally, the solution can be prepared in advance and stored until needed. Moreover, since the solution is prepared in a closed system, this avoids the potential for col~ ,.lion caused by open, colll~rolllised systems.
Accordingly, the ll~aL~llelll solution container 211 preferably comprises a sterile sealed container having a pr~fo- ....-l~ted treattnent solution sealed therein. For 25 example, the Lle~l...e.~l solution, e.g., a cryoprotectant l~ lule, can be prepared in a closed system, and sealed in the container while m~int~ining sterility. Alternatively, the container can be filled, sealed, and subsequently sterilized.
A variety of L1~ P.~I solutions can be used in accordance with the invention.
In those embodiments whel~ the treatment solution comprises a cryoprotectant, the 3 0 cryoprotectant solution or nli~lul~ includes a cryoprotectant agent and mixtures .

CA 022~9878 1999-01-22 thereof, such as, but not limited to dimethyl sulfoxide (DMSO), glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, I-erythritol, D-ribitol, D-.l~n~ Ql, D-sorbitol, I-inositol, D-lactose, choline chloride, amino acids, m~th~n- l, acet~mi-le, glycerol monoacetate, and inorganic salts.
5 Exemplary treatment solutions include those disclosed in International Publication No.

4.
In typical embodiments, DMSO is used, which is nontoxic to cells in low concentration. It is believed that, being a small molecule, DMSO freely permeates the cell and plolec~ intracellular organelles by combining with water to modify its 10 freezability and prevent damage from ice formation. The addition of plasma (e.g., to a concentration of about 20-25%) can augment the protective effect of DMSO. After the addition of DMSO, cells should be kept at 0~C until freezing, since DMSO
concentrations of about 1% are toxic at temperatures above about 4~C.
A variety of suitable treatment solution containers are known in the art. The 15 containers (as well as the seals, ~loppels, caps and/or covers) can be constructed of any material(s) compatible with the treatment solution under conditions of use, e.g., processing, sterilization, and/or storage. For example, in those embodim~nt~ wherein the tre~tm~nt solution includes a cryoprotectant such as dimethyl sulfoxide (DMSO), the container is substantially chemically inert to DMSO for the rated storage period, 2 0 e.g., up to 3 years. In some embodiments, for example, involving the plepal~lion of stem cells, leaclling (e.g., caused by the DMSO reacting with the tre~tmPnt solution container) should be minimi7ed or prevented, so that the cells are as free of extraneous (impure and possibly toxic) material as possible. Extraneous material is undesirable as, for example, it can colllproll~ise the viability of the stem cells (initially, during 2 5 subsequent h~n~lling, and/or during transfusion) and adversely impact the recipient of the cells.
Suitable materials for use as containers for storing solutions comprising DMSO
include polytetrafluorethylene (PTFE), glass, and ceramics. In one ple~lled embodiment wherein the treatment solution includes DMSO, the container comprises30 borosilicate glass.

CA 022~9878 1999-01-22 In those embodiments wherein the treatment solution container has a stopper, seal, cover, and/or cap, at least the portion of the seal, cover, and/or cap cont~.ting the solution is substantially çhemic~lly inert to the solution. For example, in an embodiment wherein the treatment solution includes DMSO, and the container includes 5 a rubber stopper, the stopper is lined or faced with PTFE.
While the treatment solution container and the associated cap or cover cont~çting the treatment solution should be substantially inert to the treatment solution (since the solution is typically stored in the container), other components of the processing set 200, e.g., receiving container 232 and conduit 203, are typically1 0 exposed to the treatment solution for lesser periods of time. Additionally, in the receiving container 232, the treatment solution is diluted. Accordingly, these other components should be resistant to the treatment solution, but can be less resistant to the solution than the treatment solution container. For example, in one embodiment wherein the treatment solution comprises a mixture including DMSO, the receiving1 5 container 232 and conduit 203 comprise DMSO resistant material such as polyvinyl chloride (PVC) plasticized with a substantially non-blood extractable plasticizer, e.g., trioctyltrimellitate or triethyleh~yl~lill~ellilate (TOTM). Additionally, suitable containers and conduits include those produced in accordance with U.S. Patent No.
4,280,497. Typically, the various containers and conduits in the processing set 200 2 0 (other than the treatment solution container) are made from plasticized PVC.In some embodiments, the tre~tmçnt solution container and/or cover is designed to be yull~;luled~ e.g., by a peneLl~Lillg connector.
If desired, the Llea~llclll solution can be ~Lal~r~ d from the treatment solution container 211 through the metering system 250 to the receiving container 232 while 2 5 m~int~ining a closed sterile system. For example, a bacterial filter that is resistant to the treatment solution can be interposed in the treatment solution fluid flow path between the connector 215 and the receiving container 232. Altelllalively, or additionally, container 211 (preferably cont~ining a predetellllined volume of treatment solution) can be pre-connçctçcl to the metelillg system 250 before use, or can be 30 conn~cte~ via sterile docking.

After the treatment solution (e.g., a cryoprotectant mixture) is passed through the metering system 250 into the receiving container 232 to provide a mixture orsuspension of white blood cells (including stem cells) and treatment solution, the cell-con1~ining mixture is further processed. Accordingly, and using Figure 3 for 5 ~fe.ellce, the white blood cells suspended in treatment solution are typically passed through conduit 204 into freezing bag 234, and bag 234 is subsequently detached and frozen. Alternatively, in some embodiments, receiving container 232 can be detached and frozen, without using a separate freezing bag.
A variety of suitable freezing bags are suitable for carrying out the invention,1 0 and are known in the art. Suitable freezing bags include those disclosed in, for example, Illlell~ional Publication WO 96/17514. In a prefelled embodiment, the freezing bag includes a plurality of collli)a~ e"~, typically delimited by heat seal.
The embodiment of the freezing bag illustrated in Figure 3 includes a major portion 234a and a minor portion 234b, wherein each portion co~ "~ icates with its 1 5 own access port 228, 229, lespecli~ely. Each portion should be provided with indicia thereon (not shown) for identi~1c~tion of the specific unit. If desired, the freezing bag can include a line of demarcation aligned to seal off portions of the bag and defining a scoreline allowing one portion to be severed, without thawing, from the other portion.
For example, the cells in the minor portion can be allocated for one use, e.g., 2 0 culturing, and the cells in the major portion can be allocated for another use, e.g., transplantation .
The freezing bag cont~ining the white blood cells/treatment solution is gradually fro~n to an extremely low te"~l)el~Lu~e (e.g., in liquid nitrogen) as is known in the art, for example, as disclosed in U.S. Patent Nos. 5,004,681 and 5,192,553, European 2 5 Patent EP 0,343,217 B1, International Publication WO 96/17514, and A. Hubel, Transfusion Medicine Reviews~ Vol. 11, 1997, pp. 224-233.
Before use (e.g., in analysis, culturing, cloning techniques, and/or transplantation), the frozen cells must be thawed. Typically, the cells are washed to remove or minimi7.e the presence of the treatment solution.
3 o The cells are thawed as is known in the art, and the freezing bag 234 from CA 022~9878 1999-01-22 processing set 200 (Figure 3) is placed in fluid co.. ~ ication with the post thaw set 300A (Figures 1 and 5) or the post thaw set 300B (Figures 6 and 7).

POST THAW SET
Two embodiments of the post thaw set, illustrated respectively in Figures 5 (post thaw set 300A) and 7 (post thaw set 300B), are described below. As will bedescribed in more detail, the post thaw set is utilized to wash the thawed cells, e.g., to remove or ~--i-~i---i~e the presence of the treatment solution.
Typically, the thawed cells are passed into the container 344A (Figure 5) or 1 o 344B (Figure 7), and wash solution, e.g., from the container 345 (Figures 5 and 7) and/or introduced via at least one port or connector such as connector 342 (Figure 5) or port 386 (Figure 7), is utilized to rinse rem~ining cells from bag 234 into container 344A or 344B. Additional wash solution can be introduced and utilized to "wash" the cells to remove or ~--ini---i~e the presence of tre~tmPnt solution. Subsequently, 1 5 container 344A or 344B is centrifuged to provide a sediment fraction comprising the thawed white cells (including the stem cells), and a supellld~llL fraction comprising wash solution and treatment solution. As will be described in more detail below, the ~upelllata~l~ fraction is typically passed from the container 344A or 344B into container 345, leaving the white cells in container 344A or 344B for further processing.
2 0 The embodiment of the post thaw set 300A illustrated in Figure 5 includes a plurality of conduits 301-308, and containers 344A, 345. The illustrated set 300A also includes two or more connectors 342, 343 (such as spike connectors and/or luer lock connectors), access ports 334-337, and a plurality of flow control devices 31.
Similar to the embodiment shown in Figure 5, the embodiment of the post thaw 2 5 set 300B shown in Figure 7 includes a plurality of conduits 351-362, as well as containers 344B and 345. The illustrated embodiment of the post thaw set 300B also includes at least one connector 343 (e.g., a spike connector and/or a luer lock connector), access ports 337 and 384-386, and a plurality of flow control devices 31.
For convenience and clarity, the container 344A (Figure 5) and the container 3 o 344B (Figure 7) will be referred to below as the "transplant container," and container CA 022~9878 1999-01-22 345 (Figures 5 and 7) will be referred to below as the "wash container." If desired, the transplant container (344A, 344B) and the wash container (345) can be similar, or even identical in configuration and/or composition. For example, the transplant container 344A and the wash container 345 shown in Figure 5 are similar in configuration. In the embodiment illustrated in Figure 7, the transplant conliildcr 344B
and the wash container 345 have configurations that are more different. Illustratively, in contrast with wash container 345, the transplant container 344B has a length longer than the width, and includes a tapered or funnel-shaped end portion.
The post thaw set can include additional components such as, but not limited to,1 0 at least one of a tre~tment solution metering system, e.g., to transfer wash solution into transplant container 344A, 344B. Other components include, but are not limited to, at least one of a vent, a container, a conduit, a transfer leg closure, a sampling arrangement, and a filter such as a bacterial filter.
Typically, the various containers and conduits in the post thaw set are flexible1 5 and are m~n-lf~ctllred from plasticized polyvinyl chloride, although other materials (having other characteristics) are suitable for callyiilg out the invention.
The emboclim~o-nt~ of the post thaw set 300A and 300B are typically utilized as follows.
The cells in the freezing bag 234 (Figure 3) are thawed, and the bag 234 is 2 0 placed in fluid co""",~ tion with post thaw set 300A (Figure 5) or 300B (Figure 7).
The system overviews illustrated in Figures 1 and 6 show freezing bag 234 in fluid co~-".,~ ication with post thaw sets 300A and 300B lespe.;lively.
For example, and referring to Figures 3, 5, and 7, the comlectol~ 343 (shown with caps) can provide fluid access with major and minor portions (234a, 234b) of 2 5 freezing bag 234 via access ports 228 and 229. If desired, e.g., in some embodiments wherein the connectors are pe~ athlg connectors, the connectors can include one or more structures such as at least one shoulder or "stop" to more efficiently monitor and/or control the depth of penetration of the connector into the bag 234.
A wash fluid can be provided in wash container 345 (Figures 5 and 7).
3 0 Alternatively or additionally, wash fluid can be introduced into the post thaw set via CA 022~9878 1999-01-22 one or more access ports or connectors, e.g., port 337 (Figures 5 and 7), ports 334, 335, 336 (Figure 5), ports 384, 385, 386 (Figure 7) and/or through connector 342(Figure 5).
The use of wash fluid can improve the efficiency of cell recovery from freezing bag 234 (Figure 3), as the fluid rinses or washes the cells from the bag 234. Typically, the wash fluid is utilized to ex~h~n~e the tl~,dlllltlll solution inside and outside the white cells. For example, the wash fluid dilutes or reduces the concentration oftreatment solution (especially DMSO) in the extracellular environment, i.e., theellvholllllellt ~ulloullding the thawed white cells in a container. The wash fluid can 1 0 also dilute the concentration of the DMSO within the cells by exch~nging with the fluid in the cells. Moreover, as will be described in more detail below, it is sometimes desirable to control the rate and/or volume of wash fluid added to the stem cell mixture so as to promote a gradual change of extracellular fluid collcellllalion and ",i"i",i~e colll~rolllising cell illLeglily by osmotic forces.
A variety of wash fluids are suitable for callyillg out the invention. In one embodiment wll~leill the thawed cells to be washed were previously frozen in a cryoprotectant mixture including DMSO, the wash fluid comprises an isotonic fluid, preferably a colloid, e.g., albumin and dextran in a saline solution. Suitable wash fluids include those disclosed in, for example, U.S. Patent No. 5,192,553, European Patent 0,343,217 Bl, and T"lel.,~ional Publication WO 96/17514.
Typically, the thawed cells are passed from freezing bag 234 into transplant container 344A (Figures 1 and 5) or transplant container 344B (Figures 6 and 7), and some of the cells rem~ining in the freezing bag 234 are rinsed from the surface of the bag 234 into 344A or 344B using wash fluid. The wash fluid can be repeatedly passed in and out of the freezing bag 234, e.g., to further improve the efficiency of cell recovery.
If desired, additional volumes of wash fluid can be introduced into the post thaw set 300A, 300B. For example, additional "fresh" wash fluid can be introduced to rinse additional cells from the freezing bag 234 into the transplant container 344A, 3 0 344B. Alternatively, or additionally, fresh wash fluid can be introduced into the CA 022~9878 1999-01-22 transplant container to wash the cells, e.g., by decreasing the DMSO concentration inside and outside of the white cells. Illustratively, additional aliquots of wash fluid can be introduced through one or more access ports or connectors as described above.
If desired, various aliquots can be introduced through different ports and/or 5 connectors.
For example, using the embodiment illustrated in Figure 5 for lefelellce, an aliquot of wash fluid can be introduced via connector 342 and passed into freezing bag 234 to rinse cells from the bag into transplant container 344A. A subsequent aliquot (e.g., for washing the cells and decreasing the intracellular and extracellular DMSO
1 0 concentration) can be introduced into container 344A via another port, e.g., port 335.
In some embodimellts, at least an initial volume of wash fluid is passed into transplant container 344A or 344B prior to the stem cells being passed from the freezing bag into the transplant container. ~ltern~tively, or additionally, wash fluid can be introduced into the transplant container already having thawed cells disposed 1 5 therein.
In some embodiment wherein the transplant container 344A or 344B has thawed cells disposed therein, it may be desirable to introduce wash fluid into the transplant container in a controlled manner, e.g., the wash fluid can be ll~l~relled into the transplant container at a controlled rate.
2 0 For example, using Figure 5 for ~rerel~ce, in an embodiment wherein flow control device 31 associated with conduit 307 comprises an adjustable flow control device (e.g., a screw clamp), the flow control device 31 can be adjusted to provide a desired flow rate. Allell~lively, or additionally, conduit 307 can have a selected inner m~ter (e.g., to provide resistance) and/or the wash container 345 can be disposed at 2 5 a selected head height relative to the transplant container 344A during use to provide a desired prede~ll~ ed controlled flow rate without m~nll~lly clamping (e.g., constricting the inner ~ m~oter ofl the conduit to control the rate.
Since the stem cells are now mixed with the wash solution, the flow rate for passing the mixture to the other containers is typically not predetermined.
3 0 Accordingly, the mixture can be passed from container 344A, to freezing bag 234, and .. . .

CA 022~9878 1999-01-22 to wash container 345 using any conventional head height and/or tubing flow resi~t~nre.
In accordance with another embodiment, e.g., using Figure 7 for reference, after the wash solution is passed into the wash container 345 (e.g., after passing through conduit 362), the wash solution is passed from the wash container 345 into the stem cell-cont~ining transplant container 344B at a controlled rate. For example, the conduit 359 can have a selected inner (li~mPter, and the wash container 345 can be disposed at a selected head height relative to the transplant container 344B during use to provide a desired predete~ ed controlled flow rate without m~ml~lly clamping a 1 0 conduit. Illustratively, while conduit 359 can have a selected inner ~i~meter in the range of about .03 inches to about .06 inches, conduits 358 and 360 can have inner m~ters of, for example, about .15 inches.
Allelllalively, or additionally (using the embodiments illustrated in Figures 5 and 7 for reference), wash fluid can be passed from wash container 345 into freezing 1 5 bag 234 to rinse out rem~ining cells and the wash fluid and cells can be passed into transplant container 344A or 344B. Once the stem cells are mixed with the wash solution, the flow rate for passing the mixture to another location (e.g., another container) is typically not predelellllined. Accordingly, the llli~lule can be passed from container 344B, to freezing bag 234, and to wash container 345 (e.g., though 2 0 conduits 353, 358, 360, and 361) using any desirable head height and conventional tubing inner di~m~ter.
In accordance with any of these embodim~nt~, after the cells have been sufficiently washed (e.g., to ~,~i,-i,..i,~ the intracellular and extracellular DMSO
collcellll~lion), and sufficiently rinsed from the rl~;Gillg bag 234 into the transplant 2 5 container 344A (post thaw set 300A, Figure 5), or 344B (post thaw set 300B, Figure 7), the freezing bag 234 is typically disconn~cted from the post thaw set, e.g., by sealing and cutting conduit 303 (post thaw set 300A) or conduit 353 (post thaw set 300B).
After the cells are washed, they are typically centrifuged in transplant container 344A or 344B to provide a supelllalalll fraction conl~lisillg wash solution and CA 022~9878 1999-01-22 treatment solution, and a se~im~nt fraction comprising the white cells (including the stem cells). The ~u~e~ nt fraction is passed from transplant container 344A (e.g., via conduits 307 and 308), or 344B (e.g., via conduits 356, 355, 354, 358, 360 and 361) into wash container 345, leaving the white cells (in a diluted concentration of cryoprotectant solution) in transplant container 344A or 344B.
The white cells in the transplant container 344A or 344B can be further processed before use. For example, the cells can be diluted to further reduce the concentration of treatment solution present and/or to provide a volume suitable for ~(imini~tration to a recipient.

The previous detailed discussion focussed on processing the white blood cell-cont~ining fluid (i.e., cont~ining the stem cells) after it had been passed into the white cell container 232 of processing set 200 (Figures 1 (or 6) and 3).
However, another embodiment of the invention includes the collection set 100 1 5 (Figures 1 (or 6) and 2) for receiving the biological fluid (e.g., cord blood) to be processed to provide the white blood cell-cont~ining fluid that is passed into the receiving container 232.
Figure 2 illustrates an embodiment of the collection set 100 in more detail. Theillustrated embodiment includes two connectors 126, 127 (such as phlebotomy needles) 2 0 in fluid cc" ,~ nir~tion with first container 123 via conduits 120-123. A first access port 124 such as an injection port, and a second access port 125 such as a connector port, are interposed between the first penetrating connectors 126, 127, and the first container 123. The illustrated embodiment also includes a vent 138, and flow control devices 31. Each of the components of the illustrated collection set embodiment is 2 5 described in further detail below.
The first container 123, hereinafter referred to as the "collection container" can be any suitable container. In the illustrated embodiment, the collection container 123 is elongated (e.g., the length is longer than the width) and includes a tapered or funnel-shaped end portion. The collection container can also have any suitable volume. In a CA 022~9878 1999-01-22 plel~llcd embodiment, the collection container has a volume of about 250 ml or less, e.g., about 200 ml or less.
The collection container 123 can be made of any suitable material, and can be substantially inflexible (e.g., colll~rising a glass bottle) or flexible. For example, the 5 collection container can be a bag made from plasticized polyvinyl chloride (PVC), e.g., PVC plasticized with dioctylphth~l~te, diethylhexylphth~l~te, or trioctyltrimellitate.
Suitable containers include those disclosed in, for example, U.S. Patent No.
4,892,537. The collection container may also be formed from polymeric materials such as polyolefins, polyurethanes, or polycarbonates.
1 0 The connectors 126, 127 are typically pe~ ing connectors such as phlebotomy needles as are known in the art. Preferably, as shown in the illustrated embodiment, the collection set includes more than one connector, e.g., to allow the set to be fluidly conn~cte~ to more than one biological fluid source, and/or to more than one access point. Either or both connectors can be used. For example, both connectors can be used at dirrelelll locations (e.g., the ends of the umbilical cord) to draw or collect more fluid from the source, or the second connector can be used to draw or collect fluid if the first connector fails to allow the continued collection of fluid (e.g., due to clogging).
The collection set includes a plurality of conduits 120-123 providing fluid 2 0 co""""~-ic~tion between elements of the set. Plcrcll~,d conduits are flexible plastic tubing, e.g., m~mlf~ctured from plasticized PVC.
If desired, the set can include one or more access ports 124, 125 such as injection ports and/or connector ports, arranged anywhere in the set. These access ports, that can be utilized to connect other components to the set, and/or used while 2 5 adding and/or removing fluid from the set, are known in the art.
In one plefe~led embodiment the collection set 100 includes a vent 138. The vent 138 may be used to pass gas or air into, or out of, the collection set. Forexample, it may be desirable to introduce gas through the vent into the collection set to recover collected biological fluid retained or trapped in a component of the collection set 100, e.g., retained in conduits 122 and 123. The retained or trapped fluid can be CA 022~9878 1999-01-22 "chased" into the collection container 123 by the gas introduced through the vent 138.
Alle~ ely, gas may be passed out of the collection set 100 via the vent 138.
Preferably, the vent 138 prevents bacteria exterior to the set from passing through the vent. For example, suitable vents typically includes a hydrophobic filter medium5 having a pore rating of less than 0.2 micrometers (,um).
In some embodiments, with or without a vent, the collection set 100 also includes a bacterial filter for passing one or more fluids th~lell~ough. For example, it may be desirable to include a bacterial filter to m~int~in a closed system in those embo-lim~-nt~ wherein one or more fluids are introduced into the set, or additional 1 0 components are connected to the set.
In an illustrative embodiment wherein the biological fluid to be handled is cordblood, the cord blood is collected and processed in collection set 100 as follows. An umbilical cord is clamped to retain cord blood therein, and one or more connectors 126, 127 such as phlebotomy needles are used to puncture the cord blood vessels, and 15 cord blood is drained theLerlom into collection container 123. Typically, collection container 123 includes anticoagulant disposed therein, to prevent coagulation of the collected blood.
If desired, gas accl-m~ t~l in the set can be passed from the set through vent 138 to recover blood retained in a component of the set such as a conduit 122.
2 0 Alternatively, or additionally, gas displaced by the blood passing through the set 100 (e.g., into collection container 123) can be passed from the set through vent 138.
In an embodiment, a sedilllelll~tion agent such as hydroxyethyl starch is added to the collection container 123, e.g., through access port 124. In some embodiment, e.g., wherein a closed system is desired, the sedimentation agent is passed through a 2 5 bacterial filter interposed in the fluid flow path between the source of the sedimenting agent, and the access port 124.
The collection container 123, including the cord blood, anticoagulant, and the sedimentation agent, is celllliruged as is known in the art (e.g., as disclosed in International Publication No. WO 96/17514), to form a ~u~elllatant fraction 3 0 comprising white blood cells (including the stem cells) and plasma, and a sediment CA 022~9878 1999-01-22 fraction colllplising red blood cells, some plasma, and the ~edim.orlt~tion agent.
After connPcting collection set 100 (Figure 2) with processing set 200 (Figures 1 and 3), the supernatant white cell-rich fraction is passed from the collection set 100 into processing set 200. Illustratively, flow control devices such as clamps associated 5 with the a~pro~liate conduits are opened or closed to allow a suitable flow path, and a pres~u,e dirr.,lelltial is created, e.g., by operating an expressor having the collection container 123 disposed therein, causing the supernatant white cell-rich fraction to pass into processing set 200 via conduit 201. The white cell-rich fraction is received in white cell container 232, and processed as described earlier.
1 0 In accordance with an embodiment of the invention, the sedimentation of the biological fluid in the collection set 100 can be optimi7ed, e.g., to improve the efficiency of centrifugation and subsequent separation of the white cell-rich supernatant fraction from the red cell-rich sedh~lll fraction.
For example, the size of the collection container 123 can be less than that of a1 5 conventional blood bag, e.g., to provide a volume of about 200 ml or less, in contrast with a volume of about 500 ml for a conventional blood bag. Since the container has a reduced volume, the container can be filled more completely, thus mi~ i n~ the potential for collapsing and/or folding over during centrifugation.
Additionally, as shown in Figures 8A and B, a support or holding device 700 can be provided to further improve the efficiency of sedi~"~ ion and centrifugalseparation of the conlpol~elll~ of the biological fluid. For example, the support or holding device 700 can l-,ini."i~ the potential for container collapse during centrifugation and/or while tlansr~llillg the container to an expressor. An advantage of mi~ g collapse during centrifugation and/or llal~rt;l is to allow, and m~int~in,2 5 efficient separation of fluid fractions. Additionally, the support or holding device 700 can allow increased exposure of the biological fluid to centrifugal force duringcentrifugation by spreading fluid along more of the length of the container. Moreover, the device 700 can improve the exposure of the container to the range of centrifugal force, e.g., while the centrifuge bucket cont~inin~ the container is extended outward.
3 0 As shown in Figures 8A and B, one embodiment of the support or holding .

CA 022~9878 1999-01-22 device 700 comprises supports 701,702 such as plates that can be placed facing the major sides of the collection container 123. The supports can be held in position by a retainer arrangement 703, such as one or more straps or bands (e.g., cont~inin~ an adhesive, or hooks and loops such as Velcro~ straps; or rubber bands). The collection container 123, interposed between the supports 701, 702 of the device 700, is subsequently placed in a centrifuge cup, and centrifuged. The container and device can be placed directly in the ce,llriruge bucket, or in a centrifuge bucket insert that is placed in the cup, with the long axis of the container arranged along the radius of centrifugation .
1 0 After centrifugation, the container and device can be placed directly in an expressor, if desired.
Since the container is preventing from collapsing, the efficiency of separation is increased by improving the distribution of fluid in the centrifugal field. Accordingly, the red cells are sedimented more efficiently, particularly in those embodimentswherein a serlimen~tion agent is utilized to accelerate the settling of the red cells.
Moreover, since the bag is prevented from collapsing after centrifugation, the device 700 assists in mi~-i"~ g the dislu,l,allce of the separation interface between the supe"lata"L and sediment fractions during transfer to the expressor and during the subsequent SUpe~ lnl ~ression. As a result, more of the white blood cells can be2 0 passed with the ~upe"~ta"l fraction into processing set 200.

ADDITIONAL SYSTEM COMPONENTS
In accordance with embodi",enls of the invention, it can be desirable to take one or more samples (e.g., of a biological fluid component) at any stage(s) of the fluid 2 5 h~-lling protocol. Samples can be obtained by lltili7.ing one or more access ports disposed anywhere in the system 1000, e.g., in sets 100, 200, 300A and/or 300B.
Alternatively, or additionally, as shown in Figure 9B and 9C, the system can include one or more sampling arrangements 500 comprising a container 2 including a resilient portion and having closed end 6 and an open end 7, and a cap 3, wherein the 3 0 arrangement 500 provides for collecting the sample. The embodiments of the CA 022~9878 1999-01-22 arrangement shown in Figures 9A and 9B also include conduits 11-13, and conduit connector 20 (Figure 9B only).
In an illustrative method of using the sampling arrangement, the container is compressed and decomplessed one or more times to allow the container 2 to fill with 5 the fluid for sampling. The container 2 cont~ining the sample therein can be separated from the set, e.g., after sealing the conduits co"",~ irating with the container, without coml)lo"~ising sterility.
The container 2 in the sampling arrangement 500, and/or container(s) 123, 232-234, 344A, 344B and/or 345, include indicia thereon for identification of the specific 1 0 sample and the source.

EXAMPLE
This Example illustrates the use of a preform~ tecl sterile solution, and the controlled addition of the solution in accordance with an embodiment of the invention.
A cryopl~lec~nl ll~ lule is plel)a,ed by combining one volume of 100%
dimethyl sulfoxide (DMSO), one volume of 10% dextran 40 (40000 MW) in water, in a precision filled system wherein a borosilicate glass ambot is filled and subsequently sealed with a teflon-faced rubber stopper having an ~hllllilllllll seal. The ambot has an internal volume of 10 ml, and contains 7 ml of the cryoprotectant mixture, and is 2 0 sealed m~int~ining the sterility of the contents, i.e., the ambot is le~ ally sterilized.
A white cell-enriched, plasma-depleted fluid, comprising stem cells, is preparedin a closed system in accordance with the invention. The receiving container having 20 ml of white cell-enriched, plasma volume-reduced fluid disposed therein is a flexible blood bag.
2 5 A cryoprotectant mixture metering system is configured as shown generally in Figure 3, and is placed in fluid coll~ ir-ation with the ambot as shown generally in Figures 4 A-E.
The receiving container has a 50 cm length of 0.035" ID microbore tubing integrally ~tt~ch~.~l thereto. Both the tubing and the receiving container are fabricated 3 0 from polyvinyl chloride resin plasticized with triethylhexyltrimellitate (TOTM).

Attached to the tubing is a vented spike. The spike includes a 0.2 micrometer bacteria retentive vent filter, a plastic base and a beveled-tip needle. The spike also includes a vent cap (for the vent filter) that is initially closed.
Using Figures 4 A-D for reference, a positioning jig 17 is slidably engaged withthe ambot wherein extensions 17(e) engage with the collar of the arnbot. The jigincludes an aperture 17(b) of sufficient size to pass the needle the~ ough. The needle is passed through the aperture and pellellates the stopper of the ambot, and the tip of the needle extends 1.3 cm into the interior of the container. The jig is slidably engaged with the handle of the spike whcleill the ends of spike handle 213 fit within slots 17(h) as side walls 17(c) move toward the spike.
By using such a jig, the tip of the spike can be reproducibly positioned inside the treatment solution container.
The ambot is inverted, and the vent is uncapped to prime the system. The walls of the receiving container are pulled apart to create a vacuum. The cryoplolectalll passes from the ambot toward the receiving container to prime the tubing. Once the cryoprotectant reaches the port of the receiving container, the vent is capped and flow stops. The receiving container, that contains the white cell-enriched fluid, is placed between two cold packs (not shown) on a commercially available agitator or shaker 240.
2 0 The ambot, as well as the jig and spike, is positioned as shown generally in Figure 4E using a stand (not shown) to provide a head height of 30 cm. The vent is uncapped, and flow resumes. The re~i~t~nt~e of the microbore tubing and the headheight provide a controlled predetermined flow rate of about 0.3 ml per minute. The flow stops autom~ti~ally when the level of cryoprotectant in the ambot reaches the 2 5 bevel of the needle. Thus, the system delivers a controlled predetermined volume of 5 ml.

All of the references cited herein, including publications, patents, and patent applications, are hereby incorporated in their entireties by refelellce.
3 0 While the invention has been described in some detail by way of illustration and CA 022~9878 1999-01-22 example, it should be understood that the invention is susceptible to various modifications and alternative forms, and is not restricted to the specific embodiments set forth. It should be understood that these specific embodiments are not intended to limit the invention but, on the contrary, the intention is to cover all modifications, 5 equivalents, and alternatives falling within the spirit and scope of the invention.

.

Claims (25)

1. A fluid metering system comprising:
a connector suitable for providing fluid communication with a first container, the first container comprising a treatment solution container having a volume oftreatment solution disposed therein;
a conduit in fluid communication with the connector, wherein the system is dimensioned for transferring from the first container intoa second container downstream of the conduit a controlled predetermined volume of the treatment solution that is less than the volume of the treatment solution in the first container.

2. A fluid metering system comprising:
a connector suitable for providing fluid communication with a first container, the first container comprising a treatment solution container and having a defined internal volume;
a conduit in fluid communication with the connector, wherein the system is dimensioned for transferring from the first container intoa second container downstream of the conduit a controlled predetermined volume of the treatment solution that is less than the internal volume of the treatment solution container.

3. A fluid metering system comprising:
a connector suitable for providing fluid communication with a first container, the first container comprising a treatment solution container having a volume oftreatment solution disposed therein;
a conduit in fluid communication with the connector, wherein the system is dimensioned for transferring from the first container intoa second container downstream of the conduit a controlled predetermined volume of the treatment solution at a controlled predetermined rate, the controlled predetermined volume being less than the volume of the treatment solution in the first container.

4. A fluid metering system comprising:
a connector suitable for providing fluid communication with a first container, the first container comprising a treatment solution container having a volume oftreatment solution disposed therein;
a conduit in fluid communication with the connector, wherein the system is dimensioned for transferring from the first container intoa second container downstream of the conduit the treatment solution at a controlled predetermined rate.

5. A fluid metering system comprising:
a conduit suitable for providing fluid communication with a first container, thefirst container comprising a treatment solution container having a volume of treatment solution disposed therein, wherein the system is dimensioned for transferring from the first container intoa second container downstream of the conduit the treatment solution at a controlled predetermined rate.

6. A fluid metering system comprising:
a conduit suitable for providing fluid communication with a first container, thefirst container comprising a treatment solution container having a volume of treatment solution disposed therein, wherein the system is dimensioned for transferring from the first container intoa second container downstream of the conduit a controlled predetermined volume of the treatment solution that is less than the volume of the treatment solution in the first container.

7. The system of claim 1 or 2, wherein the system is dimensioned for transferring the controlled predetermined volume of the treatment solution at a controlled predetermined rate.

8. The system of any one of claims 1-5, further comprising a fitting interposed between the connector and the first container.

9. The system of claim 8, wherein the fitting is dimensioned to engage with the connector and the first container.

10. The system of claim 9, wherein the fitting is re-usable.

11. The system of any one of claims 1-8, wherein the connector comprises a penetrating connector.

12. The system of any one of claims 1-7 for transferring a treatment solution including a cryoprotectant.

13. The system of any one of claims 1-8, or 11, wherein the system includes a vent.

comprising a porous medium.

14. The system of any one of claims 1-8, or 11, further comprising the fluid treatment solution container.

15. A biological fluid treatment system comprising:
a sealed terminally sterilized container;
a biological fluid treatment fluid mixture including a cryoprotectant;
wherein the sealed terminally sterilized container has the fluid mixture disposed therein.

16. The system of claim 15, wherein the sealed container has a predetermined volume of fluid mixture disposed therein.

17. The system of any preceding claim, suitable for use in a closed system.

18. A method for transferring a treatment solution comprising:
passing a controlled predetermined volume of a treatment solution through a fluid metering system.

19. A method for transferring a treatment solution comprising:
passing a treatment solution through a fluid metering system at a controlled predetermined rate.

20. A method for transferring a treatment solution comprising:
passing a controlled predetermined volume of treatment solution through the system at a controlled predetermined rate.

21. The method of any preceding claim, including automatically stopping the flow of treatment solution through the system.

22. The method of any preceding claim, wherein the treatment solution comprises a fluid mixture including a cryoprotectant.

23. The method of any preceding claim, further comprising passing the treatment solution into a container having white blood cells disposed therein.

24. The method of any preceding claim, carried out while maintaining a closed system.

25. A set for processing a white blood cell-containing fluid comprising:
a first container plasticized with triethylhexyltrimellitate a second container suitable for freezing white blood cells, wherein said second container is in fluid communication with the first container.