CA2482045A1 - Method of cryopreserving cells - Google Patents
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- CA2482045A1 CA2482045A1 CA 2482045 CA2482045A CA2482045A1 CA 2482045 A1 CA2482045 A1 CA 2482045A1 CA 2482045 CA2482045 CA 2482045 CA 2482045 A CA2482045 A CA 2482045A CA 2482045 A1 CA2482045 A1 CA 2482045A1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0278—Physical preservation processes
- A01N1/0284—Temperature processes, i.e. using a designated change in temperature over time
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
Abstract
A non-linear cryopreservation protocol and method for improving cryopreservation protocols for cells involves producing a simulation of cellular responses to a range of cooling parameters; determining optimal cooling parameters required to minimize cryoinjury to the cells; and incorporating optimal parameters into the protocol. The simulation is based on mathematical models of cellular parameters. A non-linear cryopreservation protocol for cryopreserving stem cells is also diclosed.
Description
Technical Field This application relates to methods of cryopreservation, particularly methods of cryopreserving cells and tissues.
Background Cryobiology is the study of the effects of low temperatures on biological systems. Although freezing is lethal to most living systems, cryobiologists have been able to preserve cells and tissues at a range of subzero temperatures, as low as liquid nitrogen temperatures (-196°C).
Currently, cryopreservation' can be applied to most cells in suspension, such as stem cells, other progenitor cells, red and white blood cells, sperm cells, oocytes, ova; and cellular materials derived from tissues and organs (including but not limited to pancreatic islet cells, chondrocytes, cells of neural origin, cells of hepatic origin, and cells of cardiac origin). Cryopreservation has also been used to effectively preserve tissues, such as heart valves, embryos, skin, articular cartilage, and islets of Langerhans and an increasing range of engineered tissues and tissues constructs. Although the current recovery rates of viable cells post-thaw may be sufficient for some clinical uses, recovery rates are generally considered less than optimal due to injury during the freezing process.
Cryobiology has been applied to many cell and tissue types. Recent developments in the utilization of a variety of stem cells; including cord
Background Cryobiology is the study of the effects of low temperatures on biological systems. Although freezing is lethal to most living systems, cryobiologists have been able to preserve cells and tissues at a range of subzero temperatures, as low as liquid nitrogen temperatures (-196°C).
Currently, cryopreservation' can be applied to most cells in suspension, such as stem cells, other progenitor cells, red and white blood cells, sperm cells, oocytes, ova; and cellular materials derived from tissues and organs (including but not limited to pancreatic islet cells, chondrocytes, cells of neural origin, cells of hepatic origin, and cells of cardiac origin). Cryopreservation has also been used to effectively preserve tissues, such as heart valves, embryos, skin, articular cartilage, and islets of Langerhans and an increasing range of engineered tissues and tissues constructs. Although the current recovery rates of viable cells post-thaw may be sufficient for some clinical uses, recovery rates are generally considered less than optimal due to injury during the freezing process.
Cryobiology has been applied to many cell and tissue types. Recent developments in the utilization of a variety of stem cells; including cord
-2-blood stem cells have revived interest in optimizing cryopreservation techniques for cells and tissues (D. Krause, 2002). In particular, for stem cells and other cell types which axe obtained in low numbers from donors, high recovery of these cell types is crucial. High recovery is also important in cryopreservation of engineered cells due to the high cost and length of time for manufacturing such cells. Emerging higher standards for cell and tissue banking (Guide to safety and quality assurance for organs, tissues and cells, 2'~ edition; 2004, Council of Europe Publishing, France); specifically stem cell banking, will be required to meet future needs of cell banking and therefore, optimal cryopreservation techniques are fundamental.
Currently, cryopresexvation of cells has been most successful with the use cryopreservants and cryopreservation of stem cells has been most successful with the use of dimethyl sulfoxide (DMSO). There are, however, limitations to the use of DMSO. Toxicities have been associated with infusion of stem cells preserved with DMSO (Davis et al. , 1990; Egorin et al. ; 2001; Santos et al. , 2003; Zambelli et al. , 1998). Some researchers have attempted to reduce the amount of DMSO
(Abrahamsen et al., 2002; Beaujean et al., 1998) or combine it with a non-penetrating cryopreservant, such as Hydroxyethyl staxch (HES) (Donaldson, 1996; Halle et al,, 2001; Katayama et al., 1997).
It has also been previously demonstrated that some cells can be cryopreserved without the use of a specific cryoprotectant such as DMSO. In cryopreservation', procedures, cells are generally cooled at a constant rate which is optimized for the cell type and cryopreservant.
Currently, cryopresexvation of cells has been most successful with the use cryopreservants and cryopreservation of stem cells has been most successful with the use of dimethyl sulfoxide (DMSO). There are, however, limitations to the use of DMSO. Toxicities have been associated with infusion of stem cells preserved with DMSO (Davis et al. , 1990; Egorin et al. ; 2001; Santos et al. , 2003; Zambelli et al. , 1998). Some researchers have attempted to reduce the amount of DMSO
(Abrahamsen et al., 2002; Beaujean et al., 1998) or combine it with a non-penetrating cryopreservant, such as Hydroxyethyl staxch (HES) (Donaldson, 1996; Halle et al,, 2001; Katayama et al., 1997).
It has also been previously demonstrated that some cells can be cryopreserved without the use of a specific cryoprotectant such as DMSO. In cryopreservation', procedures, cells are generally cooled at a constant rate which is optimized for the cell type and cryopreservant.
3 This optimization has typically been approached empirically by varying cooling rates and the nature and concentration of cryopreservants. In addition to cooling at a constant rate, two other techniques have been described to examine the effects of low temperatures on cells: a two-step freezing technique and a graded freezing technique. The two-step freezing technique {J . Farrant et al. , 1974) is a logical method to examine the effects of osmotic interactions on cell recovery over a broad range of subzero temperatures. In this procedure, lymphocytes were cooled rapidly to various subzero temperatures and held for various periods of time before being 1) thawed directly from that holding temperature or 2) rapidly cooled to -196°C before thawing.
McGann and Farrant later reported that the subzero temperature and the length of hold time at that temperature were important factors to consider when attempting to maximize cell survival {McGann and Farrant, 1976). The graded freezing technique was later developed by McGann and used to determine the temperature range through which slow cooling should be controlled (McGann, 1979). Samples were cooled slowly to various subzero temperatures before being either thawed directly or plunged into liquid nitrogen first and then thawed.
These experimental techniques provided insights into the effects of subzero temperatures and time; which can be used to empirically optimize a cryopreservation procedure.
There is significant interest in designing an optimized cryopreservation protocol for all cell types and tissues, which maintains cell and tissue viability but does not require toxic cryopreservants.
McGann and Farrant later reported that the subzero temperature and the length of hold time at that temperature were important factors to consider when attempting to maximize cell survival {McGann and Farrant, 1976). The graded freezing technique was later developed by McGann and used to determine the temperature range through which slow cooling should be controlled (McGann, 1979). Samples were cooled slowly to various subzero temperatures before being either thawed directly or plunged into liquid nitrogen first and then thawed.
These experimental techniques provided insights into the effects of subzero temperatures and time; which can be used to empirically optimize a cryopreservation procedure.
There is significant interest in designing an optimized cryopreservation protocol for all cell types and tissues, which maintains cell and tissue viability but does not require toxic cryopreservants.
-4-Summary of Invention This invention relates to a non-linear cryopreservation protocol for cryopreserving cells comprising determining an optimal cooling profile for maximum recovery of the cells and applying the cooling profile to the cells. The optimal cooling profile is determined using a simulation of cellular responses to cooling parameters. The cooling parameters comprise cell temperature, duration of temperature exposure, cooling level, cooling rate and presence or absence of cryopreservants. The cellular responses are determined from mathematical models of extracellular concentration parameters; intracellular concentration parameters, and cellular osmotic permeability parameters.
The cryopreservation protocol can be used with cells stored without cryopreservants. The protocol can also: be used with cells stored with cryopreservants, including r~on-penetrating cryopreservants. Such non-penetrating cryopreservants include sugars, starches, serum, or plasma.
The invention can be applied to any types of cells, including stem cells, other progenitor cells; red and white blood cells, sperm cells, ooeytes, ova, cells for research or transplant purposes, and cellular materials derived from tissues and organs (including but not limited to pancreatic islet cells, chondrocytes, cells of neural origin, cells of hepatic origin, and cells of cardiac origin). Throughout this application, cells include cells organized as tissues: Stem cells include human peripheral blood stem cells, human umbilical cord blood stem cells and stem cells derived from tissues and solid organs or other sources, including fetal and or embryonic sources. The invention is not limited to human cell types and is extendable to all mammalian and non-mammalian species.
In one embodiment of the invention, the non-linear cryopreservation protocol comprises cooling the cells to a first temperature for a first period of time, then cooling: the cells to a storage temperature for a second period of time prior to thawing.
In another embodiment of the invention, the non-linear cryopreservation protocol can be executed on cells in small samples. The invention can also be applied using a bulk freezing unit or a cryomicroscopy apparatus. The bulk freezing unit comprises a controller for executing the cryopreservation protocol, and the controller communicates with a computer to receive the cryopreservation protocol. The controller is connected to heaters surrounding the cells for maintaining the temperature of the cells, and' thermocouplers monitor the temperature of the cells.
The invention also relates to methods of optimizing cryopreservation protocols by determining: an optimal cooling profile and applying the profile to cryopreservation protocols. The invention also relates to cryopreservation protocols optimized by the method of the invention.
The invention also relates to non-linear cryopreserving protocols for cryopreserving stem cells In a specific embodiment of the invention, the method of cryopreserving stem cells comprises cooling the stem cells to a first temperature for a first period of time; then cooling the cells to-a second temperature for storing the stem cells. In one embodiment, the stem cells are cooled to a temperature between -S°C and -15°C for 1 to 3 minutes, then cooled to -196°C to store the cells.
Brief Description of Drawings Fig. 1 Simulation program user interface Fig. 2 Cell volume vs temperature at different cooling. rates Fig. 3 Maximum supercooling and maximum [KCl] as a function of cooling rate Fig. 6 TF-1 cell volume distribution Fig. 7 Boyle van't Hoff plot of TF-1 cells Fig. 8 Cell volume kinetics of TF-t cells in hypertonic solutions Fig. 9 Arrhenius plot of TF-I cells Fig. 10 chematic of CryoSimS program Fig. 11 raded freezing cooling profiles of TF-I cells Fig. 12 Simulated cell volume kinetics of TF-I cells Fig. 13 Simulated supercooling kinetics of TF-1 cells during graded freezing Fig. 14 Simulated [KCI]; kinetics of TF-1 cells during graded freezing Fig. 15 Simulated maximum supercooling of TF-1 cells using various cooling rates Fig. 16 Simulated maximum [KCI]; of TF-1 cells using various cooling rates Fig. 17 Membrane integrity-of TF-i cells in 10% DMSO
Fig. 18 Membrane integrity'of TF-1 cells using 0.2°C/rain Fig. 19 Membrane integrity of TF-1 cells using 0.5°Clmin Fig. 20 Membrane integrity of TF-1 cells using 0.9°C/rain Fig. 21 Experimental determination of two-step cooling profiles Fig. 22 Simulations of two-step cooling profiles of TF-1 cells Fig. 23 Simulated cell volume kinetics of TF-1 cells Fig. 24 Simulated supercoohng kinetics of TF-1 cells during two-step freezing Fig. 25 Simulated [KCI], kinetics of TF-1 cells during two-step freezing Fig. 26 Simulated maximum supercooling and [KCI]; of TF-1 cells held for various durations Fig. 27 Simulations of optimal plunge temperatures ranges for TF-1 cells cooled using two-step freezing Fig. 28 Membrane integrity of TF-1 cells using 3 minutes hold time g Fig. 29 Membrane integrity of TF-1 cells held at -5°C and -25°C for various durations Fig. 30 Contours of membrane integrity of TF-1 cells using two-step freezing for various durations Detailed Description of the Invention Throughout the following description, specific details are set forth in order to provide a more thorough understanding of the invention.
However, the invention may be practiced without these particulars. In other instances, well known elements have not been shown or described in detail to avoid unnecessarily obscuring the invention. Accordingly, the specification and drawings are to be regarded in an illustrative, rather than a restrictive, sense.
This invention relates to a non-linear cryopreservation protocol for cryopreserving cells comprising determining an optimal cooling profile for maximum recovery of the cells and applying the cooling profile to the cells. The optimal cooling profile is determined using a simulation of cellular responses to cooling parameters. The cooling parameters comprise cell temperature, duration of temperature exposure, cooling level; cooling rate and presence or absence of cryopreservants. The cellular responses are determined from mathematical models of extracellular concentration parameters, intracellular concentration parameters, and cellular osmotic permeability parameters.
The cryopreservation protocol can be used with cells stored without cryopreservants. The protocol can also be used with cells stored with cryopreservants, including non-penetrating cryopreservants. Such non-penetrating cryopreservants include sugars, starches, serum, or plasma.
The invention can be applied to any types of cells, including stem cells, other progenitor cells, red and white blood cells, sperm cells, oocytes, ova, cells for research or transplant purposes, and cellular materials derived from tissues and organs (including but not limited to pancreatic islet cells, chondrocytes; cells of neural origin, cells of hepatic origin, and cells of cardiac origin). Throughout this application, cells include cells organized as tissues. Stem cells include human peripheral blood stem cells, human umbilical cord blood stem cells and stem cells derived from tissues and solid organs or other sources, including fetal and or embryonic sources: The invention is not limited to human cell types and is extendable to all mammalian and non-mammalian species.In one embodiment of the invention, the non-linear cryopreservation protocol comprises cooling the cells to a first temperature for a first period of time, then cooling the cells to a storage temperature for a second period of time prior to thawing.
In another embodiment of the invention, the non-linear cryopreservation protocol can be executed on cells using a bulk freezing unit or a cryomicroscopy apparatus. The invention also relates to methods of optimizing cryopreservation protocols by determining an optimal cooling profile and applying the profile to cryopreservation protocols.
The invention also relates to cryopreservation protocols optimized by the method of the invention.
- 1~ -The invention also relates to non-linear cryopreserving protocols for cryopreserving stem cells In a specific embodiment of the invention, the method of cryopreserving stem cells comprises cooling the stem cells to a first temperature fox a first period of time; then cooling the cells to a second temperature for storing the stem cells. In one embodiment; the stem cells are cooled to a temperature between-5°C and -15°C for 1 to 3 minutes, then cooled to -196°C to store the cells.
~.7escription of Simulation 3'00l Reports of high cell recovery in the absence of permeating cryopreservants such as DMSO (J. Farrant et al.; 1974) indicate that it is feasible to eliminate such cryopreservants from cryopreservation protocols. Instead of using cryopreservants to alter the properties of solutions so that cells rnay be cooled at a constant rate (normally 1 °C/min) and achieve high recovery, in one aspect of the invention, the inventors' recent approach (Ross-Rodriguez, 2003(a); Ross-Rodriguez, 2003(b)) has been to use the properties of the intracellular and extracellular solutions of subject cells to design optimal cryopreservation protocols. A novel aspect of this approach is that the temperature profile is not constrained to be linear. Rather, the intracellular and extracellular solution properties, along with cellular osmotic properties, are used to generate a temperature profile that minimizes cryoinjury.
During the course of the inventors' research on cryoinjury and cryoprotection, the inventors have developed a mathematical model of cellular osmotic responses at low temperatures using real (nondilute) solutions assumptions for both the carrier solution and the cellular cytoplasm (McGann and Elliott; 2003). In one embodiment of the invention, this model has been implemented in a computer program as a simulation tool to calculate intracellular and extracellular parameters and properties as the temperature changes and ice forms or melts in the cells. The simulation tool generates an ideal temperature range and duration range for cooling the cells and obtaining optimal recovery of the cells upon thawing. The tool can be applied to any cell type by measuring appropriate parameters of the cells.
In some embodiments of the invention, simulations are based on changes in the composition of the extracellular solution as water is converted to ice during cooling, and the osmotic responses of cells to these changes:
a) Features of the Simulation This simulation uses nondilute solution equations in the calculations, and allows accommodation of the contribution of proteins to the solution properties of the cytoplasm. In one embodiment, the program calculates cellular responses based on a programmed temperature profile generated by specifying rates of temperature change; or by providing measured temperature profiles to simulate real cryopreservation systems. In a second embodiment, the program allows imposition of specific design criteria, including the calculation of a cooling profile that maintains a constant level of cooling in the cell, thereby minimizing exposure to the environment of concentrated solutions.
b) Determination of Solution Properties Since dilute solution assumptions do not apply as solutes concentrate in both the extracellular and intracellular compartments, nondilute descriptions of the solution properties have been utilized. The inventors have found the osmotic virial equation to be simple yet sufficient for many biological aqueous solutions. The osmotic virial equation was fit to published or measured data on freezing point as a function of concentration, and used in the simulations to describe the freezing point (TFP) of binary solutions, taking into account the non-ideal behaviour of real solutes:
Tpp =K~ w(lYI+BZ ~lYl2 +B3 ~YI23) where m is the molality of the solute, and K, , B2 and B3 are fitting constants for specific solutes in water: For electrolytes, the molality is multiplied by a dissociation parameter. The constant B3 is non-zero only for solutes with highly nonlinear behaviours, such as polymers. The following relationship was used to calculate freezing point depressions in solutions containing multiple (n) solutes (at most one of which has non-zero B3);
T~ - ~~ ' ~ m= + Bar ' ~2 + B3J ~ m= + ~ ~~Bzt + Bz; )' m1 ' m; ~ (2) i=1 .. n j=i+i :. n The last term uses Guggenheim's naive approximation to describe interactions between solutes in the solution (Elliot et al, Cryobiology 2002). In these simulations; it was assumed that the primary intracellular electrolyte is potassium chloride, and the primary extracellular electrolyte is sodium chloride. Proteins and starches exhibit highly nonlinear freezing point depression as the concentration increases; so the value of B3 in equation (2) is significant. In one case, the inventors found it necessary to include intracellular protein to more accurately describe the cytoplasm as the solution concentrates in the presence of ice (Ross-Rodriguez, 2003(x)).
c) Osmotic Transport of Water and Solutes The Johnson & Wilson model (Johnson and Wilson, 1967) was used to describe coupled water and solute transport across the plasma membrane:
_dV=LP.A.R:T~ ~Unwn~+~s'(~s-mss) dt n dSs - Ps ' (QCs ) -~ (l - O_s ) . Cs . d dt dt where V is cell volume at time t; Lp is membrane hydraulic conductivity, A is surface area of the cell; R is the gas constant; T is absolute temperature, is osmolality; and is the Staverman reflection coefficient. Superscripts a and a refer to the intracellular and extracellular compartments; respectively, and the subscripts h and s refer to nonpermeating and permeating solutes: S is the moles of solute, ~'s is the solute permeability, CS is the difference in solute concentration across the membrane, and CS is the mean solute concentration.
d) Temperature Dependence of Osmotic Parameters The Arrhenius equation wad used to describe the temperature dependence of the osmotic permeability parameters.
_E _1 1 1p ~'~ng ' a ~ T' T (4.) where LP is the hydraulic conductivity at any temperature T, Lpg is the hydraulic conductivity at a reference temperature Tg, Ea is the activation energy and R is the gas constant. A similar Arrhenius equation was used to describe the temperature dependence of the solute permeability.
Equations (1) and (2) can be made more complex as needed by using other solution thermodynamics equations from the literature. In addition, equation (2) can be expanded to the case of multiple components with non-zero B3's by adding a term, or terms analogous to the second order last term of equation (2), but of appropriate order.
There are many equations, both in the literature and to be derived in the future based on physical analysis, that can replace equation (3).
e) Computer Program far 5'imulation of Cellular Low Temperature Responses A computer program was developed (using the Delphi programming language) to perform the simulations based on either calculated or IS
measured temperature profiles. The program also includes a function to generate the temperature profile required to maintain the cytoplasm at a constant level of cooling. Parameters for equation (1) to describe solution properties for a variety of solutes are read from data files. In addition to the resulting terriperature profile, all calculated parameters are reported as a function of time to allow access to both intracellular and extracellular concentrations and fluxes.
Figure 1 shows the user interface of the program to illustrate the input parameters .
The sample output in Figure 2 shows calculated cell volumes as a function of temperature during cooling at different rates.
,~ Predictors of Cryoinjury Based on Mazur's two-factor hypothesis (Mazur et al. , 1972), the inventors used the amount of cooling as a pr~lictor of cryoinjury related to intracellular ice fo~nation, and the intracellular KCl concentration as a predictor for cryoinjury related to exposure to concentrated solutions.
Simulations allow determination of the maximum cooling and maximum intracellular KCI concentration in cells cooled at different rates down to -40°C, as shown in Figure 3.
Application of Method to Different Cell Types The invention can be applied to any types of cells, including stem cells, other progenitor cells, red and white blood cells, sperm cells, oocytes, ova, cells for research or transplant purposes; and cellular materials derived from tissues and organs (including but not limited to pancreatic islet cells, chondrocytes; cells of neural origin, cells of hepatic origin, and cells of cardiac origin): Stem cells include human peripheral blood stem cells, human umbilical cord blood stem cells and stem cells derived from tissues and solid organs or other ounces, including fetal and or embryonic sources. The invention is not limited to human cell types and is extendable to all mammalian and non-mammalian species.
Applications of this invention to different cell types utilizes information on various cell parameters;',including the osmotic permeability parameters and their temperature dependencies, solution properties of the cytoplasm, and the relationship between intracellular cooling and intracellular freezing of different cell types: Some of these properties will not change significantly between different stem cells. Solutipn properties, for example, depend primarily on the concentrations of electrolytes and proteins, which are similar for various types of cells.
Similarly, the incidence of intracellular freezing as a function of cooling is likely to be similar for different types of cells since there are likely similar mechanisms of ice nucleation in cells, whether ice is initiated by spontaneous nucleation in the cells, through aqueous pores in the plasma membrane (Acker et al., 2U01), by surface-catalysed nucleation (Toner, 1993), or by osmotic rupture of the plasma membrane (Muldew K. and McGann L.E., Biophysical Journal 66(2 Pt 1): 532-541; Feb. 1941).
Conversely, the osmotic permeability parameters and their activation energies depend strongly on 'cell type and stage of differentiation (McGrath; 1988).
Obtaining Cellular Osmotii" Properties Cellular osmotic properties can be obCained from the literature (see for example Gao et al., 1998 and Hunt et al:, 20ff3). For cell types whose osmotic properties have not yet been published, the osmotic properties can be measured as described.by the inventors in the examples and in the literature (Ross-Rodriguez, 2003(a)).
Measuring Solution Properties Calculations of osmotic transport at low temperatures require descriptions of the intracellular and extracellular solutions. Phase diagrams of even binary solutions show that solution behaviour over the range of temperature between freezing and -40°C is nonlinear, so an assumption of dilute solutions is inappropriate. The constants in equation (1) or other equations that describe nondilute behaviour are therefore required for the major intracellular and exixacellular solutes.
This information has been gathered from the literature and from the inventors' own previous experimental measurements. Intracellular and extracellular solute information for cell types can be gathered empirically according to experiments conducted by the inventors (McGann et al. , Medical and Biological Engineering and Computing, 20(1):117-20, January 1982; McGann et al., Journal of Orthopaedic Research, 6(1): 109-15, 1988; Toupin et al., Cryobiology, 26(S): 431-44, Gct. 1989; I,iu et al., -Cryobiology, 32(5): 493-502, Oct. 1995;
Gihnore et al., Animal Reproduction Science, 53(1-4.): 277-297, Oct.
1998).
1g _ In some embodiments of the invention, the inventors used proteins in cells with solution properties published for hemoglobin. In addition, fluorescent quenching (Liu-et al., 2002) can be used to measure intracellular water volume as a function of extracellular osmolality.
Also, electron spin resonance; or any other technique for specifically measuring concentration in the intracellular aqueous solution can be used to make the same measurement (Elliot et al., Cryobiology 2002).
These data allow calculation of the parameters in equation (1j for the cytoplasm. Fluorescent quenching is a relatively simple technique, so, by its application to various cell types; it can be demonstrated that solution properties of the cytoplasm do not vary significantly between types of nucleated mammalian cells.
Monitoring Cryoinjury Three in vitro methods can be applied to assess stem cell recovery after experimental treatment and to fine tune temperatures and hold times . In one embodiment, the simplest method is a single-platform viability assessment for cells that also accounts for cells lost during treatment (Yang et al., 2001). This technique, which uses 7-AAD (Molecular Probes) as the viability indicator based on membrane integrity; has been developed and implemented, in routine assessment of peripheral blood stem cells cryopreserved for transplantation (Yang, 2003). A metabolic assay, alamarBlue (Biosource International) that the inventors have used previously with other cellular systems (Acker and McGann, 2001), can be used to assess metabolic function after experimental treatment. The third assay is the standard colony growth in methycellulose to assess the ability of cells to divide and differentiate in culture. The inventors' technique for colony growth accommodates for loss of cells during experimental treatment (Yang; 2003).
Infusible Extracellular Compounds The inventors demonstrate that temperature profiles can be generated to avoid intracellular freezing and to reduce cryoinjury related to exposure to high concentrations of solutes, similar to results for human lymphocytes (Farrant et al.; 1974). However, it may not be possible to simultaneously meet these criteria for some cells, i.e. conditions required to avoid intracellular freezing may already subject the cells to lethal exposure to the solution. In this case he inventors suggest use of infusible extracellular compounds, including sugars, starches, such as Pentastarch, serum, or plasma to modify the extracellular solution properties thereby reducing the temperature of exposure of cells to the increased electrolyte concentrations.
Cryopreservation Protocol for Stem :Cells A 2-step freezing protocol used by Farrant et al. to obtain high recovery of human lymphocytes cryopreserved in serum alone (Farrant et al. , 1974) was optimized using simulation of a stem: cell line (TF-1 cells, a hematopoietic stem cell line) without cryoprotectant (Ross-Rodriguez;
2003) and validated using experimental measurements of post-thaw cell recovery. Osmotic permeability parameters were measured for the TF-1 cells and used in simulations of the 2-step cooling protocol. Maximum cooling to -40°C was used as an indicator of intracellular freezing, and maximum intracellular potassium chloride concentration used as an indicator of cryoinjury due to exposure to: the concentrated solutes.
Maximum values from t~.e simulation indicated the range of hold temperatures where cell recovery was expected to be maximal in the absence of cryoprotectants.
Experimental measurement of TF-l cell recovery using membrane integrity showed maximal recovery in the intermediate temperature range predicted by the simulations and low; recovery at intermediate temperatures outside the predicted range. The maximum recovery of TF-1 cells without cryoprotectant thawed from -196°C was equivalent to the recovery after conventional cryopreservation (cooling at 1 °C/min in the presence of 10% DMSO). In a specific embodiment, the zone of plung temperatures (-5°C to -15°C); when held for 1-3 minutes, confer comparable protection against injury (approximately 60 % viability) to the standard 10% DMSO solution (63.7~9.8%).
Results of these experiments supported the concept of using theoretical indicators of cryoinjury in the use of simulations to reduce empirical experimentation in optimization of cryopreservation protocols. These results also demonstrate the value of simulations to be used in protocol design.
Examples The following examples are intended to illustrate various embodiments of the invention and are intended to be interpreted in a non-limiting sense.
Example 1: Determination of cellular osmotic parameters of TF-l cells 1.1 Introduction Osmotic responses of cells to the formation of ice in the surrounding solution are largely dependent on the movement of water across the plasma membrane (Mazur, 1965). The formation of extracellular ice and the resulting increase in extracellular solute concentration, impose osmotic stresses on the cell (Mazur, 1972)° The osmotic parameters governing the movement of water across the membrane are specific to each .cell type. Thus different cells respond differently to anisotonic conditions. The movement of water across the membrane is faster than the movement of solutes and is the result of simple diffusion of water molecules across the plasma. membrane or the result of water movement through water channels or aquaporins. A
significant amount of the cell volume is comprised of water therefore water movement determines the cell volume. The net water movement is described using the osmotic parameters of the cell membrane.
The osmotic parameters; which govern water movement, are the hydraulic conductivity, the osmotically-inactive fraction and the Arrhenius activation energy. The hydraulic conductivity (Lp) denotes water transport across the cell membrane and thus the cell volume. Lp is a function of the rate at which water moves across the cell membrane.
Jacobs and Stewart (Jaeobs, 1932) uses he following equation, which describes the rate of cell volume change in anisotonic solutions as a function of LP (~.m/min/atm) (1) where V is the cell volume (~m3), t is the time (min), A is the cell surface area (~,m2); R is the universal gas constant (kcal/mol/K), T is the absolute temperature (K), ~e is -the e~tracellular osmolality (osmoles) and ~; is the intracellular osmolality (osmoles). The Boyle van't I-~off relationship expresses equilibrium cell volume in solutions of impermeant solutes:
~~ (1 Vb ) -I- Vb 2 :so where V~ is the equilibrium volume (~tm3), V;SO is the isotonic volume (~:m3), ~o is the isotonic osmolality (osmoles), ~c is the experimental osmolality (osmoles), and vb is ~ the osmotically-inactive fraction.
Through graphical analysis of VeqIV;so as a function of ~~/~c, vb can be determined by extrapolating he line by linear regression to the y-intercept.
The movement of water across the cell membrane is temperature dependent. The Arrhenius activation energy (Ea) for Lp is normally used to describe the temperature dependence of the hydraulic conductivity (Woods, 2000). Ea (kcal/mol) can be determined using the slope of the Arrhenius plot of the natural logarithm of Lp as a function of the inverse absolute temperature (K):
Lp -_ k . exp(RET) (3) where k is a fitting constant; R is the universal gas constant (kcal/mol/K) and T is the absolute temperature (K). Osmotic parameters are useful for computer simulations which model changes in cell volume at low temperatures and which could eventually be applied to more complex systems, such as tissues.
An electronic particle counter was used to monitor cell volume as a function of time for cells exposed to hypertonic solutions. In the past, electronic particle counters have been used for a variety of cell types:
lymphocytes (Hempling, 1977); chondrocytes (McGann, 1988);
pancreatic islet cells (Liu; 1995; Woods, 1999); human corneal endothelial, stroma, and epithelial cells (Ebertz, 2002) and selected African mammalian spermatozoa (Gilmore, 1998). As cells pass through the aperture of the electronic ' particle counter a volume of conducting fluid is displaced resulting in a current pulse, which is proportional to the cell volume. In kinetic studies, sequential measurements of cell volumes allow for the determination of cell permeability characteristics by fitting the experimental data with theoretical models. An electronic particle counter allows permeability characteristics to be obtained for osmotically slow responding cells;(Acker; 1999). A computer interfaced to a particle counter can record the volume and time of measurements, so the time evolution of cell volume distribution can be monitored. Another technique using optical measurements has also been used to study osmotic responses of other cell types (Armitage; 1984; Gao, 1994; Hubel, 1999). Acker et al. compared the two techniques and determined that even though there are no direct measurements of single cell volumes using an electronic particle counter, there was no signi~xcant difference in cell volume measurements between the two techniques (Acker, 1999).
The electronic particle counter method was used in these experiments because it provides rapid and reproducible data collection fog analysis of a population of cells in one experiment, as opposed to multiple single-cell analyses required by optical measurements.
The objective of this example was to use an electronic particle counter fitted with a cell size analyzer, to measure changes in cell volume as a function of time while exposing the cells to hypertonic solutions for TF-1 cells; as a model for hematopoietic stem cells (HSC) (Kitamura, 1989(a); Kitamura 1989(b); Marone, 2002). The osmotic parameters and temperature dependencies were then calculated from the volume measurements.
1.2 Materials & Methods TF-1 cell culture TF-1 cells (ATCC, Manassas, Virginia) were cultured at 3?°C in
The cryopreservation protocol can be used with cells stored without cryopreservants. The protocol can also: be used with cells stored with cryopreservants, including r~on-penetrating cryopreservants. Such non-penetrating cryopreservants include sugars, starches, serum, or plasma.
The invention can be applied to any types of cells, including stem cells, other progenitor cells; red and white blood cells, sperm cells, ooeytes, ova, cells for research or transplant purposes, and cellular materials derived from tissues and organs (including but not limited to pancreatic islet cells, chondrocytes, cells of neural origin, cells of hepatic origin, and cells of cardiac origin). Throughout this application, cells include cells organized as tissues: Stem cells include human peripheral blood stem cells, human umbilical cord blood stem cells and stem cells derived from tissues and solid organs or other sources, including fetal and or embryonic sources. The invention is not limited to human cell types and is extendable to all mammalian and non-mammalian species.
In one embodiment of the invention, the non-linear cryopreservation protocol comprises cooling the cells to a first temperature for a first period of time, then cooling: the cells to a storage temperature for a second period of time prior to thawing.
In another embodiment of the invention, the non-linear cryopreservation protocol can be executed on cells in small samples. The invention can also be applied using a bulk freezing unit or a cryomicroscopy apparatus. The bulk freezing unit comprises a controller for executing the cryopreservation protocol, and the controller communicates with a computer to receive the cryopreservation protocol. The controller is connected to heaters surrounding the cells for maintaining the temperature of the cells, and' thermocouplers monitor the temperature of the cells.
The invention also relates to methods of optimizing cryopreservation protocols by determining: an optimal cooling profile and applying the profile to cryopreservation protocols. The invention also relates to cryopreservation protocols optimized by the method of the invention.
The invention also relates to non-linear cryopreserving protocols for cryopreserving stem cells In a specific embodiment of the invention, the method of cryopreserving stem cells comprises cooling the stem cells to a first temperature for a first period of time; then cooling the cells to-a second temperature for storing the stem cells. In one embodiment, the stem cells are cooled to a temperature between -S°C and -15°C for 1 to 3 minutes, then cooled to -196°C to store the cells.
Brief Description of Drawings Fig. 1 Simulation program user interface Fig. 2 Cell volume vs temperature at different cooling. rates Fig. 3 Maximum supercooling and maximum [KCl] as a function of cooling rate Fig. 6 TF-1 cell volume distribution Fig. 7 Boyle van't Hoff plot of TF-1 cells Fig. 8 Cell volume kinetics of TF-t cells in hypertonic solutions Fig. 9 Arrhenius plot of TF-I cells Fig. 10 chematic of CryoSimS program Fig. 11 raded freezing cooling profiles of TF-I cells Fig. 12 Simulated cell volume kinetics of TF-I cells Fig. 13 Simulated supercooling kinetics of TF-1 cells during graded freezing Fig. 14 Simulated [KCI]; kinetics of TF-1 cells during graded freezing Fig. 15 Simulated maximum supercooling of TF-1 cells using various cooling rates Fig. 16 Simulated maximum [KCI]; of TF-1 cells using various cooling rates Fig. 17 Membrane integrity-of TF-i cells in 10% DMSO
Fig. 18 Membrane integrity'of TF-1 cells using 0.2°C/rain Fig. 19 Membrane integrity of TF-1 cells using 0.5°Clmin Fig. 20 Membrane integrity of TF-1 cells using 0.9°C/rain Fig. 21 Experimental determination of two-step cooling profiles Fig. 22 Simulations of two-step cooling profiles of TF-1 cells Fig. 23 Simulated cell volume kinetics of TF-1 cells Fig. 24 Simulated supercoohng kinetics of TF-1 cells during two-step freezing Fig. 25 Simulated [KCI], kinetics of TF-1 cells during two-step freezing Fig. 26 Simulated maximum supercooling and [KCI]; of TF-1 cells held for various durations Fig. 27 Simulations of optimal plunge temperatures ranges for TF-1 cells cooled using two-step freezing Fig. 28 Membrane integrity of TF-1 cells using 3 minutes hold time g Fig. 29 Membrane integrity of TF-1 cells held at -5°C and -25°C for various durations Fig. 30 Contours of membrane integrity of TF-1 cells using two-step freezing for various durations Detailed Description of the Invention Throughout the following description, specific details are set forth in order to provide a more thorough understanding of the invention.
However, the invention may be practiced without these particulars. In other instances, well known elements have not been shown or described in detail to avoid unnecessarily obscuring the invention. Accordingly, the specification and drawings are to be regarded in an illustrative, rather than a restrictive, sense.
This invention relates to a non-linear cryopreservation protocol for cryopreserving cells comprising determining an optimal cooling profile for maximum recovery of the cells and applying the cooling profile to the cells. The optimal cooling profile is determined using a simulation of cellular responses to cooling parameters. The cooling parameters comprise cell temperature, duration of temperature exposure, cooling level; cooling rate and presence or absence of cryopreservants. The cellular responses are determined from mathematical models of extracellular concentration parameters, intracellular concentration parameters, and cellular osmotic permeability parameters.
The cryopreservation protocol can be used with cells stored without cryopreservants. The protocol can also be used with cells stored with cryopreservants, including non-penetrating cryopreservants. Such non-penetrating cryopreservants include sugars, starches, serum, or plasma.
The invention can be applied to any types of cells, including stem cells, other progenitor cells, red and white blood cells, sperm cells, oocytes, ova, cells for research or transplant purposes, and cellular materials derived from tissues and organs (including but not limited to pancreatic islet cells, chondrocytes; cells of neural origin, cells of hepatic origin, and cells of cardiac origin). Throughout this application, cells include cells organized as tissues. Stem cells include human peripheral blood stem cells, human umbilical cord blood stem cells and stem cells derived from tissues and solid organs or other sources, including fetal and or embryonic sources: The invention is not limited to human cell types and is extendable to all mammalian and non-mammalian species.In one embodiment of the invention, the non-linear cryopreservation protocol comprises cooling the cells to a first temperature for a first period of time, then cooling the cells to a storage temperature for a second period of time prior to thawing.
In another embodiment of the invention, the non-linear cryopreservation protocol can be executed on cells using a bulk freezing unit or a cryomicroscopy apparatus. The invention also relates to methods of optimizing cryopreservation protocols by determining an optimal cooling profile and applying the profile to cryopreservation protocols.
The invention also relates to cryopreservation protocols optimized by the method of the invention.
- 1~ -The invention also relates to non-linear cryopreserving protocols for cryopreserving stem cells In a specific embodiment of the invention, the method of cryopreserving stem cells comprises cooling the stem cells to a first temperature fox a first period of time; then cooling the cells to a second temperature for storing the stem cells. In one embodiment; the stem cells are cooled to a temperature between-5°C and -15°C for 1 to 3 minutes, then cooled to -196°C to store the cells.
~.7escription of Simulation 3'00l Reports of high cell recovery in the absence of permeating cryopreservants such as DMSO (J. Farrant et al.; 1974) indicate that it is feasible to eliminate such cryopreservants from cryopreservation protocols. Instead of using cryopreservants to alter the properties of solutions so that cells rnay be cooled at a constant rate (normally 1 °C/min) and achieve high recovery, in one aspect of the invention, the inventors' recent approach (Ross-Rodriguez, 2003(a); Ross-Rodriguez, 2003(b)) has been to use the properties of the intracellular and extracellular solutions of subject cells to design optimal cryopreservation protocols. A novel aspect of this approach is that the temperature profile is not constrained to be linear. Rather, the intracellular and extracellular solution properties, along with cellular osmotic properties, are used to generate a temperature profile that minimizes cryoinjury.
During the course of the inventors' research on cryoinjury and cryoprotection, the inventors have developed a mathematical model of cellular osmotic responses at low temperatures using real (nondilute) solutions assumptions for both the carrier solution and the cellular cytoplasm (McGann and Elliott; 2003). In one embodiment of the invention, this model has been implemented in a computer program as a simulation tool to calculate intracellular and extracellular parameters and properties as the temperature changes and ice forms or melts in the cells. The simulation tool generates an ideal temperature range and duration range for cooling the cells and obtaining optimal recovery of the cells upon thawing. The tool can be applied to any cell type by measuring appropriate parameters of the cells.
In some embodiments of the invention, simulations are based on changes in the composition of the extracellular solution as water is converted to ice during cooling, and the osmotic responses of cells to these changes:
a) Features of the Simulation This simulation uses nondilute solution equations in the calculations, and allows accommodation of the contribution of proteins to the solution properties of the cytoplasm. In one embodiment, the program calculates cellular responses based on a programmed temperature profile generated by specifying rates of temperature change; or by providing measured temperature profiles to simulate real cryopreservation systems. In a second embodiment, the program allows imposition of specific design criteria, including the calculation of a cooling profile that maintains a constant level of cooling in the cell, thereby minimizing exposure to the environment of concentrated solutions.
b) Determination of Solution Properties Since dilute solution assumptions do not apply as solutes concentrate in both the extracellular and intracellular compartments, nondilute descriptions of the solution properties have been utilized. The inventors have found the osmotic virial equation to be simple yet sufficient for many biological aqueous solutions. The osmotic virial equation was fit to published or measured data on freezing point as a function of concentration, and used in the simulations to describe the freezing point (TFP) of binary solutions, taking into account the non-ideal behaviour of real solutes:
Tpp =K~ w(lYI+BZ ~lYl2 +B3 ~YI23) where m is the molality of the solute, and K, , B2 and B3 are fitting constants for specific solutes in water: For electrolytes, the molality is multiplied by a dissociation parameter. The constant B3 is non-zero only for solutes with highly nonlinear behaviours, such as polymers. The following relationship was used to calculate freezing point depressions in solutions containing multiple (n) solutes (at most one of which has non-zero B3);
T~ - ~~ ' ~ m= + Bar ' ~2 + B3J ~ m= + ~ ~~Bzt + Bz; )' m1 ' m; ~ (2) i=1 .. n j=i+i :. n The last term uses Guggenheim's naive approximation to describe interactions between solutes in the solution (Elliot et al, Cryobiology 2002). In these simulations; it was assumed that the primary intracellular electrolyte is potassium chloride, and the primary extracellular electrolyte is sodium chloride. Proteins and starches exhibit highly nonlinear freezing point depression as the concentration increases; so the value of B3 in equation (2) is significant. In one case, the inventors found it necessary to include intracellular protein to more accurately describe the cytoplasm as the solution concentrates in the presence of ice (Ross-Rodriguez, 2003(x)).
c) Osmotic Transport of Water and Solutes The Johnson & Wilson model (Johnson and Wilson, 1967) was used to describe coupled water and solute transport across the plasma membrane:
_dV=LP.A.R:T~ ~Unwn~+~s'(~s-mss) dt n dSs - Ps ' (QCs ) -~ (l - O_s ) . Cs . d dt dt where V is cell volume at time t; Lp is membrane hydraulic conductivity, A is surface area of the cell; R is the gas constant; T is absolute temperature, is osmolality; and is the Staverman reflection coefficient. Superscripts a and a refer to the intracellular and extracellular compartments; respectively, and the subscripts h and s refer to nonpermeating and permeating solutes: S is the moles of solute, ~'s is the solute permeability, CS is the difference in solute concentration across the membrane, and CS is the mean solute concentration.
d) Temperature Dependence of Osmotic Parameters The Arrhenius equation wad used to describe the temperature dependence of the osmotic permeability parameters.
_E _1 1 1p ~'~ng ' a ~ T' T (4.) where LP is the hydraulic conductivity at any temperature T, Lpg is the hydraulic conductivity at a reference temperature Tg, Ea is the activation energy and R is the gas constant. A similar Arrhenius equation was used to describe the temperature dependence of the solute permeability.
Equations (1) and (2) can be made more complex as needed by using other solution thermodynamics equations from the literature. In addition, equation (2) can be expanded to the case of multiple components with non-zero B3's by adding a term, or terms analogous to the second order last term of equation (2), but of appropriate order.
There are many equations, both in the literature and to be derived in the future based on physical analysis, that can replace equation (3).
e) Computer Program far 5'imulation of Cellular Low Temperature Responses A computer program was developed (using the Delphi programming language) to perform the simulations based on either calculated or IS
measured temperature profiles. The program also includes a function to generate the temperature profile required to maintain the cytoplasm at a constant level of cooling. Parameters for equation (1) to describe solution properties for a variety of solutes are read from data files. In addition to the resulting terriperature profile, all calculated parameters are reported as a function of time to allow access to both intracellular and extracellular concentrations and fluxes.
Figure 1 shows the user interface of the program to illustrate the input parameters .
The sample output in Figure 2 shows calculated cell volumes as a function of temperature during cooling at different rates.
,~ Predictors of Cryoinjury Based on Mazur's two-factor hypothesis (Mazur et al. , 1972), the inventors used the amount of cooling as a pr~lictor of cryoinjury related to intracellular ice fo~nation, and the intracellular KCl concentration as a predictor for cryoinjury related to exposure to concentrated solutions.
Simulations allow determination of the maximum cooling and maximum intracellular KCI concentration in cells cooled at different rates down to -40°C, as shown in Figure 3.
Application of Method to Different Cell Types The invention can be applied to any types of cells, including stem cells, other progenitor cells, red and white blood cells, sperm cells, oocytes, ova, cells for research or transplant purposes; and cellular materials derived from tissues and organs (including but not limited to pancreatic islet cells, chondrocytes; cells of neural origin, cells of hepatic origin, and cells of cardiac origin): Stem cells include human peripheral blood stem cells, human umbilical cord blood stem cells and stem cells derived from tissues and solid organs or other ounces, including fetal and or embryonic sources. The invention is not limited to human cell types and is extendable to all mammalian and non-mammalian species.
Applications of this invention to different cell types utilizes information on various cell parameters;',including the osmotic permeability parameters and their temperature dependencies, solution properties of the cytoplasm, and the relationship between intracellular cooling and intracellular freezing of different cell types: Some of these properties will not change significantly between different stem cells. Solutipn properties, for example, depend primarily on the concentrations of electrolytes and proteins, which are similar for various types of cells.
Similarly, the incidence of intracellular freezing as a function of cooling is likely to be similar for different types of cells since there are likely similar mechanisms of ice nucleation in cells, whether ice is initiated by spontaneous nucleation in the cells, through aqueous pores in the plasma membrane (Acker et al., 2U01), by surface-catalysed nucleation (Toner, 1993), or by osmotic rupture of the plasma membrane (Muldew K. and McGann L.E., Biophysical Journal 66(2 Pt 1): 532-541; Feb. 1941).
Conversely, the osmotic permeability parameters and their activation energies depend strongly on 'cell type and stage of differentiation (McGrath; 1988).
Obtaining Cellular Osmotii" Properties Cellular osmotic properties can be obCained from the literature (see for example Gao et al., 1998 and Hunt et al:, 20ff3). For cell types whose osmotic properties have not yet been published, the osmotic properties can be measured as described.by the inventors in the examples and in the literature (Ross-Rodriguez, 2003(a)).
Measuring Solution Properties Calculations of osmotic transport at low temperatures require descriptions of the intracellular and extracellular solutions. Phase diagrams of even binary solutions show that solution behaviour over the range of temperature between freezing and -40°C is nonlinear, so an assumption of dilute solutions is inappropriate. The constants in equation (1) or other equations that describe nondilute behaviour are therefore required for the major intracellular and exixacellular solutes.
This information has been gathered from the literature and from the inventors' own previous experimental measurements. Intracellular and extracellular solute information for cell types can be gathered empirically according to experiments conducted by the inventors (McGann et al. , Medical and Biological Engineering and Computing, 20(1):117-20, January 1982; McGann et al., Journal of Orthopaedic Research, 6(1): 109-15, 1988; Toupin et al., Cryobiology, 26(S): 431-44, Gct. 1989; I,iu et al., -Cryobiology, 32(5): 493-502, Oct. 1995;
Gihnore et al., Animal Reproduction Science, 53(1-4.): 277-297, Oct.
1998).
1g _ In some embodiments of the invention, the inventors used proteins in cells with solution properties published for hemoglobin. In addition, fluorescent quenching (Liu-et al., 2002) can be used to measure intracellular water volume as a function of extracellular osmolality.
Also, electron spin resonance; or any other technique for specifically measuring concentration in the intracellular aqueous solution can be used to make the same measurement (Elliot et al., Cryobiology 2002).
These data allow calculation of the parameters in equation (1j for the cytoplasm. Fluorescent quenching is a relatively simple technique, so, by its application to various cell types; it can be demonstrated that solution properties of the cytoplasm do not vary significantly between types of nucleated mammalian cells.
Monitoring Cryoinjury Three in vitro methods can be applied to assess stem cell recovery after experimental treatment and to fine tune temperatures and hold times . In one embodiment, the simplest method is a single-platform viability assessment for cells that also accounts for cells lost during treatment (Yang et al., 2001). This technique, which uses 7-AAD (Molecular Probes) as the viability indicator based on membrane integrity; has been developed and implemented, in routine assessment of peripheral blood stem cells cryopreserved for transplantation (Yang, 2003). A metabolic assay, alamarBlue (Biosource International) that the inventors have used previously with other cellular systems (Acker and McGann, 2001), can be used to assess metabolic function after experimental treatment. The third assay is the standard colony growth in methycellulose to assess the ability of cells to divide and differentiate in culture. The inventors' technique for colony growth accommodates for loss of cells during experimental treatment (Yang; 2003).
Infusible Extracellular Compounds The inventors demonstrate that temperature profiles can be generated to avoid intracellular freezing and to reduce cryoinjury related to exposure to high concentrations of solutes, similar to results for human lymphocytes (Farrant et al.; 1974). However, it may not be possible to simultaneously meet these criteria for some cells, i.e. conditions required to avoid intracellular freezing may already subject the cells to lethal exposure to the solution. In this case he inventors suggest use of infusible extracellular compounds, including sugars, starches, such as Pentastarch, serum, or plasma to modify the extracellular solution properties thereby reducing the temperature of exposure of cells to the increased electrolyte concentrations.
Cryopreservation Protocol for Stem :Cells A 2-step freezing protocol used by Farrant et al. to obtain high recovery of human lymphocytes cryopreserved in serum alone (Farrant et al. , 1974) was optimized using simulation of a stem: cell line (TF-1 cells, a hematopoietic stem cell line) without cryoprotectant (Ross-Rodriguez;
2003) and validated using experimental measurements of post-thaw cell recovery. Osmotic permeability parameters were measured for the TF-1 cells and used in simulations of the 2-step cooling protocol. Maximum cooling to -40°C was used as an indicator of intracellular freezing, and maximum intracellular potassium chloride concentration used as an indicator of cryoinjury due to exposure to: the concentrated solutes.
Maximum values from t~.e simulation indicated the range of hold temperatures where cell recovery was expected to be maximal in the absence of cryoprotectants.
Experimental measurement of TF-l cell recovery using membrane integrity showed maximal recovery in the intermediate temperature range predicted by the simulations and low; recovery at intermediate temperatures outside the predicted range. The maximum recovery of TF-1 cells without cryoprotectant thawed from -196°C was equivalent to the recovery after conventional cryopreservation (cooling at 1 °C/min in the presence of 10% DMSO). In a specific embodiment, the zone of plung temperatures (-5°C to -15°C); when held for 1-3 minutes, confer comparable protection against injury (approximately 60 % viability) to the standard 10% DMSO solution (63.7~9.8%).
Results of these experiments supported the concept of using theoretical indicators of cryoinjury in the use of simulations to reduce empirical experimentation in optimization of cryopreservation protocols. These results also demonstrate the value of simulations to be used in protocol design.
Examples The following examples are intended to illustrate various embodiments of the invention and are intended to be interpreted in a non-limiting sense.
Example 1: Determination of cellular osmotic parameters of TF-l cells 1.1 Introduction Osmotic responses of cells to the formation of ice in the surrounding solution are largely dependent on the movement of water across the plasma membrane (Mazur, 1965). The formation of extracellular ice and the resulting increase in extracellular solute concentration, impose osmotic stresses on the cell (Mazur, 1972)° The osmotic parameters governing the movement of water across the membrane are specific to each .cell type. Thus different cells respond differently to anisotonic conditions. The movement of water across the membrane is faster than the movement of solutes and is the result of simple diffusion of water molecules across the plasma. membrane or the result of water movement through water channels or aquaporins. A
significant amount of the cell volume is comprised of water therefore water movement determines the cell volume. The net water movement is described using the osmotic parameters of the cell membrane.
The osmotic parameters; which govern water movement, are the hydraulic conductivity, the osmotically-inactive fraction and the Arrhenius activation energy. The hydraulic conductivity (Lp) denotes water transport across the cell membrane and thus the cell volume. Lp is a function of the rate at which water moves across the cell membrane.
Jacobs and Stewart (Jaeobs, 1932) uses he following equation, which describes the rate of cell volume change in anisotonic solutions as a function of LP (~.m/min/atm) (1) where V is the cell volume (~m3), t is the time (min), A is the cell surface area (~,m2); R is the universal gas constant (kcal/mol/K), T is the absolute temperature (K), ~e is -the e~tracellular osmolality (osmoles) and ~; is the intracellular osmolality (osmoles). The Boyle van't I-~off relationship expresses equilibrium cell volume in solutions of impermeant solutes:
~~ (1 Vb ) -I- Vb 2 :so where V~ is the equilibrium volume (~tm3), V;SO is the isotonic volume (~:m3), ~o is the isotonic osmolality (osmoles), ~c is the experimental osmolality (osmoles), and vb is ~ the osmotically-inactive fraction.
Through graphical analysis of VeqIV;so as a function of ~~/~c, vb can be determined by extrapolating he line by linear regression to the y-intercept.
The movement of water across the cell membrane is temperature dependent. The Arrhenius activation energy (Ea) for Lp is normally used to describe the temperature dependence of the hydraulic conductivity (Woods, 2000). Ea (kcal/mol) can be determined using the slope of the Arrhenius plot of the natural logarithm of Lp as a function of the inverse absolute temperature (K):
Lp -_ k . exp(RET) (3) where k is a fitting constant; R is the universal gas constant (kcal/mol/K) and T is the absolute temperature (K). Osmotic parameters are useful for computer simulations which model changes in cell volume at low temperatures and which could eventually be applied to more complex systems, such as tissues.
An electronic particle counter was used to monitor cell volume as a function of time for cells exposed to hypertonic solutions. In the past, electronic particle counters have been used for a variety of cell types:
lymphocytes (Hempling, 1977); chondrocytes (McGann, 1988);
pancreatic islet cells (Liu; 1995; Woods, 1999); human corneal endothelial, stroma, and epithelial cells (Ebertz, 2002) and selected African mammalian spermatozoa (Gilmore, 1998). As cells pass through the aperture of the electronic ' particle counter a volume of conducting fluid is displaced resulting in a current pulse, which is proportional to the cell volume. In kinetic studies, sequential measurements of cell volumes allow for the determination of cell permeability characteristics by fitting the experimental data with theoretical models. An electronic particle counter allows permeability characteristics to be obtained for osmotically slow responding cells;(Acker; 1999). A computer interfaced to a particle counter can record the volume and time of measurements, so the time evolution of cell volume distribution can be monitored. Another technique using optical measurements has also been used to study osmotic responses of other cell types (Armitage; 1984; Gao, 1994; Hubel, 1999). Acker et al. compared the two techniques and determined that even though there are no direct measurements of single cell volumes using an electronic particle counter, there was no signi~xcant difference in cell volume measurements between the two techniques (Acker, 1999).
The electronic particle counter method was used in these experiments because it provides rapid and reproducible data collection fog analysis of a population of cells in one experiment, as opposed to multiple single-cell analyses required by optical measurements.
The objective of this example was to use an electronic particle counter fitted with a cell size analyzer, to measure changes in cell volume as a function of time while exposing the cells to hypertonic solutions for TF-1 cells; as a model for hematopoietic stem cells (HSC) (Kitamura, 1989(a); Kitamura 1989(b); Marone, 2002). The osmotic parameters and temperature dependencies were then calculated from the volume measurements.
1.2 Materials & Methods TF-1 cell culture TF-1 cells (ATCC, Manassas, Virginia) were cultured at 3?°C in
5% COZ in RPMI 1640 Medium Modified (ATCC) with 10% fetal bovine serum (FBS) (ATCC), and supplemented with 2 ng/mL recombinant human GM-CSF (Stemcell Technologies, Vancouver, Canada). Cells were cultured at a concentration between 0,1 x 106 and 1 x 106 cells/mL, according to ATCC guidelines: Prior to experiments, cells were washed twice with serum-free RPMI media and incubated overnight. TF-1 cells cultured in RPMI without FBS .and GM-CSF overnight accumulate in the G1/Go phase of the cell cycle (Kolonics, , 2001 ), resulting in a more uniform cell size distribution. Cells were then centrifuged and re-suspended in serum-free RPMI at 4 x 106 cells/mL for osmotic measurements.
Experimeptal solutions Various concentrations of phosphate-buffered saline (PBS) were used to examine the concentration-dependence of the hydraulic conductivity and the osmotically-inactive fraction. PBS solutions (1-SX) were made by diluting lOX PBS (GIBC4) with distilled water to final osmolalities of 291~6; 583~25; 861~22, 1150~17 and 1434~20 mOsm/kg respectively. Osmolalities .were measured using a freezing-point depression Osmometer (Precision Systems Inc., Natick, Massachusetts), which was calibrated using 100; 300 and 500 mOsm/kg osmometry standards (Precision Systems Inc.).
Measurements of cell volurpes The Coulter counter (ZB1, Goulter Inc., Hialeah; Florida), fitted with a pulse-height analyzer (The Great Canadian Computer Company, Spruce Grove, AB; Canada) was used to monitor cell volume as a function of time as cells passed through the 100 ~m aperture (Groves, 1969(a); Grower, 1969(b); McGann; 1982). This system has been previously used to monitor changes in cell volume for a variety of cells in suspension (Armitage; 1984; Benson; 1998; Ebertz, 2002; Hempling, 1997; Liu, 1995; Mazur; 1986; McGann, 1981; Woods, 1999; Zierger, 1999), including hematopoietic stem cells (Gao, 1998; Hubel, 1999;
McGann, 1987; Woods, 20U0):
TF-1 cells (150-200 ~;L) were injected into well-mixed hypertonic experimental solutions (10 yL). Experimental solutions were maintained at experimental temperatures using a circulating water bath with a custom insulated jacket. The current pulses proportional to the cell volumes were measured and the time recorded as the cells passed through the aperture of the Coulter counter. Experimental temperatures were measured at 4:6~0.7, 4.8~0.6, 8:1~0.711.1~0.6, 12.9~1.4, 16.4~0.5, 19.4~0.8, 23.311.2, 28.8~O.b, and 37.4~0.8°C using a Digi-Sense thermocouple thermometer (Cole Parmer, Anjou; Canada). For each experiment, three replicates were performed for each solution at each temperature. The experiments were repeated .a minimum of hree times using cells from different passages. Latex beads (15 ~.m diameter; Beckman Coulter, Miami, Florida) were used as calibrators to convert relative volumes to actual volumes in 1X PBS and in the experimental solutions.
Determination of the osmotic parameters Measurements of cell volumes as a function of time were used to determine the osmotic parameters. Least squares error its using EXCEL
Solver, was used to solve for L~ and vb, using equations l and 2 respectively. The analysis of the concentration dependence used 2-5X
PBS solutions (583-1434 W 4sm/kg) at temperatures of 4.8~0.6, 12.9~1.4, 23.3~1.2, and 37.4~0.8°C, and additional analysis with temperatures of 4.6~0.7, 8.1~0.7, 11.1+_0:6,;16.4~0.5, 19:410.8 and 28:8~0:6°C was performed for 3X PBS solutions only. Curves were fitted for Lp and Vb for each experimental, solution at each of the experimental temperatures.
The Arrhenius activation energy for Lp, described by equation 3, was fit for using linear regre sign of the natural logarithm of Lp as a function of the inverse absolute temperature in EXCEL.
Statistical analysis Statistical comparisons used a standard one-way analysis of variance (ANOVA) at 5% level of signi~canee: Estimates of Lp and vb were compared between experimental solutions (2-SX PBS) for all the experimental temperatures.
1.3 Results Isotonic volume Figure 6 shows a representative volume distribution of TF-1 cells under isotonic conditions in 1X PBS (calibration factor=7.8; mean volume=806.1 ~m3). The distribution was lognormal and narrow compared to other cell types which have a more broad distribution (unpublished data). This is the result of synchronizing the cells in Go/Gl phases of the cell cycle. For the entire data set, the isotonic volume for TF-1 cells was 776~36 pm3.
Hydraulic conductivity Changes in mean cell volume as a function of time were used to calculate the Lp. Figure 7 is a representative graph of TF-1 cells exposed _2g_ to 3X PBS at four different temperatures, which show the increase rate of cell volume shrinkage at higher temperatures, demonstrating a higher Lp.
Data from experimental solutions (2-SX PBS) at four different temperatures (4.80.6, 12.9~1.4, 23.3~1.2, and 37.410.8°C) were used to examine the concentration and temperature dependence of Lp. The L~
was determined by fitting the data to Equation 1 using the least squares method with EXCEL Solver for each experimental solution and temperature and summarized in Table 1. Values for each concentration were pooled since there was no concentration dependence (p>0.05):
Tempe~ntore ~
(~um/min/atm)4.840:62C 12.91.4C 23.31.2C 37.40:8C
2X PBS 0:08110:013 0.12210.011 0.337O.OS1 1.170.14 ' 3X PBS 0:0790.011 O.L340.020 0.3750.060 1.390.30 4X PBS 0.0?'~~0.008 O.I220.01'7 0.428O.U48 1.420.28 SX PBS 0.07610:014 0.1190:014 0.3790.036 1.500.30 pooled mean 0.0780:012 0.123f0.015 0.3880.052 1.360.26 ' TABLE 1. L~ values for TF-1 cells (mean,~ SD); p>0.05 for all values implying no concentration dependence Osmotically-inactive fraction For each sample, vb was fit to Equation 2 using the least-squares method in EXCEL Solver. Data from temperatures of 4.8~0.6, 12.9~1.4, 23.3~1.2, and 37.4~0.8°C for 2-SX PBS solutions were used in order to determine the temperature and concentration dependence of vb. The data in Table 2 show that the osmotically-inactive fraction was not dependent on concentration (p>0.05): As a result, overall data for all temperatures and concentrations were pooled. TF-1 cells had a mean vb of 0.35~0.03.
A Boyle van't Hoff plot of equilibrium volume as a function of inverse osmolality for the aggregate data is shown in Figure 8. TF-1 cells responded as, ideal osmomet~rs over a range: of 583-1434 mOsm/kg. The vb could also be determined' by extrapolating the slope back to the y-axis in which the intercept was 0.37. This value was within the error found using the least squares method;
Temperature 4.840.62C 12.911.4C 23.31.2C 37.40.8C
2X PBS 0:3980:042 ' 0:3320.058'0.3270.016 0.3210.041 3X PBS 0.3780.040 0.3830.022 0.3480.030 0.3380.050 4X PBS 0.3570.027 0.3760.051 0.3410:018 0.3300.021 SX PBS 0:3610.026 0.3$40.058 0.3710.010 0.3130.028 pooled mean 0:373+_0:034Q.3680.052 0.3470.020 0.3260.037 TABLE 2, vb values for TF'-1 cells (mean t SD); p>0.05 for all values implying no concentration dependence Arrhenius activation energy The Lp follows the Arrhenius equation over the range of experimental temperatures. The Arrhenius activation energy of Lp was determined using pooled data' from experiments based on cell volume kinetics of 2-5X PBS solutions at temperatures 4:8~0.6, 12,9~1.4, 23.3~1.2, and 37.410,8°C anal of 3X PBS solutions at temperatures 4.6~0.7, 8.1~0.7, 11.1~0.6, lfi'.4~0.5; 19.4~0.8: and 28:8~0.6°C.
Figure 9 shows Arrhenius plots of mean Lp values for all the experimental temperatures examined. The value of Ea for Lp from these data was 13.4 keal/mol. Figure 9 also demonstrates that the osmotically-inactive fraction is independent of temperature using linear regression.
1.4 Discussion The hydraulic conductivity reported in this example was found to be strongly dependent on all temperatures reported in this example, but independent of concentration, which has been previously reported for other cells types {Liu, 1995): The value for Lp was 0.342 ~m/minlatm at 20°C. The Lp is within the range reported for mammalian cells, such as rat megakarycytoporietic cells, Chinese hamster lung ~broblast cells, bovine immature oocytes; ehondrocytes, corneal endothelial, epithelial and stromal cells {McGrath, 1988). The rate of water movement is considered slow responding; thus the Coulter counter was an efficient method of monitoring changes in cell volume.
TF-1 cells follow the Boyle van't Hoff relationship and thus the cells behave as ideal osmometers. The osmotically-inactive fraction can be determined using the Boyle van't Hoff plot and the least-squares method. The vb for TF-1 cells was determined as 0.350.03 and 0.37, respectively. The value of vb reported here is within the range for a variety of mammalian cell types (0:2-0.41) (Ebertz; 2002; Gao, 1998;
Gilmore, 1998; Hempling, 197'1; Liu, 1995; McGann, 1981; McGrath, 1988). The Arrhenius: activation energy for Lp o~ 13.4 kcal/mol reported here, is within normal ranges for other types of mammalian cells (12-16 kcal/mol) (Ebertz, 2002; Hempling, 1977; Liu, 1995). It has been reported that cells with an Ea of <6 kcallmol for I,p, are fast responding, and may exhibit channel-mediated water transport (Elmoazzen, 2002).
Also, cells with an Ea >i0 kcal/mol for Lp, such as the TF-1 cells, are slow responding and may transport water by solubility-diffusion through the plasma membrane. The high Ea for Lp indicates that the water permeability of the plasma membrane of TF-1 cells is highly dependent on temperature. However, the Ea for Lp alone may not be enough to negate the presence of aqueous pores and further analysis is required of slow responding cells to explore the possibility of other types of pores (Elmoazzen, 2002).
The osmotic parameters reported here for TF-1 cells are comparable with those previously reported for H$Cs. The value reported here for Lp is comparable with he Lp previously reported for cord blood CD34+ cells of 0.168~0.03 ~,m/atm/min at 20°C (Hunt, 2'003), indicating that the rate of water movement is similar for TF-1 cells and for other HSC. Based on Lp values reported by Hunt et al. at two temperatures (Hunt, 2003), Ea for L~ in cord blood CD34+ cells were calculated to be 18.8'kcal/mol. The Ea for Lp also denotes a slow-responding cell. The vb is comparable to that previously reported for both bone marrow hematopoietic CD34+ cells of 0:205 (Goo, 1998) and umbilical cord blood CD34+ cells of 0.32 (Hunt, 2003) and 0.27~0.01 (Hunt, 2003). The average size of TF-l cells (776~36 ~.m3) was higher than that previously reported for both bone marrow CD34+ cells of 345 pm3 (Goo, 1998) and umbilical cord blood CD34+ cells of 274~13 ~.m3 (Hunt, 2003). This indicates that although there are differences in cell volume between TF-1 cells and CD34+ cells from patient samples; the osmotic parameters for both cell types are comparable.
The parameters determined in this example are sufficient to be used in the mathematical analysis of the TF-1 cellular responses to low temperatures and ultimately in designing a cryopreservation procedure speei~c to these cells. The parameters summarized in Table 3 were subsequently used in simulations described in Example 2. The simulations model the cellular responses to ice formation in the extracellular solution based on how the cells responded osmotically to hypertonic solutions. It is also possible to use the osmotic parameters from other cell types to model their cellular response to low temperatures.
Isotonic Volume ' 776 ~.m Inactive Fraction 0.350 Lp (20C) 0:342 wm/min/atm Activation Energy for L"~; 13,4 kcallmol Isotonic osmolality 0.301 osm/kg TABLE 3. Osmotic parameters for TF-1 cells used in simulations.
Example 2: Simulations of cellular responses to low temperatures 2.1 Introduction The freezing of cells in suspension has largely been approached empirically. However, simulations have been used to mathematically predict cellular responses to low temperatures for a variety of cell types:
bovine erythrocytes (Leibo, 1976); yeast (Schwartz, 1983); hamster ova (Shabana, 1988); haanster pancreatic islet cells (Liu, 1995); and epithelial, endothelial and stroma cells (Ebertz, 2002). Modeling is based on the theoretical response of the cell to a changing extracellular environment.
The cellular responses to! the formation of extracellular ice in surrounding solution are largely dependent on the movement of water across the plasma membrane: Ea~tracellular ice formation increases the concentration of solutes in the residual liquid, resulting in osmotic efflux of water from the cell. The. ,properties of the cell membrane, specifically the osmotic parameters, govern the rate of change of cell volume.
Osmotic parameters can be used in simulations to theoretically model cellular responses to low temperatures:
Simulations reduce the time and expense- involved with empirical experiments. Simulations also provide a means to analyze changes in cell volume prior to empirical: experimentation: Simulations provide insight into intracellular osmolalities; concentrations and rates of water movement. These results can then be used for comparisons between cryopreservation protocols and for comparison between different cell types which may be necessary if attempting to cryopreserve a heterogeneous cell population or a tissue. Ultimately, simulations allow for unlimited theoretical protocols to be explored by controlling cooling and warming rate, experimental temperatures, and the components of the intracellular and extracellu.lar comparCments for any cell type for which the osmotic parameters are known.
Mazur has previously used simulations to explore the effects of solutions, osmotics and temperatures on cellular systems (Mazur, 1963).
Mazur reported the rate of water loss from the cell and the change in permeability with temperature as the parameters necessary to predict changes in volume of intracellular water with temperature. Furthermore, predictions about the probability of intracellular ice formation can be made based on the amount of intracellular water and the temperature of the cell. Subsequently, Mazur indicated that rates greater than 1 °C/min may generate a supercooled cytoplasm in yeast: (Mazur, 1963; Mazur, 1977). Supercooling is tl~e amount a solution can be cooled below its freezing point without ice forming and it used as an indicator of the potential for intracellular ice formation (Mazur, 1972). Intracellular supercooling is the extent to which a cell is cooled below the phase-change temperature before the formation of intracellular ice. Mazur also reported that there was a 10°C limit to supercooling above which the risk of intracellular ice is increased (Mazur, 1963; Mazur, 1977). Cooling rates of approximately 1 °C/min should reduce the amount of supercooling therefore they reduce the risk of intracellular ice. Diller further examined the probability of intracellular ice formation based on the synergistic interaction of cooling rates and supercooling (Diner, 1975). During cooling, the cell attempts to maintain equilibrium across the plasma membrane either through, osmotic dehydration or the formation of ice. Therefore; with no supercooling, there is a high probability that the cell will dehydrate; whereas with greater than 10°C
supercooling, the cell may form intracellular ice (Diller, 1975). Ebertz also reported the use of supercooling as an indicator for intracellular ice formation for simulations an corneal endothelial, epithelial and stromal cells (Diller, 1975).
Based on Mazur et al.'s 'two-factor hypothesis', solution effects injury must also be considered along with intracellular ice formation injury, when attempting to determine the optimal cooling rate for cryopreserving a cell type (Mazur; 1972): During slow cooling, cell injury is due to prolonged exposure to high solute concentrations, as a result of cell dehydration due to extracellular ice formation. This work represents a novel approach of combining supercooling with intracellular electrolyte concentratio~a in the presence of extracellular ice, to use as indicators of cryoinjury.
The objective of these simulations was to theoretically determine the cellular responses of TF-1 cells at various stages of the graded freezing protocol for comparison with experimental data: Simulations were performed using the osmotic parameters of TF-1 cells reported in Example 1 (Table 3).. Maximum levels of intracellular electrolyte concentrations ([KCI]i) and of supercooling were examined upon cooling the cells to -40°C; as indicators for solution effects injury and intracellular ice formation injury, respectively:
2.2 Calculations of low-temperature responses Methods The GryoSimS pr~:gram (Dr. Locksley McGann; University of Alberta; Canada) was used to perform the simulations and Figure is a representative image of the program interface. The program uses the phase diagrams of the components of the intracellular and extracellular solutions, the osmotic characteristics of the cell membrane and the temperature dependencies of the parameters. The simulations axe calculations of the cellular osmotic responses to the concentration of solutes in the residual liquid in the presence of ice at low temperatures.
Ice nucleation is assumed to be at the freezing point of the extracellular solution. Phase diagrams were used to calculate concentrations in the liquid phase for sodium chloride (NaCI)-H20 (Wolf, 1982) and potassium chloride (KCI)-H20 (Wolf; 1982) for the extracellular and intracellular compartments, respeeti~ely. The amount of intracellular protein has been reported to be more than half the dry weight of the cell (Alberts, 1994). It has also been reported that red blood cells possess approximately 0.0073 mol of hemoglobin per kg of intracellular water (7.3 mmolal) (Dick, 1958; Savitz, 1964; Williams; 1959). Since the intracellular protein content is unknown for TF-1 ceps to the inventors' knowledge, the inventors used half the molality of hemoglobin in red blood cells (3.65 mmolal) for the simulations. The hydraulic conductivity (Lp) was used to calculate osmotic cellular responses to changes in the extracellular conditions. The Arrhenius activation energy (Ea) for Lp was used to describe the temperature dependency of hydraulic conductivity. The numerical values of the hydraulic conductivity and the Ea for Lp from Example l (Table 3); were extrapolated to lower subzero temperatures.
Temperature profiles To explore the role of low and high cooling rates typically used to cryopreserve cells, the inventors simulated' the empirical procedure of the graded freezing protocols. Graded freezing provides insights into the two types of freezing injury which can affect cell recovery: solution effects and intracellular ice formation (McGann, 1979). The graded freezing technique involved cooling (ie. I °Clmin) the samples to various subzero temperatures before being either thawed directly in a 37 °C water bath or plunged into liquid nitrogen first and then thawed (McGann, 1979). With this procedure it is possible to separate injury sustained during the initial cooling phase to subzero experimental temperatures; from that sustained upon further cooling to storage temperatures. Various cooling rates can also be used to explore the effect of time spent during cooling on cell recovery. Simulations were performed in which cells with no cryopreservant were cooled to various subzero temperatures ranging from -4°C to -30°C at cooling rates ranging from 0.2°C/min to 100°Clmin; prior to being plunged to -40°C
at 325°C/min to model graded freezing (Ebertz, 2002(x)).
In the experimental .procedure, the samples are first placed in a -3°C methanol bath from 0°C and allowed to equilibrate prior further cooling to the various experimental subzero temperatures. The temperature profile of this equilibration is governed by Fourier's Law.
Fourier's Law describes the rate of heat transfer which depends on the temperature distribution of the system (Incropera, 2002):
aT ~ & . oT
~t ~1) where dTldt is the rate of change in temperature of the sample with time, k is the fitting constant, and DT is the difference in temperature between the bath and the sample. The constant was determined by monitoring the cooling prol 1e of a sample taken from 0°C and placed in a -3°C
methanol bath with a Type T thermocouple (Omega, Laval, Canada,). This profile was then fitted to a curve using equation 1. The constant was then used in simulations to model the equilibration step of the graded freezing procedure. Simulating the equilibration step using Fourier's Law is a novel approach.
2.3 Results Cooling profiles Cells were cooled to -3°C from 0°G (k=7) and then cooled at 0.2., 0.5, 1.0; 5.0, 10, 20; or 100°C/min to plunge temperatures ranging from -4°C to -30°C, prior to being cooled rapidly to -40°C.
Figure 11 is a representative temperature cooling profile for equilibration and cooling at 1 °C/min to various subzero experimental temperatures. The cooling profiles were similar for all cooling rates except that the length of time needed to reach the intermediate temperatures decreased with increasing cooling rate.
Cell volume during cooling Figure 12 is a representative graph for 1 °C/min demonstrating the changes in cell volume as a function of temperature. The data showed that cells did not reach the same volume when cooled to -3°C prior to rapid cooling to -40°C, as did cells cooled to other subzero temperatures. The cell volumes for the other temperatures were very close to the values obtained for -30°C, which was the minimal cell volume recorded. This may indicate that ~ cells cooled to -3°C may have a greater amount of supercooling at low subzero temperatures due to the higher water content than cells cooled to the other temperatures. The results not shown for 0.2 and 0.5°C/min demonstrated similar changes in cell volume. The results not shown for 1.0, 5.0, 10, 20, or 100°C/min demonstrated less of a reduction in cell volume when cooled to the subzero temperatures.
Supercooling & [KCl]i during cooling Supercooling was calculated in cells cooled to the plunge temperatures prior to being plunged to -40°C (325°C/min) fox all cooling rates. A representative graph demonstrating supercooling as a function of temperature for cells cooled at 1°C/min is shown in Figure 13a. At low cooling rates, supercpoling is greater than 10°C for cells initially cooled to -3°C prior to plunging. Also, at the higher cooling rates such as 100°C/min, supercooling was also seen at plunge temperatures of -
Experimeptal solutions Various concentrations of phosphate-buffered saline (PBS) were used to examine the concentration-dependence of the hydraulic conductivity and the osmotically-inactive fraction. PBS solutions (1-SX) were made by diluting lOX PBS (GIBC4) with distilled water to final osmolalities of 291~6; 583~25; 861~22, 1150~17 and 1434~20 mOsm/kg respectively. Osmolalities .were measured using a freezing-point depression Osmometer (Precision Systems Inc., Natick, Massachusetts), which was calibrated using 100; 300 and 500 mOsm/kg osmometry standards (Precision Systems Inc.).
Measurements of cell volurpes The Coulter counter (ZB1, Goulter Inc., Hialeah; Florida), fitted with a pulse-height analyzer (The Great Canadian Computer Company, Spruce Grove, AB; Canada) was used to monitor cell volume as a function of time as cells passed through the 100 ~m aperture (Groves, 1969(a); Grower, 1969(b); McGann; 1982). This system has been previously used to monitor changes in cell volume for a variety of cells in suspension (Armitage; 1984; Benson; 1998; Ebertz, 2002; Hempling, 1997; Liu, 1995; Mazur; 1986; McGann, 1981; Woods, 1999; Zierger, 1999), including hematopoietic stem cells (Gao, 1998; Hubel, 1999;
McGann, 1987; Woods, 20U0):
TF-1 cells (150-200 ~;L) were injected into well-mixed hypertonic experimental solutions (10 yL). Experimental solutions were maintained at experimental temperatures using a circulating water bath with a custom insulated jacket. The current pulses proportional to the cell volumes were measured and the time recorded as the cells passed through the aperture of the Coulter counter. Experimental temperatures were measured at 4:6~0.7, 4.8~0.6, 8:1~0.711.1~0.6, 12.9~1.4, 16.4~0.5, 19.4~0.8, 23.311.2, 28.8~O.b, and 37.4~0.8°C using a Digi-Sense thermocouple thermometer (Cole Parmer, Anjou; Canada). For each experiment, three replicates were performed for each solution at each temperature. The experiments were repeated .a minimum of hree times using cells from different passages. Latex beads (15 ~.m diameter; Beckman Coulter, Miami, Florida) were used as calibrators to convert relative volumes to actual volumes in 1X PBS and in the experimental solutions.
Determination of the osmotic parameters Measurements of cell volumes as a function of time were used to determine the osmotic parameters. Least squares error its using EXCEL
Solver, was used to solve for L~ and vb, using equations l and 2 respectively. The analysis of the concentration dependence used 2-5X
PBS solutions (583-1434 W 4sm/kg) at temperatures of 4.8~0.6, 12.9~1.4, 23.3~1.2, and 37.4~0.8°C, and additional analysis with temperatures of 4.6~0.7, 8.1~0.7, 11.1+_0:6,;16.4~0.5, 19:410.8 and 28:8~0:6°C was performed for 3X PBS solutions only. Curves were fitted for Lp and Vb for each experimental, solution at each of the experimental temperatures.
The Arrhenius activation energy for Lp, described by equation 3, was fit for using linear regre sign of the natural logarithm of Lp as a function of the inverse absolute temperature in EXCEL.
Statistical analysis Statistical comparisons used a standard one-way analysis of variance (ANOVA) at 5% level of signi~canee: Estimates of Lp and vb were compared between experimental solutions (2-SX PBS) for all the experimental temperatures.
1.3 Results Isotonic volume Figure 6 shows a representative volume distribution of TF-1 cells under isotonic conditions in 1X PBS (calibration factor=7.8; mean volume=806.1 ~m3). The distribution was lognormal and narrow compared to other cell types which have a more broad distribution (unpublished data). This is the result of synchronizing the cells in Go/Gl phases of the cell cycle. For the entire data set, the isotonic volume for TF-1 cells was 776~36 pm3.
Hydraulic conductivity Changes in mean cell volume as a function of time were used to calculate the Lp. Figure 7 is a representative graph of TF-1 cells exposed _2g_ to 3X PBS at four different temperatures, which show the increase rate of cell volume shrinkage at higher temperatures, demonstrating a higher Lp.
Data from experimental solutions (2-SX PBS) at four different temperatures (4.80.6, 12.9~1.4, 23.3~1.2, and 37.410.8°C) were used to examine the concentration and temperature dependence of Lp. The L~
was determined by fitting the data to Equation 1 using the least squares method with EXCEL Solver for each experimental solution and temperature and summarized in Table 1. Values for each concentration were pooled since there was no concentration dependence (p>0.05):
Tempe~ntore ~
(~um/min/atm)4.840:62C 12.91.4C 23.31.2C 37.40:8C
2X PBS 0:08110:013 0.12210.011 0.337O.OS1 1.170.14 ' 3X PBS 0:0790.011 O.L340.020 0.3750.060 1.390.30 4X PBS 0.0?'~~0.008 O.I220.01'7 0.428O.U48 1.420.28 SX PBS 0.07610:014 0.1190:014 0.3790.036 1.500.30 pooled mean 0.0780:012 0.123f0.015 0.3880.052 1.360.26 ' TABLE 1. L~ values for TF-1 cells (mean,~ SD); p>0.05 for all values implying no concentration dependence Osmotically-inactive fraction For each sample, vb was fit to Equation 2 using the least-squares method in EXCEL Solver. Data from temperatures of 4.8~0.6, 12.9~1.4, 23.3~1.2, and 37.4~0.8°C for 2-SX PBS solutions were used in order to determine the temperature and concentration dependence of vb. The data in Table 2 show that the osmotically-inactive fraction was not dependent on concentration (p>0.05): As a result, overall data for all temperatures and concentrations were pooled. TF-1 cells had a mean vb of 0.35~0.03.
A Boyle van't Hoff plot of equilibrium volume as a function of inverse osmolality for the aggregate data is shown in Figure 8. TF-1 cells responded as, ideal osmomet~rs over a range: of 583-1434 mOsm/kg. The vb could also be determined' by extrapolating the slope back to the y-axis in which the intercept was 0.37. This value was within the error found using the least squares method;
Temperature 4.840.62C 12.911.4C 23.31.2C 37.40.8C
2X PBS 0:3980:042 ' 0:3320.058'0.3270.016 0.3210.041 3X PBS 0.3780.040 0.3830.022 0.3480.030 0.3380.050 4X PBS 0.3570.027 0.3760.051 0.3410:018 0.3300.021 SX PBS 0:3610.026 0.3$40.058 0.3710.010 0.3130.028 pooled mean 0:373+_0:034Q.3680.052 0.3470.020 0.3260.037 TABLE 2, vb values for TF'-1 cells (mean t SD); p>0.05 for all values implying no concentration dependence Arrhenius activation energy The Lp follows the Arrhenius equation over the range of experimental temperatures. The Arrhenius activation energy of Lp was determined using pooled data' from experiments based on cell volume kinetics of 2-5X PBS solutions at temperatures 4:8~0.6, 12,9~1.4, 23.3~1.2, and 37.410,8°C anal of 3X PBS solutions at temperatures 4.6~0.7, 8.1~0.7, 11.1~0.6, lfi'.4~0.5; 19.4~0.8: and 28:8~0.6°C.
Figure 9 shows Arrhenius plots of mean Lp values for all the experimental temperatures examined. The value of Ea for Lp from these data was 13.4 keal/mol. Figure 9 also demonstrates that the osmotically-inactive fraction is independent of temperature using linear regression.
1.4 Discussion The hydraulic conductivity reported in this example was found to be strongly dependent on all temperatures reported in this example, but independent of concentration, which has been previously reported for other cells types {Liu, 1995): The value for Lp was 0.342 ~m/minlatm at 20°C. The Lp is within the range reported for mammalian cells, such as rat megakarycytoporietic cells, Chinese hamster lung ~broblast cells, bovine immature oocytes; ehondrocytes, corneal endothelial, epithelial and stromal cells {McGrath, 1988). The rate of water movement is considered slow responding; thus the Coulter counter was an efficient method of monitoring changes in cell volume.
TF-1 cells follow the Boyle van't Hoff relationship and thus the cells behave as ideal osmometers. The osmotically-inactive fraction can be determined using the Boyle van't Hoff plot and the least-squares method. The vb for TF-1 cells was determined as 0.350.03 and 0.37, respectively. The value of vb reported here is within the range for a variety of mammalian cell types (0:2-0.41) (Ebertz; 2002; Gao, 1998;
Gilmore, 1998; Hempling, 197'1; Liu, 1995; McGann, 1981; McGrath, 1988). The Arrhenius: activation energy for Lp o~ 13.4 kcal/mol reported here, is within normal ranges for other types of mammalian cells (12-16 kcal/mol) (Ebertz, 2002; Hempling, 1977; Liu, 1995). It has been reported that cells with an Ea of <6 kcallmol for I,p, are fast responding, and may exhibit channel-mediated water transport (Elmoazzen, 2002).
Also, cells with an Ea >i0 kcal/mol for Lp, such as the TF-1 cells, are slow responding and may transport water by solubility-diffusion through the plasma membrane. The high Ea for Lp indicates that the water permeability of the plasma membrane of TF-1 cells is highly dependent on temperature. However, the Ea for Lp alone may not be enough to negate the presence of aqueous pores and further analysis is required of slow responding cells to explore the possibility of other types of pores (Elmoazzen, 2002).
The osmotic parameters reported here for TF-1 cells are comparable with those previously reported for H$Cs. The value reported here for Lp is comparable with he Lp previously reported for cord blood CD34+ cells of 0.168~0.03 ~,m/atm/min at 20°C (Hunt, 2'003), indicating that the rate of water movement is similar for TF-1 cells and for other HSC. Based on Lp values reported by Hunt et al. at two temperatures (Hunt, 2003), Ea for L~ in cord blood CD34+ cells were calculated to be 18.8'kcal/mol. The Ea for Lp also denotes a slow-responding cell. The vb is comparable to that previously reported for both bone marrow hematopoietic CD34+ cells of 0:205 (Goo, 1998) and umbilical cord blood CD34+ cells of 0.32 (Hunt, 2003) and 0.27~0.01 (Hunt, 2003). The average size of TF-l cells (776~36 ~.m3) was higher than that previously reported for both bone marrow CD34+ cells of 345 pm3 (Goo, 1998) and umbilical cord blood CD34+ cells of 274~13 ~.m3 (Hunt, 2003). This indicates that although there are differences in cell volume between TF-1 cells and CD34+ cells from patient samples; the osmotic parameters for both cell types are comparable.
The parameters determined in this example are sufficient to be used in the mathematical analysis of the TF-1 cellular responses to low temperatures and ultimately in designing a cryopreservation procedure speei~c to these cells. The parameters summarized in Table 3 were subsequently used in simulations described in Example 2. The simulations model the cellular responses to ice formation in the extracellular solution based on how the cells responded osmotically to hypertonic solutions. It is also possible to use the osmotic parameters from other cell types to model their cellular response to low temperatures.
Isotonic Volume ' 776 ~.m Inactive Fraction 0.350 Lp (20C) 0:342 wm/min/atm Activation Energy for L"~; 13,4 kcallmol Isotonic osmolality 0.301 osm/kg TABLE 3. Osmotic parameters for TF-1 cells used in simulations.
Example 2: Simulations of cellular responses to low temperatures 2.1 Introduction The freezing of cells in suspension has largely been approached empirically. However, simulations have been used to mathematically predict cellular responses to low temperatures for a variety of cell types:
bovine erythrocytes (Leibo, 1976); yeast (Schwartz, 1983); hamster ova (Shabana, 1988); haanster pancreatic islet cells (Liu, 1995); and epithelial, endothelial and stroma cells (Ebertz, 2002). Modeling is based on the theoretical response of the cell to a changing extracellular environment.
The cellular responses to! the formation of extracellular ice in surrounding solution are largely dependent on the movement of water across the plasma membrane: Ea~tracellular ice formation increases the concentration of solutes in the residual liquid, resulting in osmotic efflux of water from the cell. The. ,properties of the cell membrane, specifically the osmotic parameters, govern the rate of change of cell volume.
Osmotic parameters can be used in simulations to theoretically model cellular responses to low temperatures:
Simulations reduce the time and expense- involved with empirical experiments. Simulations also provide a means to analyze changes in cell volume prior to empirical: experimentation: Simulations provide insight into intracellular osmolalities; concentrations and rates of water movement. These results can then be used for comparisons between cryopreservation protocols and for comparison between different cell types which may be necessary if attempting to cryopreserve a heterogeneous cell population or a tissue. Ultimately, simulations allow for unlimited theoretical protocols to be explored by controlling cooling and warming rate, experimental temperatures, and the components of the intracellular and extracellu.lar comparCments for any cell type for which the osmotic parameters are known.
Mazur has previously used simulations to explore the effects of solutions, osmotics and temperatures on cellular systems (Mazur, 1963).
Mazur reported the rate of water loss from the cell and the change in permeability with temperature as the parameters necessary to predict changes in volume of intracellular water with temperature. Furthermore, predictions about the probability of intracellular ice formation can be made based on the amount of intracellular water and the temperature of the cell. Subsequently, Mazur indicated that rates greater than 1 °C/min may generate a supercooled cytoplasm in yeast: (Mazur, 1963; Mazur, 1977). Supercooling is tl~e amount a solution can be cooled below its freezing point without ice forming and it used as an indicator of the potential for intracellular ice formation (Mazur, 1972). Intracellular supercooling is the extent to which a cell is cooled below the phase-change temperature before the formation of intracellular ice. Mazur also reported that there was a 10°C limit to supercooling above which the risk of intracellular ice is increased (Mazur, 1963; Mazur, 1977). Cooling rates of approximately 1 °C/min should reduce the amount of supercooling therefore they reduce the risk of intracellular ice. Diller further examined the probability of intracellular ice formation based on the synergistic interaction of cooling rates and supercooling (Diner, 1975). During cooling, the cell attempts to maintain equilibrium across the plasma membrane either through, osmotic dehydration or the formation of ice. Therefore; with no supercooling, there is a high probability that the cell will dehydrate; whereas with greater than 10°C
supercooling, the cell may form intracellular ice (Diller, 1975). Ebertz also reported the use of supercooling as an indicator for intracellular ice formation for simulations an corneal endothelial, epithelial and stromal cells (Diller, 1975).
Based on Mazur et al.'s 'two-factor hypothesis', solution effects injury must also be considered along with intracellular ice formation injury, when attempting to determine the optimal cooling rate for cryopreserving a cell type (Mazur; 1972): During slow cooling, cell injury is due to prolonged exposure to high solute concentrations, as a result of cell dehydration due to extracellular ice formation. This work represents a novel approach of combining supercooling with intracellular electrolyte concentratio~a in the presence of extracellular ice, to use as indicators of cryoinjury.
The objective of these simulations was to theoretically determine the cellular responses of TF-1 cells at various stages of the graded freezing protocol for comparison with experimental data: Simulations were performed using the osmotic parameters of TF-1 cells reported in Example 1 (Table 3).. Maximum levels of intracellular electrolyte concentrations ([KCI]i) and of supercooling were examined upon cooling the cells to -40°C; as indicators for solution effects injury and intracellular ice formation injury, respectively:
2.2 Calculations of low-temperature responses Methods The GryoSimS pr~:gram (Dr. Locksley McGann; University of Alberta; Canada) was used to perform the simulations and Figure is a representative image of the program interface. The program uses the phase diagrams of the components of the intracellular and extracellular solutions, the osmotic characteristics of the cell membrane and the temperature dependencies of the parameters. The simulations axe calculations of the cellular osmotic responses to the concentration of solutes in the residual liquid in the presence of ice at low temperatures.
Ice nucleation is assumed to be at the freezing point of the extracellular solution. Phase diagrams were used to calculate concentrations in the liquid phase for sodium chloride (NaCI)-H20 (Wolf, 1982) and potassium chloride (KCI)-H20 (Wolf; 1982) for the extracellular and intracellular compartments, respeeti~ely. The amount of intracellular protein has been reported to be more than half the dry weight of the cell (Alberts, 1994). It has also been reported that red blood cells possess approximately 0.0073 mol of hemoglobin per kg of intracellular water (7.3 mmolal) (Dick, 1958; Savitz, 1964; Williams; 1959). Since the intracellular protein content is unknown for TF-1 ceps to the inventors' knowledge, the inventors used half the molality of hemoglobin in red blood cells (3.65 mmolal) for the simulations. The hydraulic conductivity (Lp) was used to calculate osmotic cellular responses to changes in the extracellular conditions. The Arrhenius activation energy (Ea) for Lp was used to describe the temperature dependency of hydraulic conductivity. The numerical values of the hydraulic conductivity and the Ea for Lp from Example l (Table 3); were extrapolated to lower subzero temperatures.
Temperature profiles To explore the role of low and high cooling rates typically used to cryopreserve cells, the inventors simulated' the empirical procedure of the graded freezing protocols. Graded freezing provides insights into the two types of freezing injury which can affect cell recovery: solution effects and intracellular ice formation (McGann, 1979). The graded freezing technique involved cooling (ie. I °Clmin) the samples to various subzero temperatures before being either thawed directly in a 37 °C water bath or plunged into liquid nitrogen first and then thawed (McGann, 1979). With this procedure it is possible to separate injury sustained during the initial cooling phase to subzero experimental temperatures; from that sustained upon further cooling to storage temperatures. Various cooling rates can also be used to explore the effect of time spent during cooling on cell recovery. Simulations were performed in which cells with no cryopreservant were cooled to various subzero temperatures ranging from -4°C to -30°C at cooling rates ranging from 0.2°C/min to 100°Clmin; prior to being plunged to -40°C
at 325°C/min to model graded freezing (Ebertz, 2002(x)).
In the experimental .procedure, the samples are first placed in a -3°C methanol bath from 0°C and allowed to equilibrate prior further cooling to the various experimental subzero temperatures. The temperature profile of this equilibration is governed by Fourier's Law.
Fourier's Law describes the rate of heat transfer which depends on the temperature distribution of the system (Incropera, 2002):
aT ~ & . oT
~t ~1) where dTldt is the rate of change in temperature of the sample with time, k is the fitting constant, and DT is the difference in temperature between the bath and the sample. The constant was determined by monitoring the cooling prol 1e of a sample taken from 0°C and placed in a -3°C
methanol bath with a Type T thermocouple (Omega, Laval, Canada,). This profile was then fitted to a curve using equation 1. The constant was then used in simulations to model the equilibration step of the graded freezing procedure. Simulating the equilibration step using Fourier's Law is a novel approach.
2.3 Results Cooling profiles Cells were cooled to -3°C from 0°G (k=7) and then cooled at 0.2., 0.5, 1.0; 5.0, 10, 20; or 100°C/min to plunge temperatures ranging from -4°C to -30°C, prior to being cooled rapidly to -40°C.
Figure 11 is a representative temperature cooling profile for equilibration and cooling at 1 °C/min to various subzero experimental temperatures. The cooling profiles were similar for all cooling rates except that the length of time needed to reach the intermediate temperatures decreased with increasing cooling rate.
Cell volume during cooling Figure 12 is a representative graph for 1 °C/min demonstrating the changes in cell volume as a function of temperature. The data showed that cells did not reach the same volume when cooled to -3°C prior to rapid cooling to -40°C, as did cells cooled to other subzero temperatures. The cell volumes for the other temperatures were very close to the values obtained for -30°C, which was the minimal cell volume recorded. This may indicate that ~ cells cooled to -3°C may have a greater amount of supercooling at low subzero temperatures due to the higher water content than cells cooled to the other temperatures. The results not shown for 0.2 and 0.5°C/min demonstrated similar changes in cell volume. The results not shown for 1.0, 5.0, 10, 20, or 100°C/min demonstrated less of a reduction in cell volume when cooled to the subzero temperatures.
Supercooling & [KCl]i during cooling Supercooling was calculated in cells cooled to the plunge temperatures prior to being plunged to -40°C (325°C/min) fox all cooling rates. A representative graph demonstrating supercooling as a function of temperature for cells cooled at 1°C/min is shown in Figure 13a. At low cooling rates, supercpoling is greater than 10°C for cells initially cooled to -3°C prior to plunging. Also, at the higher cooling rates such as 100°C/min, supercooling was also seen at plunge temperatures of -
6°C
through -12°C, respectively (data shown for 100°C/min in Figure 13b ).
.: L
A representative graph demonstrating [KCl]i as a function of temperature for cells cooled at 1 °C/min is shown in Figure 14a. Cells cooled to increasingly lower subzero plunge temperatures showed increasing concentrations of {KCl);, with the highest concentration for cells cooled to -30°C at 1°C/min. This correlates with the decrease in cell volume reported in the previous section. This gradual increase in [KCI]i demonstrates the potential for increased solution effects upon cooling to the lower subzero temperatures: Results for 0.2°Clmin and 0.5°C/min were not shown however were similar to 1 °Clmin data, but over a longer period of time. Figure 14b shows the [KCl]i as a function of temperature for cells cooled at 100°C/min. This demonstrates the increase of [KCl];
is minimized with higher cooling rates:
Maximum supercooling and [KCI]; during cooling The maximum amount of supercooling was calculated as the highest amount of supercooling which occurred throughout the cooling profile for each plunge temperature. Figure 13a and 13b shows supercooling as a function of temperature for TF-1 cells cooled at 1.0°C/min and at 100°C/min respectively; with arrows indicating where the maximum supercooling was determined for the various plunge temperatures. The maximum supercooling for each cooling rate was then summarized and graphed as a function of plunge temperature (Figure 15).
Similar patterns of high supercooling (27°C) at -3°C were demonstrated for all the cooling rates; with: a decrease to below 10°C at approximately -4°C for cells cooled at 02, 0.5; and 1.0°C/min and at approximately -13 °C for cells cooled at 100°C/min. Intracellular ice formation thus only appears to play a role in freezing 'injury for cells cooled to high subzero temperatures for low cooling rates and intermediate subzero temperatures for higher cooling rates.
The maximum amount of [KCl]a was calculated as the highest concentration of KCl which occurred throughout the cooling profile for each plunge temperature. Figure 14a and 14b shows the [KCI]; as a function of temperature for TF-1 cells cooled at 1.0°CJmin and at 100°C/min respectively, with arrows indicating where the maximum [KCl]; was determined for the various plunge temperatures. The maximum [KCl]; for each cooling rate was then summarized and graphed as a function of plunge temperature (Figure 16). Similar patterns of an increase in [KCl]; are demonstrated for all cooling rates, which is consistent with the change in cell volume. Cooling rates of 0.2, 0.5, and 1.0°C/min showed the sharpest increase in [KCl]; at the higher subzero temperatures, compared with higher cooling rates that showed a gradual increase, which is consistent with the cell dehydrating sufficient to maintain equilibrium. Thus at the low cooling rates examined, cells were exposed to comparably ' high solute conditions at high subzero temperatures. However, there was increased time spent exposed to the solutes for the 0.2°C/min :compared with the 1.0°C/min, so one would expect that 0.2°C/min would have a lower cell recovery because cells would have been exposed to increasingly high solute concentrations for a longer period of time. The optimal temperature for plunging the cells after the initial cooling phase is a function of temperature and the amount of time spent cooling to that temperature, which influences [KCl]; and supercooling.
Example 3: Experimental assessments of simulation outcomes 3:1 Introduction In order for simulations to be used in cryopreservation, it is necessary to test the predictions of simulations empirically. The objective of this example was to conduct graded freezing experiments with '1'F-1 cells and compare the cell survival outcomes with the theoretical predictions put :forward in Example 2 based on degrees of supercooling and intracellular electrolyte concentration ([KCl];).
Membrane integrity was used as an assay for freeze-thaw injury.
Membrane integrity has been used as an indicator of cell damage during freezing, as it has been shown that the membrane is a site of freezing-thawing injury (Acker; 2001). Also, it has been shown that there is a correlation between intracellular freezing and membrane damage for cells in suspension (Acker; 2001; Mazur; 1965).
3.2 Materials & Methods TF-1 cell culture TF-1 cells (ATCC; Manassas, Virginia) were grown at 37°C in 5%
C02 in RPMI 1640 Medium Modified (ATCG) with 10% fetal bovine serum (FBS) (ATCC), and supplemented with 2 ng/mL recombinant human GM-CSF (Stemcell -Technologies, Vancouver, Canada). Cells were maintained between 0:1 x 106 and l x 106 cells/rnL, according to ATCC guidelines. Prior to experiments; cells were washed twice with serum-free RPMI media and incubated overnight to synchronize the cells (Kolonics, 2001). Cells were then centrifuged and re-suspended at a concentration of 4 x 106IrnL, which was necessary for the viability assessment program to be used:
Experimental solutions TF-1 cells were re-suspended in serum-free RPMI prior to the graded freezing experiments. In order to compare the results with the clinical standard, TF-1 cells were also re-suspended in 10%
DMSO/RPMI at 4°C; prior to freezing experiments.
Graded freezing experiments Samples of 0.2 mL cell suspension; in serum-free RPMI or 10%
DMSO/RPMI, in glass tubes (Fisher, Edmonton, Canada) were cooled in a 0°C ice bath fox 5 minutes. Control samples were removed and either warmed in a 37°C water bath or plunged into liquid nitrogen (325°C/min;
(Ebertz, 2002(a)). Experimental samples were transferred into a methanol bath preset at -3°C and allowed to equilibrate for 5 minutes prior to ice nucleation with cold forceps. After 5 minutes, the bath cooled at 0.2, 0:5, or 0.9°C/min to -40°C. The cooling rates were monitored using a Type T thermocouple (Omega; Laval, Canada). Samples were then removed at -3; -6, -9, -12; -15; -20, -30, and -40°C and either thawed directly in a 37°C water bath or plunged into liquid nitrogen. Samples were kept in liquid nitrogen for a minimum of 1 hour prior to being thawed in a 37°C water bath. Duplicate samples were used for both the direct thaw and the plunge conditions at each experimental temperature.
Each experiment was repeated in triplicate for each cooling rate and experimental solution. Samples in 10%DMSOfRPMI were only cooled using 0.9°C/min.
Viability assessment Cell viability was assessed by a membrane integrity assay. The assay was performed by incubating cells with SYTO~ 13 (Molecular Probes, Eugene, Oregon) and ethidium bromide (EB) (Sigma, Mississauga, Canada) (Yang; 1998). Syto 13 permeates the cell membrane of all cehs and complexes with DNA and fluoresces green under UV exposure. EB penetrates cells with a damaged plasma membrane and also complexes with DNA fluorescing red under UV
conditions. The dual stain allows for differentiation between cells with and without intact plasma membranes.
The SytolEB stain was prepared using 40 p,I, of 2.5 mM EB stock solution and IO p.L of 5 mM SYTO~ 13 stock solution mixed with 350 ~.L phosphate-buffered saline (PBS): Final concentrations were 0.25 mM
EB and 0.125 mM Syto. Twenty p.L of stain was added to 200 ~,L each sample, mixed, and allowed to incubate for 2 minutes at room temperature: Fluorescent images were captured using a Leitz Dialux 22 fluorescence (440-480 nmj microscope (Leitz, Germany) fitted with a PIXERA Viewfinder Pro digital camera (Pixera Corporation, Los Gatos, CA, USA} digital camera. The Viability Assessment Program (The Great Canadian Computer Company, Spruce Grove, Canada), which counts red versus green pixels was used to quantify cell membrane integrity from digital images (Jomha; 2003). This method measures membrane integrity of the cell remaining after experimental treatment.
3:3 Results Conventional cryopreservation protocol with DMSO
The standard for cryopreserving HSCs is to cool the cells at 1 °C/min in 10% DMSO. TF-1 cells were cooled at 0:9°Clmin in 10% DMSO/RPMI
to various temperatures down to -40°C, prior to being thawed directly or plunged into liquid nitrogen (Figure 17). The maximum percentage of membrane integrity was 63.79.8%, when samples were cooled to -12°C
to -15°C, prior to being plunged into liquid nitrogen: The results were comparable with that previously reported for cryopreserving HSCs with 10% DMSO cooling at 1:0°C/min (7915% (Hunt, 2003); 67.42.0%
(Yang, 2003). This experiment was limited by the cooling capacity of the methanol bath:
Graded freezing with no cryapreservant using various cooling rates TF-1 cells were suspended in serum-free RPMI and cooled at 0.2°C/min to various temperatures up to -20°C, prior to being thawed directly or plunged into liquid nitrogen. Cells thawed directly from the subzero plunge temperatures showed a ' S0% decrease in membrane integrity by -12°C, indicating that a major portion of cells were damaged prior to being plunged into liquid nitrogen (Figure 18). However, damage at higher subzero temperatures occurred as a result of the plunge into liquid nitxogen. There was limited cell recovery when the cells were plunged into liquid nitrogen at all plunge temperatures. The maximum recovery of 24.2tS.5% was seen for TF-1 cells plunged at -3°C:
Figure 19 shows the membrane integrity as a function of plunge temperature for TF-1 cells cooled at 0.5°C/min to -40°C, prior to being plunged into liquid nitrogen: TF-1 cells demonstrated similar membrane integrity for both thaw and plunge samples as with 0:2°C/min. The maximum recovery of 28.25.8% was obtained between -3°C and -9°C.
Membrane integrity as a function of plunge temperature for TF-1 cells cooled at 0.9°C/min to -40°C, prior to being thawed directly or plunged into liquid nitrogen is shown in Figure 20. Results were also similar to those for 0.2°C/min and 0.5°C/min. TF-1 cells showed maximum recovery of 27.80:8% at -9°C. This experiment was limited by the cooling capacity of the methanol bath; which had a maximum cooling rate of 0.9°C/min.
Data from all three cooling rates demonstrated a 50% decline in membrane integrity for cells'thawed directly from the plunge temperature at -12°C. This indicates that cells were damaged prior to being plunged into liquid nitrogen, possibly due to solution effects. However, there was a significant difference between the membrane integrity for cells directly thawed and hose further plunged into liquid nitrogen. Due to the high cooling rate upon plunging into liquid nitrogen (325°C/min), this indicates that intracellular ice formation may play a role in damage at these temperatures. There does appear to be a zone of subzero plunge temperatures (-3°C to -9°C), which confers some protection against injury during the plunge into liquid nitrogen for all the cooling rates.
This would constitute an optimal subzero plunge temperature range for these cooling rates.
3.4 Discussion Discussion of experimental'data The conventional cryopreservation protocol of using 10%DMSO
solution yielded comparable results with other HSCs cryopreservation reports. The experimental results for cryopreserving TF-1 cells without cryopreservants were significantly lower than the standard. The data showed that the membrane integrity between the various cooling rates was within standard error mean of each other. TF-1 cells responded similarly to exposure to the various subzero plunge temperatures and to subsequent plunging into liquid nitrogen.
Comparison of theoretical and experimental results Simulations from Example 2 predicted that there was no difference in maximum [KCl]i, between the cooling rates, however the time spent exposed to these elevated concentrations may cause additional cryoinjury.
The experimental results demonstrated that there was not a significant difference in membrane integrity between the cooling rates of 0.2, 0.5, and 0.9°C/min, which is consistent with the theoretical results based solely on [KCl];. Simulations did indicate that there was potential for increased exposure time to the solutes with the lower cooling rate (0.2°C/min), however the experimental data demonstrated that the percentages of membrane integrity were within error for all the cooling rates. Therefore, tile increased exposure time for the lower cooling rates was not significant.
For all cooling rates, simulations predicted a progressive increase in [KCl]i upon cooling to lower temperatures. Based on Lovelock's work, the inventors predicted that salt concentrations of greater than 3 M
would be damaging to the cells (Lovelock, 1953) and the experimental data demonstrated that there was a decrease in membrane integrity (~60%) at this concentration for all the cooling rates. The experimental data also demonstrated a progressive decline in membrane integrity for cells thawed directly from subzero plunge temperatures. At low subzero plunge temperatures (<-20°C), cells directly thawed had low percentages of membrane integrity (<20%). Therefore, the exposure time coupled with the concentration of solutes may have been signiricant variables for freezing injury.
Simulations also predicted that cells cooled to -3°C prior to being cooled at 325°C/rnin to -40°C would have a high degree of supercooling (27°C). The experimental results demonstrated that upon cooling to -3°C
prior to plunging into liquid nitrogen, TF-1 cells had a relatively high percentage of membrane integrity. This indicates that although intracellular ice formation may have played a role in membrane damage at this plunge temperature; there was another source of damage upon cooling to lower temperatures, where solutions effects were present.
Also, the range of plunge temperatures between -3°C and -9°C, which demonstrated the highest viability, had high variations in maximum supercooling (2°C to 27°C) and in maximum [KCl]; (2 to 4 1V.~.
This alludes to the complex interactions between these two types of injury at subzero temperatures.
Example 4: Theoretical design of a cryopreservation protocol 4.~. Introduction The cellular responses to the formation of ice in surrounding solution are largely dependent on the movement of water across the plasma membrane. Ice formation causes osmotic stress on the cell membrane forcing water out of the cell to maintain equilibrium with the extracellular solution. The properties of the cell membrane, specifically the osmotic parameters, govern these changes in cell volume. The osmotic parameters can be used in simulations to theoretically model cellular responses to: low temperatures. Simulations also provide precise results regarding changes in cell volume and the amount of supercooling.
These results can then be used for comparisons between cryopreservation protocols and for comparison between different cell types which may be present in one tissue: Ultimately; simulations allow for unlimited theoretical protocols to be explored by controlling cooling and warming rate; plunge temperatures, and the components of the intracellular and extracellular compartments for any cell type for which the osmotic parameters are known.
To distinguish between the two types of,injury, solution effects and intracellular ice formation, the inventors simulated the empirical procedure of two-step freezing. The two-step freezing technidue was developed by Farrant et al. and has provided a logical method to examine the effects of freezing injury on cell recovery due to non-linear cooling rates and to exposure to a range of subzero temperatures (Farrant, 1974).
In their procedure, samples were cooled at an uncontrolled non-linear cooling rate to various subzero plunge temperatures by being transferred to a preset bath before being 1 ) thawed directly from that holding temperature or 2) plunged to 196°C before thawing. McGann and Farrant later reported the subzero plunge temperatures and the length of hold time -at that temperature were important variables to consider when attempting to maximize cell survival (McGann, 1976).
The objective of this example was to further the inventors' understanding of the theoretical responses of TF-1 cells, a model for hematopoietic stem cells (HSC) (Kitamura; 1989(a); Marone, 2002), to subzero plunge temperatures aid to hold times at those temperatures.
Simulations were done using the osmotic parameters of TF-1 cells reported in Example 1. The objective of these simulations was to theoretically determine the conditions of TF-1 cells at various stages of a freezing protocol. Maximum levels of intracellular electrolyte concentrations ([KCl]i) aid; of supercooling were examined upon cooling the cells to -40°C, as indicators for solution effects injury and intracellular ice formation injury, respectively.
4.2 Simulations of two-step freezing protocol Methods Simulations were performed according to those done in Example 2 using the osmotic parameters of TF-1 cells (Table 3) in the C~~yoSimS
program (Dr. Locksley IVIcCrann; University of Alberta, Canada). The simulations were based on a two-step freezing technique, which has been used to examine the effects of high solute concentrations and intracellular ice formation on cell survival during freezing (Farrant, 1977). The cryopreservation protocol was defined by assigning a starting temperature and then varying the cooling rates; based on typical two-step freezing procedures. Supercooling :and [KCl]i were used as indicators of potential intracellular ice formation and solution effects; respectively.
Temperature profiles The two-step freezing technique involved rapidly cooling the samples to various subzero plunge temperatures before either being thawed directly in a 37°C water bath or plunged into liquid nitrogen first and then thawed (McGann; 1979). For the simulations, cells were cooled using a temperature profile derived from Fourier's Law. Fourier's Law describes the rate of heat transfer which depends on the temperature distribution of the system, previously described in Example 2 (Incropera, 2002). The ~Ztting constant was determined by monitoring the cooling profile of a sample taken from room temperature and exposed to the experimental subzero temperature with a Type T thermocouple (4mega, Laval, Canada). Figure 2l is a representative cooling profile of a sample cooled from room temperature to -15°C. This prafzle was then fitted to a curve and the equation was then used in simulations. The variations between the experimental and :fitted curves were due to the latent heat of fusion.
Simulations were performed in which cells with no cryopreservant were cooled to various subzero plunge temperatures ranging from -3 °C
to -40°C and held at that temperature for 0:3, 0.5, 0.7, 1, 2, 3, 5, 7, or minutes, prior to being plunged to -40°C (325°Clmin) (Ebertz, 2002).
4e3 Results Changes in cell volume during cooling Figure 22 shows simulation results of temperature as a function of time for TF-1 cells cooled to various subzero plunge temperatures ranging from -3 °C to -30°C, held for various hold times (minutes); prior to being plunged to -40°C
(325°C/min). Based on the results from the simulations; the hold times were grouped according to similarities of changes in cell volume, [KCl]i, and supercooling: hold times of 1 minute or less will be represented by the 0.5 minute data; hold times between 2 and 5 minutes will be represented by the 3 minute data; and hold times of between ? and 10 minutes will be represented by the 10 minute data. Figure 23 demonstrates the changes in cell volume as a function of temperature upon cooling to various subzero plyge temperatures ranging from -3 °C
to -30°C, held for a duration, prior to being plunged to -40°C
(325°C/min). The data shown is for (a) 0.5 min., (b) 3 min., and (c) 10 min. hold times. Cells showed a progressive decrease in cell volume upon cooling. Cells only held for 0.5 minutes at the subzero temperature did not reach the same volumes as those held for 3 or 10 minutes at -3°C
and -35°C. This data suggests that the cells have not had sufficient amount of time to dehydrate with a hold time of 0.5 minutes, as opposed to greater than 3 minutes, for both high and low subzero plunge temperatures. This data also indicates that the cells would have a higher amount of supercooling at these outlying plunge temperatures due to the lack of cellular dehydration. Also, with lower concentrations of [KCl]i, it is possible that the cells would not be subjected to high solution effects.
Supercooling during cooling Figure 24 demonstrates the changes in supercooling as a function of temperature upon cooling to various subzero plunge temperatures ranging from -3°C to -30°C, prior to being plunged to -40°C
(325°Clmin). Data shown are for (a) 0.5 min., (b) 3 min., and (c) 10 min. hold times. Supercooling of up to 10°C occurs for all the hold times down to -12°C. This suggests that supercooling plays a key role in potential injury during freezing to lower subzero plunge temperatures. At these lower temperatures, cells were exposed to increasingly supercooled conditions up to 30°C at -30°C.
[KCl]; during cooling Figure 25 demonstrates the changes in [KCl]; as a function of temperature upon cooling to various subzero plunge temperatures ranging from -3°C to -30°C, prior to being plunged to -40°C
(325°C/min). The data shown is for (a) 0.5 min., (b) 3 min., and (c) 10 min. hold times.
Cells cooled to lower subzero plunge temperatures showed increasing concentrations of [KCl];, with the highest concentration for cells cooled to -30°C and held for greater than 3 minutes. This correlates with the gradual decrease in cell volume reported in the previous section. This gradual increase in [IfCI]; demonstrates the potential for increased solution effects upon cooling to the lower subzero plunge temperatures.
The data show similar concentrations of [KCl]; at all plunge temperatures except ~ at -3°C and below -30°C.
Maximum supercooling and [KCI]~ during cooling The maximum amount of supercooling was calculated as the highest amount of supercooling which occurred throughout the cooling profile for each plunge temperature. Figure 24a supercooling as a function of teyperature for TF-1 cells with arrows indicating where the maximum supercooling was determined for the various plunge temperatures. The maximum amount of supercooling was calculated and graphed as a function of plunge temperature (Figure 26). Data is shown for (a) 0.5 min., (b): 3 min., and (c) 10 min. hold times. The maximum supercooling obtained appears to be the primary contributor to potential injury, which suggests that a target plunge temperature between -6°C to -12°C would lead to high levels of survival because the supercooling does not exceed 10°C. Cells held for 0.5 minutes have a more narrow range of optimal plunge temperatures, limited by the amount of supercooling.
These results correlate with the lack of cellular dehydration discussed in the previous sections.
The maximum amount of [KCl]; was calculated as the highest concentration of KCl which occurred throughout the cooling profile for each plunge temperature. Figure 25a shows the [KCl]; as a function of temperature for TF-1 cells, with arrows indicating where the maximum [KCl]; was determined for the various plunge temperatures. The levels of maximum [KCl]; for cells held for 0.5; 3 and 10 minutes gradually increase from -3°C to -20°C (Figure 26). The slope between -3°C and -6°C varies from cells held for 0.5 minutes and 3 to 10 minutes, suggesting that at plunge temperatures between -3°C and -6°C, there may be a difference in cell recovery between U:5 minutes and 3 to 10 minutes.
For all the hold times, based on the temperature range set by the 10°C
limit to supercooling, the data suggests that the lower [KCl]; levels would result in better cell recovery. Figure 27 shows the plunge temperature ranges for cells held for 3 minutes based on 10°C supercooling and 3 M
[KCl];, This range varies between 0.5 minute hold time and the 3 and 10 minute hold time. However, a target plunge temperature of approximately -6°C should result in the highest cell recovery for all the hold times.
These simulations suggest that supercooling plays a key role in two-step freezing and the effects of increasing solute concentrations are secondary. The optimal temperature for plunging the cells after the initial cooling phase is a function of the amount of time spent to cool to a specific temperature, which influences [KCl]i and supercooling.
Example 5: Experimental correlation and optimization of a theoreticall'~-desi ned cryopreservation protocol 5.1 Introduction The simulations performed in Example 4 predicted that subzero plunge temperature and time spent at that temperature were critical variables in the optimization of cryopreservation protocols. In order for simulations to be used in cryopreservation; it is necessary to test the predictions of simulations empirically. The purpose of this example was to explore the range of subzero plunge temperatures and time spent at those temperatures. Two-step freezing experiments were conducted with TF-1 cells and compared with the cell survival outcomes that were theoretically predicted in Example 4. Membrane integrity was used as an assay fox freeze-thaw injury. Membrane integrity has been used as an indicator of cell damage during freezing, as it has been shown that the membrane is a site of freezing-thawing injury (Acker, 2001): Also, it has been shown that there is a correlation between intracellular freezing and membrane damage for cells in suspension (Acker, 2001; Mazur, 1965).
5.2 Materials & Methods TF-1 cell culture TF-1 cells (ATCC; Manassas, Virginia) were grown at 37°C in 5%
COZ in RPMI 1640 Medium Modified (ATCC) with 10% fetal bovine serum (FBS) (ATCC), and supplemented with 2 ng/mL recombinant human GM-CSF (Stemcell Technologies, Vancouver, Canada). Cells were maintained between 0.1 x 106 and 1 x cells/mL; according to ATCC
guidelines. Prior to experiments, cells were washed twice with serum-free RPMI media and incubated overnight. Cells were then centrifuged and re-suspended at a concentration of 4 x 106 cells/mL, which was necessary for the viability assessment program to be used.
Experimental solutions TF-1 cells were re-suspended in serum-free RPMI prior to the two-step freezing experiments.
Two-step freezing experiments Samples of 0.2 mL cell suspension; in serum-free RPMI, in glass tubes were allowed to equilibrate at room temperature for 5 minutes.
Control samples were either warmed in a 37°C water bath or plunged into - SQ
liquid nitrogen. Experimental samples were individually transferred into a methanol bath preset at -3, -6, -9, -12, -15, -20, -30, and -40°C and allowed to ' equilibrate for 2 minutes at that temperature prior to ice nucleation with cold forceps. After nucleation, samples were allowed to equilibrate for 3 minutes before either being thawed directly in a 37°C
water bath or plunged into liquid nitrogen. Samples were kept in liquid nitrogen for a minimum of 1 hour prior to being thawed in a 37°C water bath. Duplicate samples were used for both the direct thaw and the plunge conditions at each plunge temperature. Each experiment was repeated in triplicate.
The two-step freezing experiments were repeated with varying hold times. Cells were cooled to -5, -7, -9, -12, -15, and -25°C and allowed to equilibrate for 2 minutes prior to ice nucleation with cold forceps. After nucleation, samples were allowed to equilibrate for 0.5 or minutes before either being thawed directly in a 37°C water bath or plunged into liquid nitrogen. Samples were kept in liquid nitrogen for a minimum of 1 hour prior to being thawed in a 37°C water bath. Duplicate samples were used for both the direct thaw and the plunge conditions at each plunge temperature. Each experiment was repeated in triplicate.
Viability assessment Cell viability was assessed by a membrane integrity assay. The assay was performed by incubating cells with SYT'O~ 13 (Molecular Probes, Eugene, Oregon) and ethidium bromide (EB) (Sigma, Mississauga, Canada) (Yang, 1998). Syto 13 permeates the cell membrane of all cells and complexes with DNA and it fluoresces green under UV exposure. EB penetrates cells with a damaged plasma membrane and also complexes with DNA fluorescing red under Uil conditions. The dual stain allows for differentiation between cells with and without intact plasma membranes.
The Syto/EB stain was prepared using 40 p.I, of 2.5 mM EB stock solution and 10 p;L of 5 mM Syto~ 13 stock solution mixed with 350 p.I, 1 X phosphate-buffered saline (PB S). Final concentrations were 0.25 mM
EB and 0.125 mM Syto. Twenty p.L, of stain was added to each sample and allowed to incubate for 2 minutes at room temperature. Fluorescent images were captured using a Leitz Dialux 22 fluorescence (440-480 nm) microscope (Leitz, Germany) fitted with a PIXERA DiRactor (Pixera Corporation; Los Gatos, CA, USA) digital camera. The Viability Assessment Program (The Great Canadian Computer Company, Spruce Grove, Canada), which counts red versus green pixels was used to quantify cell membrane integrity from digital images (Jomha, 2003). This method measures membrane integrity of the cell remaining after experimental treatment.
5.3 Results Varying plunge temperature TF-1 cells were suspended in serum-free RPMI to various plunge temperatures up to -40°C and held for 3 minutes, prior to being thawed directly or plunged into liquid nitrogen (Figure 28). Overall, cells demonstrated a higher percentage of membrane integrity than cells cooled at 0.2°C/min to 0.9°C/min presented in Example 3. Cells plunged into liquid nitrogen showed comparable results for membrane integrity to cells directly thawed from temperatures ranging from -1S°C to -40°C.
Cells thawed directly from the plunge temperatures showed a 50 % decrease in membrane integrity by -17°C, indicating that a major portion of cells were damaged prior to being plunged into liquid nitrogen (Figure 28).
However, this membrane damage occurred at a higher subzero plunge temperature for cells cooled at 0.9°C/min. The maximum recovery of 58.86.5% was seen for TF- cells cooled to -I2°C or -15°C before being plunged into liquid nitrogen. These results were higher than those previously reported in Example 3 of approximately 28.2%. Furthermore, these results were comparable with those shown for cooling at 0:9°C/min in 10% DMSOIRPMI of 63:7% (Example 3).
Varying experimental hold time TF-1 cells were cooled to -5, -7, -9, -12, -15, or -25°C and allowed to equilibrate for 2 minutes prior to ice nucleation with cold forceps.
After nucleation, samples were allowed to equilibrate fox varying times (0.3, 0.5, 0.7, 1, 2, 3, 5; 7; and 10 minutes) before either being thawed directly in a 37°C water bath or plunged into liquid nitrogen. Figure 29a shows the membrane integrity of TF-1 cells as a function of hold time for cells cooled to -5°C, held and plunged into liquid nitrogen. Comparable results of membrane integrity were obtained for cells held at -7°C to -9°C
(results not shown). However there was minimal membrane integrity for cells held below -25°C (data shown in Figure 29b). Cells that were directly thawed from the subzero plunge temperatures after being held showed progressive decrease in membrane integrity based on reduced temperature and increased duration of hold time (Figure 30a). Results indicated a high percentage of membrane integrity of SS to 60%, when cells were held for 1-S minutes at high subzero plunge temperatures.
Cooled cells from room temperature to -5°C and -7°C and held for 1-3 minutes, prior to plunging into liquid nitrogen resulted in the highest percentage ofunembrane integrity of approximately 60% (Figure 30b). A
hold time of greater than 5 minutes resulted in a marked decrease in cell survival. This data indicates that there is a zone of subzero plunge temperatures (-5°C' to -15°C); when held for 2-3 minutes, which confers protection against injury comparable to DM50.
5.4 Correlation with theoretically-designed protocol Discussion of experimental results The experimental results for cryopreserving TF-1 cells without cryopreservants indicate that cells can be cryopreserved without DMSO.
This data indicates that there is a zone of subzero plunge temperatures (-5°C to -15°C), when held for l-3 minutes, which confers comparable protection against injury to the standard 10% DMSO/RPMI solution, previously reported in Example 3. This range would constitute an optimal subzero temperature range for these hold times based on experimental results.
Comparison of theoretical and experimental results Simulations were done based on an empirical approach to cryopreservation, two-step freezing, which can be used to examine the role of exposure to subzero plunge temperatures and exposure time. The cooling rates used in, the two-step freezing protocol axe governed by Fourier's Law and were determined experimentally in Example 4. Cells were exposed to increasingly supercooled conditions up to 40°C at a plunge temperature of -40°C. Supercooling appears to be the primary contributor to potential freezing injury. This research supported the upper limit of 10°C supercooling, previously reported by Mazur. The proposed target plunge temperature was suggested to be between -4°C
and -12°C, as supercooling was restricted to less than 10°C, which is comparable to the range determined empirically. Also, based on levels of intracellular KCl ([KCl];), it was suggested that the higher subzero plunge temperature would have the lowest potential for solution effects based on the lack of cell dehydration, which was also supported by this data.
Two-step freezing experiments demonstrated a high percentage of membrane integrity for TF-1 cells when cells were cooled to between -5°C and -12°C and held for 1-5 minutes. These plunge temperatures corresponded with the theoretical values of 5°C to 10°C supercooling, which suggested that a certain amount of supercooling is necessary to achieve a higher viability (Diner, 1975). However, this also supports the belief that excessive supercooling may lead to damage as a result of intracellular ice formation.
Based on the simulations, the duration of time the cells were held at the subzero plunge temperature was also considered an important factor. When cells were held for 0.5 minutes; they did not have sufficient time to dehydrate and reach the same volume as cells held for greater than 2 minutes. This excess intracellular water may have caused damage by forming ice upon subsequent cooling. According to the two-step freezing experiments, cells held for 2 minutes at -5°C and for S
minutes at -12°C, had the highest cell recovery. Those held for 10 minutes may have been exposed to high concentrations of solutes for a duration which was damaging. Simulations from Example 4 predicted that there was no difference in [KCl]; concentrations and supereooling between hold times down to -25°C. The experimental results demonstrated that the differences in membrane integrity between he hold times may depend on the duration of exposure, which is consistent with the theoretical results.
For all the hold times; simulations predicted a progressive increase in [KCl]i upon cooling to lower plunge temperatures down to -25°C for cells held for 0.5 minutes. The experimental results demonstrated a progressive decline in membrane integrity for cells thawed directly from subzero plunge temperatures. At low subzero plunge temperatures (<-20°C), cells directly thawed had low percentages of membrane integrity (<30%). Therefore, either the exposure time and/or the concentration of solutes may have been significant variables for freezing injury.
As will be apparent to those skilled in the art in the light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without departing from the spirit or scope thereof.
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through -12°C, respectively (data shown for 100°C/min in Figure 13b ).
.: L
A representative graph demonstrating [KCl]i as a function of temperature for cells cooled at 1 °C/min is shown in Figure 14a. Cells cooled to increasingly lower subzero plunge temperatures showed increasing concentrations of {KCl);, with the highest concentration for cells cooled to -30°C at 1°C/min. This correlates with the decrease in cell volume reported in the previous section. This gradual increase in [KCI]i demonstrates the potential for increased solution effects upon cooling to the lower subzero temperatures: Results for 0.2°Clmin and 0.5°C/min were not shown however were similar to 1 °Clmin data, but over a longer period of time. Figure 14b shows the [KCl]i as a function of temperature for cells cooled at 100°C/min. This demonstrates the increase of [KCl];
is minimized with higher cooling rates:
Maximum supercooling and [KCI]; during cooling The maximum amount of supercooling was calculated as the highest amount of supercooling which occurred throughout the cooling profile for each plunge temperature. Figure 13a and 13b shows supercooling as a function of temperature for TF-1 cells cooled at 1.0°C/min and at 100°C/min respectively; with arrows indicating where the maximum supercooling was determined for the various plunge temperatures. The maximum supercooling for each cooling rate was then summarized and graphed as a function of plunge temperature (Figure 15).
Similar patterns of high supercooling (27°C) at -3°C were demonstrated for all the cooling rates; with: a decrease to below 10°C at approximately -4°C for cells cooled at 02, 0.5; and 1.0°C/min and at approximately -13 °C for cells cooled at 100°C/min. Intracellular ice formation thus only appears to play a role in freezing 'injury for cells cooled to high subzero temperatures for low cooling rates and intermediate subzero temperatures for higher cooling rates.
The maximum amount of [KCl]a was calculated as the highest concentration of KCl which occurred throughout the cooling profile for each plunge temperature. Figure 14a and 14b shows the [KCI]; as a function of temperature for TF-1 cells cooled at 1.0°CJmin and at 100°C/min respectively, with arrows indicating where the maximum [KCl]; was determined for the various plunge temperatures. The maximum [KCl]; for each cooling rate was then summarized and graphed as a function of plunge temperature (Figure 16). Similar patterns of an increase in [KCl]; are demonstrated for all cooling rates, which is consistent with the change in cell volume. Cooling rates of 0.2, 0.5, and 1.0°C/min showed the sharpest increase in [KCl]; at the higher subzero temperatures, compared with higher cooling rates that showed a gradual increase, which is consistent with the cell dehydrating sufficient to maintain equilibrium. Thus at the low cooling rates examined, cells were exposed to comparably ' high solute conditions at high subzero temperatures. However, there was increased time spent exposed to the solutes for the 0.2°C/min :compared with the 1.0°C/min, so one would expect that 0.2°C/min would have a lower cell recovery because cells would have been exposed to increasingly high solute concentrations for a longer period of time. The optimal temperature for plunging the cells after the initial cooling phase is a function of temperature and the amount of time spent cooling to that temperature, which influences [KCl]; and supercooling.
Example 3: Experimental assessments of simulation outcomes 3:1 Introduction In order for simulations to be used in cryopreservation, it is necessary to test the predictions of simulations empirically. The objective of this example was to conduct graded freezing experiments with '1'F-1 cells and compare the cell survival outcomes with the theoretical predictions put :forward in Example 2 based on degrees of supercooling and intracellular electrolyte concentration ([KCl];).
Membrane integrity was used as an assay for freeze-thaw injury.
Membrane integrity has been used as an indicator of cell damage during freezing, as it has been shown that the membrane is a site of freezing-thawing injury (Acker; 2001). Also, it has been shown that there is a correlation between intracellular freezing and membrane damage for cells in suspension (Acker; 2001; Mazur; 1965).
3.2 Materials & Methods TF-1 cell culture TF-1 cells (ATCC; Manassas, Virginia) were grown at 37°C in 5%
C02 in RPMI 1640 Medium Modified (ATCG) with 10% fetal bovine serum (FBS) (ATCC), and supplemented with 2 ng/mL recombinant human GM-CSF (Stemcell -Technologies, Vancouver, Canada). Cells were maintained between 0:1 x 106 and l x 106 cells/rnL, according to ATCC guidelines. Prior to experiments; cells were washed twice with serum-free RPMI media and incubated overnight to synchronize the cells (Kolonics, 2001). Cells were then centrifuged and re-suspended at a concentration of 4 x 106IrnL, which was necessary for the viability assessment program to be used:
Experimental solutions TF-1 cells were re-suspended in serum-free RPMI prior to the graded freezing experiments. In order to compare the results with the clinical standard, TF-1 cells were also re-suspended in 10%
DMSO/RPMI at 4°C; prior to freezing experiments.
Graded freezing experiments Samples of 0.2 mL cell suspension; in serum-free RPMI or 10%
DMSO/RPMI, in glass tubes (Fisher, Edmonton, Canada) were cooled in a 0°C ice bath fox 5 minutes. Control samples were removed and either warmed in a 37°C water bath or plunged into liquid nitrogen (325°C/min;
(Ebertz, 2002(a)). Experimental samples were transferred into a methanol bath preset at -3°C and allowed to equilibrate for 5 minutes prior to ice nucleation with cold forceps. After 5 minutes, the bath cooled at 0.2, 0:5, or 0.9°C/min to -40°C. The cooling rates were monitored using a Type T thermocouple (Omega; Laval, Canada). Samples were then removed at -3; -6, -9, -12; -15; -20, -30, and -40°C and either thawed directly in a 37°C water bath or plunged into liquid nitrogen. Samples were kept in liquid nitrogen for a minimum of 1 hour prior to being thawed in a 37°C water bath. Duplicate samples were used for both the direct thaw and the plunge conditions at each experimental temperature.
Each experiment was repeated in triplicate for each cooling rate and experimental solution. Samples in 10%DMSOfRPMI were only cooled using 0.9°C/min.
Viability assessment Cell viability was assessed by a membrane integrity assay. The assay was performed by incubating cells with SYTO~ 13 (Molecular Probes, Eugene, Oregon) and ethidium bromide (EB) (Sigma, Mississauga, Canada) (Yang; 1998). Syto 13 permeates the cell membrane of all cehs and complexes with DNA and fluoresces green under UV exposure. EB penetrates cells with a damaged plasma membrane and also complexes with DNA fluorescing red under UV
conditions. The dual stain allows for differentiation between cells with and without intact plasma membranes.
The SytolEB stain was prepared using 40 p,I, of 2.5 mM EB stock solution and IO p.L of 5 mM SYTO~ 13 stock solution mixed with 350 ~.L phosphate-buffered saline (PBS): Final concentrations were 0.25 mM
EB and 0.125 mM Syto. Twenty p.L of stain was added to 200 ~,L each sample, mixed, and allowed to incubate for 2 minutes at room temperature: Fluorescent images were captured using a Leitz Dialux 22 fluorescence (440-480 nmj microscope (Leitz, Germany) fitted with a PIXERA Viewfinder Pro digital camera (Pixera Corporation, Los Gatos, CA, USA} digital camera. The Viability Assessment Program (The Great Canadian Computer Company, Spruce Grove, Canada), which counts red versus green pixels was used to quantify cell membrane integrity from digital images (Jomha; 2003). This method measures membrane integrity of the cell remaining after experimental treatment.
3:3 Results Conventional cryopreservation protocol with DMSO
The standard for cryopreserving HSCs is to cool the cells at 1 °C/min in 10% DMSO. TF-1 cells were cooled at 0:9°Clmin in 10% DMSO/RPMI
to various temperatures down to -40°C, prior to being thawed directly or plunged into liquid nitrogen (Figure 17). The maximum percentage of membrane integrity was 63.79.8%, when samples were cooled to -12°C
to -15°C, prior to being plunged into liquid nitrogen: The results were comparable with that previously reported for cryopreserving HSCs with 10% DMSO cooling at 1:0°C/min (7915% (Hunt, 2003); 67.42.0%
(Yang, 2003). This experiment was limited by the cooling capacity of the methanol bath:
Graded freezing with no cryapreservant using various cooling rates TF-1 cells were suspended in serum-free RPMI and cooled at 0.2°C/min to various temperatures up to -20°C, prior to being thawed directly or plunged into liquid nitrogen. Cells thawed directly from the subzero plunge temperatures showed a ' S0% decrease in membrane integrity by -12°C, indicating that a major portion of cells were damaged prior to being plunged into liquid nitrogen (Figure 18). However, damage at higher subzero temperatures occurred as a result of the plunge into liquid nitxogen. There was limited cell recovery when the cells were plunged into liquid nitrogen at all plunge temperatures. The maximum recovery of 24.2tS.5% was seen for TF-1 cells plunged at -3°C:
Figure 19 shows the membrane integrity as a function of plunge temperature for TF-1 cells cooled at 0.5°C/min to -40°C, prior to being plunged into liquid nitrogen: TF-1 cells demonstrated similar membrane integrity for both thaw and plunge samples as with 0:2°C/min. The maximum recovery of 28.25.8% was obtained between -3°C and -9°C.
Membrane integrity as a function of plunge temperature for TF-1 cells cooled at 0.9°C/min to -40°C, prior to being thawed directly or plunged into liquid nitrogen is shown in Figure 20. Results were also similar to those for 0.2°C/min and 0.5°C/min. TF-1 cells showed maximum recovery of 27.80:8% at -9°C. This experiment was limited by the cooling capacity of the methanol bath; which had a maximum cooling rate of 0.9°C/min.
Data from all three cooling rates demonstrated a 50% decline in membrane integrity for cells'thawed directly from the plunge temperature at -12°C. This indicates that cells were damaged prior to being plunged into liquid nitrogen, possibly due to solution effects. However, there was a significant difference between the membrane integrity for cells directly thawed and hose further plunged into liquid nitrogen. Due to the high cooling rate upon plunging into liquid nitrogen (325°C/min), this indicates that intracellular ice formation may play a role in damage at these temperatures. There does appear to be a zone of subzero plunge temperatures (-3°C to -9°C), which confers some protection against injury during the plunge into liquid nitrogen for all the cooling rates.
This would constitute an optimal subzero plunge temperature range for these cooling rates.
3.4 Discussion Discussion of experimental'data The conventional cryopreservation protocol of using 10%DMSO
solution yielded comparable results with other HSCs cryopreservation reports. The experimental results for cryopreserving TF-1 cells without cryopreservants were significantly lower than the standard. The data showed that the membrane integrity between the various cooling rates was within standard error mean of each other. TF-1 cells responded similarly to exposure to the various subzero plunge temperatures and to subsequent plunging into liquid nitrogen.
Comparison of theoretical and experimental results Simulations from Example 2 predicted that there was no difference in maximum [KCl]i, between the cooling rates, however the time spent exposed to these elevated concentrations may cause additional cryoinjury.
The experimental results demonstrated that there was not a significant difference in membrane integrity between the cooling rates of 0.2, 0.5, and 0.9°C/min, which is consistent with the theoretical results based solely on [KCl];. Simulations did indicate that there was potential for increased exposure time to the solutes with the lower cooling rate (0.2°C/min), however the experimental data demonstrated that the percentages of membrane integrity were within error for all the cooling rates. Therefore, tile increased exposure time for the lower cooling rates was not significant.
For all cooling rates, simulations predicted a progressive increase in [KCl]i upon cooling to lower temperatures. Based on Lovelock's work, the inventors predicted that salt concentrations of greater than 3 M
would be damaging to the cells (Lovelock, 1953) and the experimental data demonstrated that there was a decrease in membrane integrity (~60%) at this concentration for all the cooling rates. The experimental data also demonstrated a progressive decline in membrane integrity for cells thawed directly from subzero plunge temperatures. At low subzero plunge temperatures (<-20°C), cells directly thawed had low percentages of membrane integrity (<20%). Therefore, the exposure time coupled with the concentration of solutes may have been signiricant variables for freezing injury.
Simulations also predicted that cells cooled to -3°C prior to being cooled at 325°C/rnin to -40°C would have a high degree of supercooling (27°C). The experimental results demonstrated that upon cooling to -3°C
prior to plunging into liquid nitrogen, TF-1 cells had a relatively high percentage of membrane integrity. This indicates that although intracellular ice formation may have played a role in membrane damage at this plunge temperature; there was another source of damage upon cooling to lower temperatures, where solutions effects were present.
Also, the range of plunge temperatures between -3°C and -9°C, which demonstrated the highest viability, had high variations in maximum supercooling (2°C to 27°C) and in maximum [KCl]; (2 to 4 1V.~.
This alludes to the complex interactions between these two types of injury at subzero temperatures.
Example 4: Theoretical design of a cryopreservation protocol 4.~. Introduction The cellular responses to the formation of ice in surrounding solution are largely dependent on the movement of water across the plasma membrane. Ice formation causes osmotic stress on the cell membrane forcing water out of the cell to maintain equilibrium with the extracellular solution. The properties of the cell membrane, specifically the osmotic parameters, govern these changes in cell volume. The osmotic parameters can be used in simulations to theoretically model cellular responses to: low temperatures. Simulations also provide precise results regarding changes in cell volume and the amount of supercooling.
These results can then be used for comparisons between cryopreservation protocols and for comparison between different cell types which may be present in one tissue: Ultimately; simulations allow for unlimited theoretical protocols to be explored by controlling cooling and warming rate; plunge temperatures, and the components of the intracellular and extracellular compartments for any cell type for which the osmotic parameters are known.
To distinguish between the two types of,injury, solution effects and intracellular ice formation, the inventors simulated the empirical procedure of two-step freezing. The two-step freezing technidue was developed by Farrant et al. and has provided a logical method to examine the effects of freezing injury on cell recovery due to non-linear cooling rates and to exposure to a range of subzero temperatures (Farrant, 1974).
In their procedure, samples were cooled at an uncontrolled non-linear cooling rate to various subzero plunge temperatures by being transferred to a preset bath before being 1 ) thawed directly from that holding temperature or 2) plunged to 196°C before thawing. McGann and Farrant later reported the subzero plunge temperatures and the length of hold time -at that temperature were important variables to consider when attempting to maximize cell survival (McGann, 1976).
The objective of this example was to further the inventors' understanding of the theoretical responses of TF-1 cells, a model for hematopoietic stem cells (HSC) (Kitamura; 1989(a); Marone, 2002), to subzero plunge temperatures aid to hold times at those temperatures.
Simulations were done using the osmotic parameters of TF-1 cells reported in Example 1. The objective of these simulations was to theoretically determine the conditions of TF-1 cells at various stages of a freezing protocol. Maximum levels of intracellular electrolyte concentrations ([KCl]i) aid; of supercooling were examined upon cooling the cells to -40°C, as indicators for solution effects injury and intracellular ice formation injury, respectively.
4.2 Simulations of two-step freezing protocol Methods Simulations were performed according to those done in Example 2 using the osmotic parameters of TF-1 cells (Table 3) in the C~~yoSimS
program (Dr. Locksley IVIcCrann; University of Alberta, Canada). The simulations were based on a two-step freezing technique, which has been used to examine the effects of high solute concentrations and intracellular ice formation on cell survival during freezing (Farrant, 1977). The cryopreservation protocol was defined by assigning a starting temperature and then varying the cooling rates; based on typical two-step freezing procedures. Supercooling :and [KCl]i were used as indicators of potential intracellular ice formation and solution effects; respectively.
Temperature profiles The two-step freezing technique involved rapidly cooling the samples to various subzero plunge temperatures before either being thawed directly in a 37°C water bath or plunged into liquid nitrogen first and then thawed (McGann; 1979). For the simulations, cells were cooled using a temperature profile derived from Fourier's Law. Fourier's Law describes the rate of heat transfer which depends on the temperature distribution of the system, previously described in Example 2 (Incropera, 2002). The ~Ztting constant was determined by monitoring the cooling profile of a sample taken from room temperature and exposed to the experimental subzero temperature with a Type T thermocouple (4mega, Laval, Canada). Figure 2l is a representative cooling profile of a sample cooled from room temperature to -15°C. This prafzle was then fitted to a curve and the equation was then used in simulations. The variations between the experimental and :fitted curves were due to the latent heat of fusion.
Simulations were performed in which cells with no cryopreservant were cooled to various subzero plunge temperatures ranging from -3 °C
to -40°C and held at that temperature for 0:3, 0.5, 0.7, 1, 2, 3, 5, 7, or minutes, prior to being plunged to -40°C (325°Clmin) (Ebertz, 2002).
4e3 Results Changes in cell volume during cooling Figure 22 shows simulation results of temperature as a function of time for TF-1 cells cooled to various subzero plunge temperatures ranging from -3 °C to -30°C, held for various hold times (minutes); prior to being plunged to -40°C
(325°C/min). Based on the results from the simulations; the hold times were grouped according to similarities of changes in cell volume, [KCl]i, and supercooling: hold times of 1 minute or less will be represented by the 0.5 minute data; hold times between 2 and 5 minutes will be represented by the 3 minute data; and hold times of between ? and 10 minutes will be represented by the 10 minute data. Figure 23 demonstrates the changes in cell volume as a function of temperature upon cooling to various subzero plyge temperatures ranging from -3 °C
to -30°C, held for a duration, prior to being plunged to -40°C
(325°C/min). The data shown is for (a) 0.5 min., (b) 3 min., and (c) 10 min. hold times. Cells showed a progressive decrease in cell volume upon cooling. Cells only held for 0.5 minutes at the subzero temperature did not reach the same volumes as those held for 3 or 10 minutes at -3°C
and -35°C. This data suggests that the cells have not had sufficient amount of time to dehydrate with a hold time of 0.5 minutes, as opposed to greater than 3 minutes, for both high and low subzero plunge temperatures. This data also indicates that the cells would have a higher amount of supercooling at these outlying plunge temperatures due to the lack of cellular dehydration. Also, with lower concentrations of [KCl]i, it is possible that the cells would not be subjected to high solution effects.
Supercooling during cooling Figure 24 demonstrates the changes in supercooling as a function of temperature upon cooling to various subzero plunge temperatures ranging from -3°C to -30°C, prior to being plunged to -40°C
(325°Clmin). Data shown are for (a) 0.5 min., (b) 3 min., and (c) 10 min. hold times. Supercooling of up to 10°C occurs for all the hold times down to -12°C. This suggests that supercooling plays a key role in potential injury during freezing to lower subzero plunge temperatures. At these lower temperatures, cells were exposed to increasingly supercooled conditions up to 30°C at -30°C.
[KCl]; during cooling Figure 25 demonstrates the changes in [KCl]; as a function of temperature upon cooling to various subzero plunge temperatures ranging from -3°C to -30°C, prior to being plunged to -40°C
(325°C/min). The data shown is for (a) 0.5 min., (b) 3 min., and (c) 10 min. hold times.
Cells cooled to lower subzero plunge temperatures showed increasing concentrations of [KCl];, with the highest concentration for cells cooled to -30°C and held for greater than 3 minutes. This correlates with the gradual decrease in cell volume reported in the previous section. This gradual increase in [IfCI]; demonstrates the potential for increased solution effects upon cooling to the lower subzero plunge temperatures.
The data show similar concentrations of [KCl]; at all plunge temperatures except ~ at -3°C and below -30°C.
Maximum supercooling and [KCI]~ during cooling The maximum amount of supercooling was calculated as the highest amount of supercooling which occurred throughout the cooling profile for each plunge temperature. Figure 24a supercooling as a function of teyperature for TF-1 cells with arrows indicating where the maximum supercooling was determined for the various plunge temperatures. The maximum amount of supercooling was calculated and graphed as a function of plunge temperature (Figure 26). Data is shown for (a) 0.5 min., (b): 3 min., and (c) 10 min. hold times. The maximum supercooling obtained appears to be the primary contributor to potential injury, which suggests that a target plunge temperature between -6°C to -12°C would lead to high levels of survival because the supercooling does not exceed 10°C. Cells held for 0.5 minutes have a more narrow range of optimal plunge temperatures, limited by the amount of supercooling.
These results correlate with the lack of cellular dehydration discussed in the previous sections.
The maximum amount of [KCl]; was calculated as the highest concentration of KCl which occurred throughout the cooling profile for each plunge temperature. Figure 25a shows the [KCl]; as a function of temperature for TF-1 cells, with arrows indicating where the maximum [KCl]; was determined for the various plunge temperatures. The levels of maximum [KCl]; for cells held for 0.5; 3 and 10 minutes gradually increase from -3°C to -20°C (Figure 26). The slope between -3°C and -6°C varies from cells held for 0.5 minutes and 3 to 10 minutes, suggesting that at plunge temperatures between -3°C and -6°C, there may be a difference in cell recovery between U:5 minutes and 3 to 10 minutes.
For all the hold times, based on the temperature range set by the 10°C
limit to supercooling, the data suggests that the lower [KCl]; levels would result in better cell recovery. Figure 27 shows the plunge temperature ranges for cells held for 3 minutes based on 10°C supercooling and 3 M
[KCl];, This range varies between 0.5 minute hold time and the 3 and 10 minute hold time. However, a target plunge temperature of approximately -6°C should result in the highest cell recovery for all the hold times.
These simulations suggest that supercooling plays a key role in two-step freezing and the effects of increasing solute concentrations are secondary. The optimal temperature for plunging the cells after the initial cooling phase is a function of the amount of time spent to cool to a specific temperature, which influences [KCl]i and supercooling.
Example 5: Experimental correlation and optimization of a theoreticall'~-desi ned cryopreservation protocol 5.1 Introduction The simulations performed in Example 4 predicted that subzero plunge temperature and time spent at that temperature were critical variables in the optimization of cryopreservation protocols. In order for simulations to be used in cryopreservation; it is necessary to test the predictions of simulations empirically. The purpose of this example was to explore the range of subzero plunge temperatures and time spent at those temperatures. Two-step freezing experiments were conducted with TF-1 cells and compared with the cell survival outcomes that were theoretically predicted in Example 4. Membrane integrity was used as an assay fox freeze-thaw injury. Membrane integrity has been used as an indicator of cell damage during freezing, as it has been shown that the membrane is a site of freezing-thawing injury (Acker, 2001): Also, it has been shown that there is a correlation between intracellular freezing and membrane damage for cells in suspension (Acker, 2001; Mazur, 1965).
5.2 Materials & Methods TF-1 cell culture TF-1 cells (ATCC; Manassas, Virginia) were grown at 37°C in 5%
COZ in RPMI 1640 Medium Modified (ATCC) with 10% fetal bovine serum (FBS) (ATCC), and supplemented with 2 ng/mL recombinant human GM-CSF (Stemcell Technologies, Vancouver, Canada). Cells were maintained between 0.1 x 106 and 1 x cells/mL; according to ATCC
guidelines. Prior to experiments, cells were washed twice with serum-free RPMI media and incubated overnight. Cells were then centrifuged and re-suspended at a concentration of 4 x 106 cells/mL, which was necessary for the viability assessment program to be used.
Experimental solutions TF-1 cells were re-suspended in serum-free RPMI prior to the two-step freezing experiments.
Two-step freezing experiments Samples of 0.2 mL cell suspension; in serum-free RPMI, in glass tubes were allowed to equilibrate at room temperature for 5 minutes.
Control samples were either warmed in a 37°C water bath or plunged into - SQ
liquid nitrogen. Experimental samples were individually transferred into a methanol bath preset at -3, -6, -9, -12, -15, -20, -30, and -40°C and allowed to ' equilibrate for 2 minutes at that temperature prior to ice nucleation with cold forceps. After nucleation, samples were allowed to equilibrate for 3 minutes before either being thawed directly in a 37°C
water bath or plunged into liquid nitrogen. Samples were kept in liquid nitrogen for a minimum of 1 hour prior to being thawed in a 37°C water bath. Duplicate samples were used for both the direct thaw and the plunge conditions at each plunge temperature. Each experiment was repeated in triplicate.
The two-step freezing experiments were repeated with varying hold times. Cells were cooled to -5, -7, -9, -12, -15, and -25°C and allowed to equilibrate for 2 minutes prior to ice nucleation with cold forceps. After nucleation, samples were allowed to equilibrate for 0.5 or minutes before either being thawed directly in a 37°C water bath or plunged into liquid nitrogen. Samples were kept in liquid nitrogen for a minimum of 1 hour prior to being thawed in a 37°C water bath. Duplicate samples were used for both the direct thaw and the plunge conditions at each plunge temperature. Each experiment was repeated in triplicate.
Viability assessment Cell viability was assessed by a membrane integrity assay. The assay was performed by incubating cells with SYT'O~ 13 (Molecular Probes, Eugene, Oregon) and ethidium bromide (EB) (Sigma, Mississauga, Canada) (Yang, 1998). Syto 13 permeates the cell membrane of all cells and complexes with DNA and it fluoresces green under UV exposure. EB penetrates cells with a damaged plasma membrane and also complexes with DNA fluorescing red under Uil conditions. The dual stain allows for differentiation between cells with and without intact plasma membranes.
The Syto/EB stain was prepared using 40 p.I, of 2.5 mM EB stock solution and 10 p;L of 5 mM Syto~ 13 stock solution mixed with 350 p.I, 1 X phosphate-buffered saline (PB S). Final concentrations were 0.25 mM
EB and 0.125 mM Syto. Twenty p.L, of stain was added to each sample and allowed to incubate for 2 minutes at room temperature. Fluorescent images were captured using a Leitz Dialux 22 fluorescence (440-480 nm) microscope (Leitz, Germany) fitted with a PIXERA DiRactor (Pixera Corporation; Los Gatos, CA, USA) digital camera. The Viability Assessment Program (The Great Canadian Computer Company, Spruce Grove, Canada), which counts red versus green pixels was used to quantify cell membrane integrity from digital images (Jomha, 2003). This method measures membrane integrity of the cell remaining after experimental treatment.
5.3 Results Varying plunge temperature TF-1 cells were suspended in serum-free RPMI to various plunge temperatures up to -40°C and held for 3 minutes, prior to being thawed directly or plunged into liquid nitrogen (Figure 28). Overall, cells demonstrated a higher percentage of membrane integrity than cells cooled at 0.2°C/min to 0.9°C/min presented in Example 3. Cells plunged into liquid nitrogen showed comparable results for membrane integrity to cells directly thawed from temperatures ranging from -1S°C to -40°C.
Cells thawed directly from the plunge temperatures showed a 50 % decrease in membrane integrity by -17°C, indicating that a major portion of cells were damaged prior to being plunged into liquid nitrogen (Figure 28).
However, this membrane damage occurred at a higher subzero plunge temperature for cells cooled at 0.9°C/min. The maximum recovery of 58.86.5% was seen for TF- cells cooled to -I2°C or -15°C before being plunged into liquid nitrogen. These results were higher than those previously reported in Example 3 of approximately 28.2%. Furthermore, these results were comparable with those shown for cooling at 0:9°C/min in 10% DMSOIRPMI of 63:7% (Example 3).
Varying experimental hold time TF-1 cells were cooled to -5, -7, -9, -12, -15, or -25°C and allowed to equilibrate for 2 minutes prior to ice nucleation with cold forceps.
After nucleation, samples were allowed to equilibrate fox varying times (0.3, 0.5, 0.7, 1, 2, 3, 5; 7; and 10 minutes) before either being thawed directly in a 37°C water bath or plunged into liquid nitrogen. Figure 29a shows the membrane integrity of TF-1 cells as a function of hold time for cells cooled to -5°C, held and plunged into liquid nitrogen. Comparable results of membrane integrity were obtained for cells held at -7°C to -9°C
(results not shown). However there was minimal membrane integrity for cells held below -25°C (data shown in Figure 29b). Cells that were directly thawed from the subzero plunge temperatures after being held showed progressive decrease in membrane integrity based on reduced temperature and increased duration of hold time (Figure 30a). Results indicated a high percentage of membrane integrity of SS to 60%, when cells were held for 1-S minutes at high subzero plunge temperatures.
Cooled cells from room temperature to -5°C and -7°C and held for 1-3 minutes, prior to plunging into liquid nitrogen resulted in the highest percentage ofunembrane integrity of approximately 60% (Figure 30b). A
hold time of greater than 5 minutes resulted in a marked decrease in cell survival. This data indicates that there is a zone of subzero plunge temperatures (-5°C' to -15°C); when held for 2-3 minutes, which confers protection against injury comparable to DM50.
5.4 Correlation with theoretically-designed protocol Discussion of experimental results The experimental results for cryopreserving TF-1 cells without cryopreservants indicate that cells can be cryopreserved without DMSO.
This data indicates that there is a zone of subzero plunge temperatures (-5°C to -15°C), when held for l-3 minutes, which confers comparable protection against injury to the standard 10% DMSO/RPMI solution, previously reported in Example 3. This range would constitute an optimal subzero temperature range for these hold times based on experimental results.
Comparison of theoretical and experimental results Simulations were done based on an empirical approach to cryopreservation, two-step freezing, which can be used to examine the role of exposure to subzero plunge temperatures and exposure time. The cooling rates used in, the two-step freezing protocol axe governed by Fourier's Law and were determined experimentally in Example 4. Cells were exposed to increasingly supercooled conditions up to 40°C at a plunge temperature of -40°C. Supercooling appears to be the primary contributor to potential freezing injury. This research supported the upper limit of 10°C supercooling, previously reported by Mazur. The proposed target plunge temperature was suggested to be between -4°C
and -12°C, as supercooling was restricted to less than 10°C, which is comparable to the range determined empirically. Also, based on levels of intracellular KCl ([KCl];), it was suggested that the higher subzero plunge temperature would have the lowest potential for solution effects based on the lack of cell dehydration, which was also supported by this data.
Two-step freezing experiments demonstrated a high percentage of membrane integrity for TF-1 cells when cells were cooled to between -5°C and -12°C and held for 1-5 minutes. These plunge temperatures corresponded with the theoretical values of 5°C to 10°C supercooling, which suggested that a certain amount of supercooling is necessary to achieve a higher viability (Diner, 1975). However, this also supports the belief that excessive supercooling may lead to damage as a result of intracellular ice formation.
Based on the simulations, the duration of time the cells were held at the subzero plunge temperature was also considered an important factor. When cells were held for 0.5 minutes; they did not have sufficient time to dehydrate and reach the same volume as cells held for greater than 2 minutes. This excess intracellular water may have caused damage by forming ice upon subsequent cooling. According to the two-step freezing experiments, cells held for 2 minutes at -5°C and for S
minutes at -12°C, had the highest cell recovery. Those held for 10 minutes may have been exposed to high concentrations of solutes for a duration which was damaging. Simulations from Example 4 predicted that there was no difference in [KCl]; concentrations and supereooling between hold times down to -25°C. The experimental results demonstrated that the differences in membrane integrity between he hold times may depend on the duration of exposure, which is consistent with the theoretical results.
For all the hold times; simulations predicted a progressive increase in [KCl]i upon cooling to lower plunge temperatures down to -25°C for cells held for 0.5 minutes. The experimental results demonstrated a progressive decline in membrane integrity for cells thawed directly from subzero plunge temperatures. At low subzero plunge temperatures (<-20°C), cells directly thawed had low percentages of membrane integrity (<30%). Therefore, either the exposure time and/or the concentration of solutes may have been significant variables for freezing injury.
As will be apparent to those skilled in the art in the light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without departing from the spirit or scope thereof.
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Claims (40)
1. A non-linear cryopreservation protocol for cryopreserving cells comprising determining an optimal cooling profile for maximum recovery of the cells and applying the cooling profile to the cells.
2. A non-linear cryopreservation protocol according to aspect 1, wherein the optimal cooling profile is determined using a simulation of cellular responses to cooling parameters.
3. A non-linear cryopreservation protocol according to aspect 2, wherein the cooling parameters comprise cell temperature, duration of temperature exposure, cooling level, cooling rate and presence or absence of cryopreservants.
4. A non-linear cryopreservation protocol according to aspect 2, wherein the cellular responses are determined from mathematical models of extracellular concentration parameters, intracellular concentration parameters, and cellular osmotic permeability parameters.
5. A non-linear cryopreservation protocol according to aspect 1, wherein the cryopreservation protocol comprises cooling the cells to a first temperature for a first period of time, then cooling the cells to a storage temperature for a second period of time prior to thawing.
6. A non-linear cryopreservation protocol according to aspect 1, wherein the cryopreservation protocol is used with cells stored without cryopreservants.
7. A non-linear cryopreservation protocol according to aspect 1, wherein the cryopreservation protocol is used with cells stored with non-penetrating cryopreservants.
8. A non-linear cryopreservation protocol according to aspect 7, wherein the non-penetrating cryopreservants comprise sugars, starches, serum, or plasma.
9. A non-linear cryopreservation protocol according to aspect 1, wherein the cells comprise stem cells, other progenitor cells, red and white blood cells, sperm cells, oocytes, ova, cells for research or transplant purposes, cellular materials derived from tissues and organs, pancreatic islet cells, chondrocytes, cells of neural origin, cells of hepatic origin, and cells of cardiac origin.
10. A non-linear cryopreservation protocol according to aspect 9, wherein the stem cells comprise peripheral blood stem cells, human umbilical cord blood stem cells and stem cells derived from tissues and solid organs or other sources, including fetal and or embryonic sources.
11. A non-linear cryopreservation protocol according to aspect 1, wherein the non-linear cryopreservation protocol is executed on the cells using a bulk freezing unit.
12. A non-linear cryopreservation protocol according to aspect 1, wherein the non-linear cryopreservation protocol is executed on the cells using a cryomicroscopy apparatus.
13. A method for optimizing a cryopreservation protocol for cryopreserving cells, the method comprising:
a. producing a simulation of cellular responses to a range of cooling parameters;
b. based on information derived from the simulation of cellular responses, determining an optimal cooling profile; and c. incorporating the cooling profile into the cryopreservation protocol.
a. producing a simulation of cellular responses to a range of cooling parameters;
b. based on information derived from the simulation of cellular responses, determining an optimal cooling profile; and c. incorporating the cooling profile into the cryopreservation protocol.
14. A method according to aspect 13, wherein the cooling parameters comprise cell temperatures, temperature exposure duration periods, cooling rates, cooling levels, and presence or absence of cryopreservants.
15. A method according to aspect 13, wherein the simulation of cellular responses comprises calculating cellular responses using mathematical models of extracellular solution concentration parameters, intracellular solution concentration parameters, and cellular osmotic permeability parameters.
16. A non-linear cryopreservation protocol optimized according to aspect 13.
17. A method for optimizing a cryopreservation protocol for cryopreserving cells wherein the cells are cooled to a first temperature for a first period of time, then cooled to a second temperature at which the cells are stored for a second period of time before the cells are thawed, the method comprising:
a. producing a simulation of cellular responses to a range of cooling parameters;
b. based on information derived from the simulation of cellular responses, determining an optimal first temperature, first period of time, and cooling level required to minimize cryoinjury to the cells; and c. incorporating the optimal first temperature, optimal first period of time, and optimal cooling level into the non-linear cryopreservation protocol.
a. producing a simulation of cellular responses to a range of cooling parameters;
b. based on information derived from the simulation of cellular responses, determining an optimal first temperature, first period of time, and cooling level required to minimize cryoinjury to the cells; and c. incorporating the optimal first temperature, optimal first period of time, and optimal cooling level into the non-linear cryopreservation protocol.
18. A method according to aspect 17, wherein the cooling parameters comprise cell temperatures, temperature exposure duration periods, cooling rates, cooling levels, and presence or absence of cryopreservants.
19. A method according to aspect 17, wherein the simulation of cellular responses comprises calculating cellular responses using mathematical models of extracellular solution concentration parameters, intracellular solution concentration parameters, and cellular osmotic permeability parameters.
20. A non-linear cryopreservation protocol optimized according to aspect 17.
21. A non-linear cryopreservation protocol for cryopreserving stem cells comprising determining an optimal cooling profile for maximum recovery of the stem cells and applying the cooling profile to the stem cells.
22. A non-linear cryopreservation protocol according to aspect 21, wherein the optimal cooling profile is determined using a simulation of cellular responses to cooling parameters.
23. A non-linear cryopreservation protocol according to aspect 22, wherein the cooling parameters comprise cell temperature, duration of temperature exposure, cooling level, cooling rate and presence or absence of cryopreservants.
24. A non-linear cryopreservation protocol according to aspect 22, wherein the cellular responses are determined from mathematical models of extracellular concentration parameters, intracellular concentration parameters, and cellular osmotic permeability parameters.
25. A non-linear cryopreservation protocol according to aspect 21, wherein the cryopreservation protocol comprises cooling the stem cells to a first temperature for a first period of time, then cooling the stem cells to a storage temperature for a second period of time prior to thawing.
26. A non-linear cryopreservation protocol according to aspect 21, wherein the cryopreservation protocol is used with stem cells stored with non-penetrating cryopreservants.
27. A non-linear cryopreservation protocol according to aspect 26, wherein the non-penetrating cryopreservants comprise sugars, starches, serum, or plasma.
28. A cryopreservation protocol for cryopreserving stem cells, the protocol comprising:
a. Cooling the stem cells to a first temperature for a first period of time; and b. Cooling the stem cells to a second temperature for storing the cells for a second period of time.
a. Cooling the stem cells to a first temperature for a first period of time; and b. Cooling the stem cells to a second temperature for storing the cells for a second period of time.
29. A cryopreservation protocol for cryopreserving stem cells according to aspect 28, wherein the first temperature is between 0°C and -60°C, and the cells are cooled at a rate between 10°C/minute to 30°C/minute.
30. A cryopreservation protocol for cryopreserving stem cells according to aspect 28, wherein the first temperature is between -5°C and -15°C; and the first time period is between 1 and 30 minutes.
31. A cryopreservation protocol for cryopreserving stem cells according to aspect 30, wherein the second temperature is between -60°C and -196°C.
32. A non-linear cryopreservation protocol according to aspect 5, wherein the first temperature is between -2°C and -40°C, and the cells are cooled at a rate between 10°C/minute and 100°C/minute, and the second temperature is below -60°C.
33. A non-linear cryopreservation protocol according to aspect 32, wherein the second temperature is between -60°C and -196°C.
34. A non-linear cryopreservation protocol according to aspect 17, wherein the first temperature is between -2°C and -40°C, and the cells are cooled at a rate between 10°C/minute and 100°C/minute, and the second temperature is below -60°C.
35. A non-linear cryopreservation protocol according to aspect 34, wherein the second temperature is between -60°C and -196°C.
36. A non-linear cryopreservation protocol according to aspect 25, wherein the first temperature is between -2°C and -40°C, and the cells are cooled at a rate between 10°C/minute and 100°C/minute, and the second temperature is below -60°C.
37. A non-linear cryopreservation protocol according to aspect 36, wherein the second temperature is between -60°C and -196°C.
38. A method for optimizing cryopreservation protocols of cells comprising any new, useful, and inventive feature, combination of features or sub-combination of features described or clearly inferred herein.
39. A cryopreservation protocol comprising any new, useful, and inventive feature, combination of features or sub-combination of features described or clearly inferred herein.
40. An apparatus for cryopreserving cells comprising any new, useful, and inventive feature, combination of features or sub-combination of features described or clearly inferred herein.
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| CA2482045A CA2482045C (en) | 2004-09-17 | 2004-09-17 | Method of cryopreserving cells |
| US11/228,388 US20060063141A1 (en) | 2004-09-17 | 2005-09-19 | Method of cryopreserving cells |
| US12/656,600 US9078429B2 (en) | 2004-09-17 | 2010-02-04 | Method of cryopreserving stem cells |
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| US8758988B2 (en) | 2009-10-19 | 2014-06-24 | The Governors Of The University Of Alberta | Cryopreservation of articular cartilage |
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| US8758988B2 (en) | 2009-10-19 | 2014-06-24 | The Governors Of The University Of Alberta | Cryopreservation of articular cartilage |
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