Patent Application
20190313632
The present invention is directed to the fields of cryobiology and cryopreservation.
Current long term storage of cell stocks routinely requires the use of liquid nitrogen (LN2), because commonly used cryopreservation media containing cell membrane permeating cryoprotectants are thermally unstable when frozen at non-cryogenic temperatures, e.g. −80° C., thereby leading to ice recrystallization and causing progressive loss of cell viability over time under the storage conditions provided by most laboratory deep freezers. The dependency on LN2 for cell storage significantly increases operational expense and raises numerous issues related to impaired working efficiency and safety.
Two general approaches are widely used in cryopreservation: equilibrium (slow freezing) and non-equilibrium (vitrification) cooling procedures. The vitrification method, as well as its “slow vitrification” variant, not only introduces cell osmotic damage and toxicity due to the use of high concentrations (typically 40-50% v/v) of permeating cryoprotectant but requires a supply of LN2 or other cryogenic liquids to achieve and maintain vitrification of both intracellular and extracellular solutions at cryogenic temperatures, e.g. the saturation temperature of LN2 at one atmosphere pressure (−196° C.) or LN2 vapor (typically −120° C.). For the slow freezing approach, cells are first loaded with a relatively low concentration (typically 10% v/v) of cryoprotectant and then slowly cooled to an intermediate non-cryogenic temperature, e.g. −80° C. in a deep freezer. During cooling, ice precipitation gradually increases solute concentrations, such that, after reaching the intermediate temperature, the residual solution containing the cells is highly concentrated and in a viscous liquid state. The extracellular ice in such a partially frozen system is unstable, and the small ice crystals formed during cooling spontaneously begin to merge and form larger crystals to minimize their surface energy and become progressively distributed throughout the sample. Such events, so-called recrystallization, either cause severe mechanical damage to cells that contact the emerging large crystals or introduce lethal intracellular ice formation. Even though this cell damaging process is quite slow (typically occurring over weeks rather than hours), it is progressive even at temperatures as low as −80° C. Demonstrated by numerous publications, in either scientific research articles or cryopreservation medium product manuals, current storage in −80° C. deep freezers is only suitable for temporary or short-term purposes of use. Accordingly, to achieve long-term storage of cells after they have been slowly frozen at −80° C., it has been necessary to have a second step in which the samples are cooled to cryogenic temperatures.
Antifreeze proteins and certain small molecules are able to quench ice recrystallization by inducing thermal hysteresis, but this process only occurs over a temperature range just below the melting point of ice and is ineffective at lower temperatures.
Various polymers and associated methods have been developed to improve post-thaw cell viability and functionality by increasing solution viscosity or improving cell membrane stability after storage in liquid nitrogen. However, currently, there has been no significant improvement on the low survival rate of many cell types that are highly valuable for both research and biomedical applications after long-term storage at −80° C. by using these polymers and methods. The application of these widely used polymers for cryopreservation, e.g. polyvinylpyrrolidone (PVP), Hydroxyethyl starch (HES), etc are inadequate in prevention of recrystallization of cryoprotectant solutions at non-cryogenic temperatures.
The present invention is directed to media for preserving cells at non-cryogenic freezing temperatures comprising a macromolecule that forms compact three-dimensional structures that are spherical in shape when dissolved in an aqueous liquid. The invention is also directed to medium-cellular suspensions comprising a medium of the present invention with the cells suspended in the medium. The invention is further directed to methods of using the medium of the present invention to preserve cells. At non-cryogenic freezing temperatures the compact and spherical structures are concentrated in an unfrozen portion of the medium with the cells and this crowding effect prevents ice recrystallization during storage at non-cryogenic temperatures.
In certain aspects of the invention, the medium comprises a hydrophilic and nontoxic macromolecule, an aqueous liquid, and a cryoprotectant. The macromolecule may be at a concentration in the medium equal to or greater than about 20% (w/v), about 25% (w/v) or greater, about 35% (w/v) or greater or about 50% (w/v) or greater.
In certain aspects of the invention, the cryoprotectant is at a concentration equal to or greater than about 20% of the concentration of the macromolecule in the medium, equal to or greater than about 50% of the concentration of the macromolecule in the medium, equal to or greater than about 75% of the concentration of the macromolecule in the medium or equal to or greater than about 100% of the concentration of the macromolecule in the medium.
In certain aspects of the invention the macromolecule is a polymer. The polymer may comprise molecules that form the compact three-dimensional structures that are approximately spherical in shape when dissolved in the aqueous liquid. In such embodiments, the polymer may be selected from the group consisting of spherical hydrophilic polysaccharides, polymerized cyclodextrin or saccharides, globular proteins or spheroproteins, spherical glycoproteins formed by attaching oligosaccharide chains to those globular proteins, other derivatives of those globular proteins or combinations thereof. The polymer may be a hydrophilic polysaccharide and may be formed by the copolymerization of sucrose and epichlorohydrin.
In certain aspects of the invention, the cryoprotectant is selected from the group consisting of dimethyl sulphoxide (DMSO), glycerol, ethylene glycol, propanediol, and combinations thereof. In certain aspects of the invention, the aqueous liquid is selected from the group consisting of a cell culture medium, a nutritious medium, a saline and combinations thereof. The aqueous liquid may be selected from the group consisting of serums, FBS (fetal bovine serum), DMEM (Dulbecco's Modified Eagle Medium), HEPES (4-(2-hyroxyethyl)-1-pierazineethanesulfonic acid), FHM (flushing-holding medium), PBS (phosphate-buffered saline), DPBS (Dulbecco's phosphate-buffered saline), RPMI (Roswell Park Memorial Institute medium), BF5 medium, EX-CELL medium, Lysogeny broth (LB) medium, CaCl2 aqueous solution, NaCl aqueous solutions, KCl aqueous solutions and combinations thereof.
In certain aspects of the invention, the suspended cells are eukaryotic cells. The eukaryotic cells may be mammalian cells. The mammalian cells may be selected from the group consisting of murine cells, porcine cells, human cells, and combinations thereof. The mammalian cells may be selected from the group consisting of stem cells, somatic cells, reproduction cells and combinations thereof. In other aspects of the invention, the suspended cells are prokaryotic cells.
In certain aspects of the invention, the compact approximately spherical structures are about 100 nm (nanometer) or less in their widest dimension, comprise structures ranging from about 1 to 50 nm in their widest dimension or comprise structures ranging from about 5 nm to 10 nm in their widest dimension.
In certain aspects of the invention, the medium is substantially free of serum, animal proteins or human proteins.
Certain aspects of the invention are directed to a method for preserving cells at non-cryogenic freezing temperatures that includes providing a cryopreservation medium comprising a hydrophilic and nontoxic macromolecule, a cryoprotectant, and an aqueous liquid. In certain embodiments, the macromolecule is at a concentration in the medium greater than 10% (w/v), and the macromolecule forms a highly compact approximately spherical structure when dissolved in the aqueous liquid. The cells are added to the medium to form a medium-cellular suspension. The medium-cellular suspension is cooled to a non-cryogenic freezing temperature, wherein the non-cryogenic freezing temperature is about −85° C. or higher. The medium-cellular suspension may be maintained at or near the non-cryogenic freezing temperature, or a different non-cryogenic freezing temperature, for a time period longer than three weeks while maintaining post-thaw cell survival rates of the cells equal to or about the same as would be obtained for storage of the cells in liquid nitrogen for the same period of time. In certain aspects of the method, the macromolecule is a polymer.
In certain aspects of the method, the concentration of the polymer or other macromolecule in the cryopreservation medium ranges from 10% to the polymer's solubility in the aqueous liquid, or ranges from 20% to 50%.
In certain aspects of the method, the cells added to the medium are in a first suspension of cells, wherein the volumetric ratio of the cryopreservation medium to the first suspension of the cells is from 10:1 and 1:5, or is from 3:2 to 1:5.
In certain aspects of the invention, the medium-cellular suspension is stored for a time period of about three weeks and extending up to at least one year while maintaining post-thaw cell survival rates of the cells equal to or about the same as would be obtained for storage of the cells in liquid nitrogen for such period of time. In certain embodiments the time period is one year or more, 5 years or more, or 10 years or more.
In certain embodiments, the post-thaw cell survival rate is equal to or greater than about 70% of that obtained for storage of the cells in liquid nitrogen for the same period of time.
In certain embodiments, the non-cryogenic temperature ranges from −100° C. to −20° C., ranges from −85° C. to −65° C., or ranges from −80° C. to −75° C.
In certain embodiments, the medium-cellular suspension is cooled at a rate of about 0.01° C./min to 1000° C./min, a rate of about 0.1 to 10° C./min, or a rate of about 0.5 to 1° C./min.
In certain embodiments, after the cooling step, the medium-cellular suspension is partially frozen and the macromolecule is at a concentration of at least 25% (w/v) in an unfrozen portion of the medium-cellular suspension, or is at a concentration of at least 40% (w/v).
Additional aspects of the invention, together with the advantages and novel features appurtenant thereto, will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned from the practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.
The present invention is directed to a medium for preserving cells at non-cryogenic freezing temperatures. The medium comprises a hydrophilic and nontoxic polymer or other macromolecule, an aqueous liquid, and a cryoprotectant. The molecules of the polymer or other macromolecule form compact three-dimensional structures that are spherical in shape when dissolved in the aqueous liquid.
As described in more detail in the Examples, it was surprisingly found that when cells are suspended in the medium of the present invention, the resulting mixture, also referred to herein as the medium-cellular suspension, can be stored at non-cryogenic freezing temperatures for unexpectedly long periods of time with results similar to those obtained with storage at cryogenic temperatures with liquid nitrogen. It is believed this is due to a macromolecular crowding effect discovered to result from the highly compact and mechanically strong three-dimensional structure formed by the macromolecules in the medium of the present invention. At a non-cryogenic freezing temperature the three-dimensional structures also occupy a large portion of, or are highly concentrated in, the unfrozen portion of the medium of the medium-cellular suspension. The unfrozen portion of the medium is in phase equilibrium with ice crystals formed during freezing, along with the cells, and this crowding effect prevents ice recrystallization during storage at the non-cryogenic freezing temperatures. In certain embodiments, the concentration of the polymer or other macromolecule in the unfrozen portion of the medium of the medium-cellular suspension is at least about 25% (w/v), at least about 35% (w/v), at least about 40% (w/v) or any value or range therein.
This newly discovered macro-molecular crowding effect is supported by the simulation described in more detail in Example 1. As shown in
Example 2 provides scanning electron microscopy (SEM) evidence of the lowered ice recrystallization resulting from the macromolecular crowding effect. As shown in
The macromolecule of the present invention may be any hydrophilic and nontoxic macromolecule that forms a compact three-dimensional structure that is spherical in shape when dissolved in the aqueous liquid. The compact structures are preferably about 100 nm (nanometer) or less in their widest dimension. In certain embodiments, the compact structures include structures ranging from about 1 to 50 nm in their widest dimension, from about 5 to 10 nm in their widest dimension, or any value or range therebetween. It should be understood that not all of the macromolecules contained in the medium must be within the desired ranges. The term “spherical” does not require that the macromolecules form structures that are a perfect sphere. Rather the macromolecules form structures that are generally spherical in shape.
In certain embodiments, the macromolecule is a polymer. The polymer may be a hydrophilic polysaccharide or similar structured macromolecule having molecules that form compact three-dimensional structures that are spherical in shape when dissolved in an aqueous liquid. Suitable macromolecules include spherical hydrophilic polysaccharides, polymerization of cyclodextrin or any saccharides to form large spherical molecules, globular proteins or spheroproteins (e.g. albumins, such as bovine serum albumin (BSA)), spherical glycoproteins formed by attaching oligosaccharide chains to those globular proteins or other derivatives of those globular proteins. Hydrophilic nanoparticles can also be suitable macromolecules. One suitable polymer is a polymer formed by the copolymerization of sucrose and epichlorohydrin, without any ionized groups, such as that sold under the brand name FICOLL by GE Healthcare Bio-Sciences AB.
In certain embodiments the macromolecule has a molecular weight from about 300,000 to about 500,000, preferably 400,000, such as that sold under the brand name Ficoll 400. In other embodiments, the macromolecule has a molecular weight from about 60,000 to about 80,000, preferably about 70,000, such as that sold under the brand name Ficoll 70.
In certain embodiments of the medium, before addition of cells, the polymer or other macromolecule is present at a concentration greater than about 10% (w/v) or greater, about 20% (w/v) or greater, about 25% (w/v) or greater, about 35% (w/v) or greater, or about 50% (w/v) or greater, or any range or value therein. In certain embodiments, the polymer or other macromolecule is at a concentration up to the solubility of the polymer (or other macromolecule) in the aqueous liquid or water.
The cryoprotectant can be any cryoprotectant known in the art. Preferably the cryoprotectant is a cell permeating small organic molecule. Cryoprotectants suitable for use in the present invention include dimethyl sulphoxide (DMSO), glycerol, ethylene glycol, propanediol, and combinations thereof.
The medium of the present invention allows use of lower amounts of the cryoprotectant to be used than in standard cryopreservation media. The cryoprotectant can be present in the medium at a concentration equal to or greater than about 20% of the concentration of the polymer (or other macromolecule) in the medium, equal to or greater than about 50% of the concentration of said polymer in the medium, equal to or greater than about 75% of the concentration of said polymer in the medium, or equal to or greater than about 100% of the concentration of said polymer in the medium, or any value or range therein. The volumetric ratio of the polymer (or other macromolecule) and aqueous liquid to the cryoprotectant is from 10:1 to 1:1, from 5:1 to 1:1, or any range or values therebetween.
The aqueous liquid can be any aqueous liquid suitable for use in suspending cells, and can be a liquid selected from the group consisting of a cell culture medium, a nutritious medium, a saline and combinations thereof. Aqueous liquids suitable for use with the present invention include serums, FBS (fetal bovine serum), DMEM (Dulbecco's Modified Eagle Medium), HEPES (4-(2-hyroxyethyl)-1-pierazineethanesulfonic acid), FHM (flushing-holding medium), PBS (phosphate-buffered saline), DPBS (Dulbecco's phosphate-buffered saline), RPMI (Roswell Park Memorial Institute medium), BF5 medium, EX-CELL medium, Lysogeny broth (LB) medium, CaCl2 aqueous solution, NaCl aqueous solutions, KCl aqueous solutions and combinations thereof. The medium may be substantially free of serum, animal proteins or human proteins.
The medium of the present invention is suitable for use with any types of cells. For example, the suspended cells can be eukaryotic cells. The eukaryotic cells may be mammalian cells, such as mammalian cells selected from the group consisting of murine cells, porcine cells, human cells, and combinations thereof. The mammalian cells can be any type of cell, including cells selected from the group consisting of stem cells, somatic cells, reproduction cells and combinations thereof. Reproduction cells may include, for example, embryos and oocytes. Other eukaryotic cells that may be used included insect cells. In other embodiments the calls may be prokaryotic cells. The prokaryotic cells may be bacteria, such as E. coli, Streptococcus and Staphylococcus.
The cells may be separated into single cells or may be in clumps. Cells may be added as isolated cells or in a suspension. The term “cells” may also encompass other cellular materials comprising multiple cells, including tissues.
For suspensions of mammalian cells, their cell concentration may be in the range of 105 to 106 cells per 0.5-1 ml cell suspension sample. For oocytes and embryos, the cell density (number) is low, typically around 102 to 105 cells in one sample (sample volume is around 0.25-0.5 ml), because of the difficulty in obtaining millions of embryos or oocytes. In certain embodiments, it may be possible to preserve only several hundred embryos or oocytes in one sample (e.g. a 0.5 ml straw containing embryos or oocytes), and in some embodiments, the number of embryos or oocytes in the sample is around 20. Prokaryotes, e.g. E. coli, can be available in high density. Prior to freezing, the cell concentration can reach 109-10 cells per ml.
The present invention is also directed to methods for preserving cells at a non-cryogenic freezing temperature in a medium of a present invention. When used herein, the term non-cryogenic freezing temperature can be any temperature above the saturation temperature of LN2 at one atmosphere pressure (−196° C.) or LN2 vapor (typically −120° C.). Non-cryogenic freezing generally occurs in freezer set at −80° C., with temperatures that can go as low as −85° C., but can also rise above −80° C. due to temperature variations that can result from opening the freezer door or placing unfrozen materials into the freezer. The medium of the present invention also allows the cells to be maintained frozen by dry ice while maintaining acceptable cell survival rates. Suitable non-cryogenic freezing temperatures can include temperatures from about −100° C. to −20° C., about −85° C. to −65° C., or about −80° C. to −75° C., and any values and ranges therebetween.
The process includes providing a cryopreservation medium comprising a hydrophilic and nontoxic macromolecule, a cryoprotectant, and an aqueous liquid, wherein the macromolecule forms a highly compact spherical structure when dissolved in said aqueous liquid, adding said medium to cell suspensions, or adding cells or cell suspensions to said medium, or in any order of adding part of said medium or cell suspension, to form a medium-cellular suspension, and cooling the medium-cellular suspension to a non-cryogenic freezing temperature.
The total concentration of cells in the medium-cellular suspension prior to freezing can vary widely depending on the intended use, as will be readily understood to those in skilled in the art. In certain embodiments, the concentration of cells in the cryopreservation medium prior to freezing is single or sparsely distributed cells in the whole system, 102-4 cells/ml, 105-6 cells/ml, 107 or more cells/ml, or even a whole tissue or any value or range therebetween. In certain embodiments, the cells are added as cellular suspension, and the volumetric ratio of the cryopreservation medium to the suspension of cells can range from about 10:1 to about 1:5, about 2:1 to about 1:2, about 3:2 to 1:1, or any value and range therebetween.
The cooling step will generally involve slow cooling. In such embodiments, the medium-cellular suspension can be cooled at a rate of about 0.01° C./min to about 1000° C./min, about 0.1 to about 10° C./min, about 0.5 to 1° C./min, or any value or range therebetween.
As discussed in more detail above and in the examples, the polymer or other macromolecule is concentrated in an unfrozen portion of the medium-cellular suspension after the cooling step. In certain embodiments, the concentration of the polymer or other macromolecule in the unfrozen portion of the medium is at least about 25% (w/v), at least about 35% (w/v), at least about 40% (w/v) or any value or range therein.
The medium-cellular suspension can be maintained at a non-cryogenic freezing temperature for long periods of time. It should be understood that although the medium-cellular suspension can be maintained at or near the original non-cryogenic freezing temperature for the entire time it is frozen, the temperature can vary between different non-cryogenic freezing temperatures during the freeing period. During storage, the medium-cellular suspension can also be cooled to cryogenic freezing temperatures for periods of time and warmed up back to non-cryogenic temperature range for the rest of the period of time (e.g. in the case that cells are stored in liquid nitrogen by one user and then stored in deep freezers by another user; or cells are stored in liquid nitrogen, but warmed and shipped in dry ice box (above −78° C.)).
As discussed above, the medium-cellular suspension can be maintained at a non-cryogenic freezing temperature for surprisingly long periods of time, while maintaining post-thaw cell survival rates of the cells about the same as would be obtained for storage of the cells in liquid nitrogen for the same period of time. Consistent with the present invention, the medium-cellular suspension can be maintained at non-cryogenic freezing temperatures for over three weeks, about three weeks and extending up to at least one year, about one year or more, about 5 years or more, or about 10 years or more, and any time period or range of time periods therein.
As demonstrated in the Examples, at the end of the freezing period, the cells stored in the medium-cellular suspension have a post-thaw cell survival rate about the same as would be obtained for storage of said cells in liquid nitrogen for the same period of time. The cell survival rate can be at least about 80%, at least about 90%, about 100% or higher, and any value or range therebetween, of the cell survival rate for cells stored in liquid nitrogen for the same period of time. Without using the medium of the present invention, for example using solely 10% DMSO as the cryoprotectant, 5% to 30% (of the number of those survived from storage in liquid nitrogen) cells (depending on cell types, see
The polymer (or other macromolecule) and cryoprotectant can be combined with each other and the liquid in any order that allows the polymer to be dissolved in the desired concentration. In certain embodiments, the polymer is first dissolved in the aqueous liquid to form a first mixture, and the cryoprotectant is then added, or that order can be reversed. In other embodiments, the cryoprotectant and polymer are added simultaneously or small amounts of each can be added until the desired ratios are reached. The volumetric ratio of the Ficoll/aqueous liquid to cryoprotectant can range from about 10:1 to 1:5, about 5:1 to 1:1, about 2:1 to 1:1, or any values or ranges therebetween.
The present invention demonstrates that addition of a hydrophilic and nontoxic macromolecule of the present invention to typical cryopreservation solutions significantly improves system thermal stability at non-cryogenic freezing temperatures. It is believed this occurs through macromolecular crowding effects achieved by the macromolecule after slow freezing procedures. Accordingly, using the cryopreservation medium of the present invention provides reliable cryopreservation of various kinds of cells at −80° C. for at least one year, with the post-thaw viability, plating efficiency, and full retention of cell phenotype comparable to that achieved with LN2 storage. These results achieved with the medium of the present invention illustrate the practicability of a non-cryogenic cell storage method that completely eliminates the need of LN2.
Certain aspects of the present invention are illustrated by the following non-limiting examples.
Three simulation boxes for molecular systems including Ficoll 70-DMSO-water, sucrose-DMSO-water and DMSO-water were prepared for the molecular dynamics simulations. For all these systems, the systematic temperature was fixed as −80° C. The concentration of each component is determined by premeasured phase diagram of these ternary systems. For the Ficoll 70-DMSO-water system with the Ficoll and DMSO mass ratio as 1:1, the phase diagram determines that at −80° C., the concentrations of Ficoll, DMSO and liquid water are to be 35%, 35% and 30% (w/w) to reach phase equilibrium with solid water (ice phase). In other words, for the medium of the present invention, after it mixes with DMSO and a cell suspension, the new mixture is slowly cooled to −80° C., and the Ficoll concentration is then significantly increased.
Importantly, as demonstrated in
According to phase diagrams, for the sucrose-DMSO-water system with sucrose and DMSO weight ratio as 1:1, the sucrose, DMSO, and water concentrations are 36%, 36% and 28% (w/w), respectively, when they reach phase equilibrium with the ice phase; for the DMSO-water system with DMSO and water weight ratio as 1:9 prior freezing (as in widely used cryopreservation methods), their phase equilibrium concentrations are 58% and 42% respectively. The dimension of the simulation boxes of these three cases, Ficoll-DMSO-water (
To demonstrate the thermal stability of above systems, a typical cubic ice nucleus, presented as a group of 512 water molecules forming a 10 nm cube, is artificially placed at the center of each simulation box, as shown in
The activity of the liquid water molecules surrounding the ice nucleus was measured through the root-mean-square (RMS) distance of atomic positions, along with the final equilibrated structures of these three systems, and the results are shown in
Scanning electron microscopy (SEM) was performed to demonstrate the capability of a medium of the present invention in significantly improving the thermal stability of ice crystals in samples.
In one embodiment of the medium of the present invention, a solution of 20% (w/v) Ficoll 70 and 20% (v/v) DMSO in DMEM medium was gradually mixed with a substitute of cell suspension, e.g. DMEM medium without any cells, with a volume ratio of 3:2, and transferred to 1.5 ml cryovials. These vials were then loaded into a widely used freezing container (Nalgene Mr. Frosty), which was mounted into a −80° C. freezer to provide an approx. 1° C./min cooling rate until the next day. These vials were then transferred into a precooled and sealed vial container in the freezer and stored for 5 weeks. For comparison, the control group as the solution of 10% (v/v) DMSO in DMEM medium was also cooled and stored in the same approach and for the same period of time. At the end of 5 weeks, the cryovials were directly plunged into LN2 to fix the ice crystal morphology and were fractured inside LN2. The fixed and fractured samples covered by LN2 in a copper stage and were then transferred into the vacuum chamber of a SEM system. After the LN2 evaporated and chamber pressure was lower than 10−2 Pa, the surface of the samples were scanned by the SEM to observe the ice morphology for the fracture surface.
Crowding of Ficoll, a compact, spherical, hydrophilic polysaccharide, in aqueous solutions can restrict microscale diffusion and limit structural reconfiguration of macromolecules. To explore its potential in regulating ice recrystallization, differential scanning calorimetry (DSC) was used to examine the thermal stability of aqueous solutions of DMSO and Ficoll (Ficoll 70 and Ficoll 400) at non-cryogenic temperatures. For comparison, similar ternary systems containing DMSO and polyvinylpyrrolidone (PVP) or sucrose, representing commonly used polymers and small molecules used for cryopreservation, were also tested. To model the residual solutions at the end of a slow freezing process, highly concentrated solutions comprised of a 1:1 weight ratio of one of the non-permeating solutes and the permeating cryoprotectant DMSO were prepared with the same total solute concentration (50% w/w). A standard DSC procedure to detect vitrification and devitrification was followed using a Pyris Diamond DSC (Perkin-Elmer Corp). A volume of 8 μl of each model solution was sealed in a standard 10 μl aluminum crucible (Perkin-Elmer Corp) designed for liquid samples and then loaded in the sample holder of the DSC machine. All samples were cooled to −160° C. from 1° C. at 100° C./min to achieve complete vitrification, which was confirmed by continuous heat capacity change near −130° C. for all the samples during the cooling and following warming procedures, and none of the samples experienced any crystallization during the cooling process. After being held at −160° C. for 1 min, the samples were heated to 20° C. at a warming rate of 10° C./min. Devitrification was detected in all the samples and the onset temperatures for the corresponding exothermic curves were determined as the values of devitrification temperatures using the Pyris™ thermal analytic software provided by Perkin-Elmer Corp. Table 1 shows the devitrification temperatures (Td) of the highly concentrated solutions modeling the unfrozen residual portion of the aqueous solutions containing one polymer (or sucrose) and DMSO at the end of a slow freezing process. The total solute weight percentage for each solution is fixed as 50% w/w.
Since recrystallization involves a spontaneous process that generates no detectable latent heat release, the thermal stabilities of these model solutions were assessed by their devitrification temperatures (Td). This approach is possible because Td is always lower than, but close to, the temperature at which recrystallization begins. Accordingly, if the Td measured for any of these model solutions during slow warming is higher than −80° C., as observed for solutions containing Ficoll 70 (Td, −67° C.) or Ficoll 400 (Td, −75.7° C.), it is considered to be thermally stable and no recrystallization will occur at −80° C. (Table 1). Of the two Ficoll polymers, Ficoll 70 appeared to be superior at providing a potentially useful cryopreservation medium, since at a 1:1 weight ratio with DMSO it demonstrated the higher Td value. These results suggested that initiating slow freezing with lower concentrations (e.g. 10% for each) of Ficoll 70 and DMSO, to achieve sufficient macromolecular crowding by cooling to −80° C. when the cryopreservation medium is much more concentrated, potentially prevents recrystallization in extracellular solutions and hence realize long-term storage of cells at −80° C.
The ability of the medium of the present invention to preserve the viability and pluripotent features of the O2K line of porcine induced pluripotent stem cells (iPSC) during long-term storage in a commercial deep freezer was examined. The O2K line of porcine iPSC is a naïve-type of pluripotent stem cell, dependent upon leukemia inhibitory factor (LIF) and STAT3 signaling for self-renewal, which can be dispersed into single cells without significant loss of viability. For routine maintenance, O2K piPSC were cultured either on a laminin (Gibco) coated substratum or irradiated mouse embryonic fibroblasts feeder on six-well culture plates (Nunc) in N2B27 (Gibco) medium, supplemented with three inhibitors (CHIR99021 (Stemgent), PD032591 (Stemgent), and PD173074), 2 μg/ml doxycycline (Stemgent), and 1000 unit/ml human LIF (Millipore). O2K piPSC were passaged every three days after dispersing with Accutase (Millipore) for 7 min at 37° C. Cell colonies were dispersed to single cells with a cell detachment solution sold under the tradename Accutase® by Innovative Cell Technologies, Inc. Dissociated cells were collected by centrifugation (200×g for 5 min) and resuspended in chilled culture medium. Different embodiments of the medium of the present invention were prepared as: 10% (w/v) Ficoll 70 and 20% (v/v) DMSO, 20% (w/v) Ficoll 70 and 20% (v/v) DMSO, 30% (w/v) Ficoll and 20% (v/v) DMSO. These media were based on either FBS (a serum) or Dulbecco's Modified Eagle Medium (DMEM/F12, serum-free), and in another word, excluding Ficoll 70 and DMSO, the liquid portion of the media is either FBS or DMEM. Each of these media was added drop-wise to a suspension of cells in their culture media (total volume ratio between the cell suspension and all added medium drops is approximately 3:2). After mixture, the final concentration in the medium-cellular suspension of DMSO was approximately 10% v/v and Ficoll 70 as 5%, 10% or 15% w/v prior to freezing, respectively. Such a mixing procedure is to modify the cell damage caused by osmotic damage generated by directly mixing the whole cryopreservation media with cell suspensions. The cryovials were then placed into a freezing box (Mr. Frosty, Nalgene), as widely used for current cryopreservation of many cell types. The latter was placed overnight into a −80° C. freezer to provide an approximately 1° C./min cooling rate. On the following day, the vials were stored in the −80° C. freezer for two weeks. The control groups were cells treated with a similar procedure to achieve the same final concentration of DMSO (10%), except that the cryopreservation medium was based on FBS alone and contained no Ficoll, as generally used for stem cell LN2 storage. These control samples were cooled by the same slow freezing procedure and then stored at −80° C. (as a negative control) or in a LN2 dewar (as a positive control).
For thawing, all cryovials were rapidly warmed in a 37° C. water bath for approximately 1 min until the ice mass disappeared. The medium-cellular suspension was then transferred to a 15 ml centrifuge tube and slowly mixed with 5 ml of warmed culture medium. After centrifugation (200×g for 5 min), the supernatant solution was removed, and cell pellets resuspended in 1 ml fresh culture medium.
Thawed and cultured cells were plated in a 6-well plate and cultured overnight. After the first medium change, images of adherent colonies were acquired over five different areas within each well. Plating efficiency was estimated as colonies/number of initially plated cells×100%. Colonies were then fully dispersed by Accutase, and total cell numbers were assessed by using a TC10 automated cell counter (Bio-Rad).
The results after two weeks of storage are shown in
Only the cells cryopreserved at −80° C. within the medium-cellular suspension comprising the mixture of cell suspension and the 20% Ficoll and 20% DMSO medium (final post-mixture and prior-freezing Ficoll concentration is 10% as shown in
The results for O2K porcine iPSC thawed after 2, 5, 10 and 58 weeks are shown in
Even by the end of week 2, the ability of the cells cryopreserved without using the medium of present invention at −80° C. to attach, proliferate and provide colonies (red bars) had fallen significantly relative to the other two treatments. These declines were progressive over storage time, such that, at 10 weeks, recovery was very low, and no colonies at all formed after 58 weeks of storage, results consistent with the concept that the recrystallization process causes progressive rather than immediate cell damage. By contrast, the porcine iPSC stored at −80° C. within the mixture of cell suspension and said embodiment of the medium of the present invention showed no apparent decrease in either plating efficiency (
For the tests of pluripotency of the thawed cells, after thawing, cells were allowed to establish colonies, passaged and grown on coverslips. Specimens were fixed in 4% v/v paraformaldehyde in PBS for 15 min at room temperature, washed, and exposed to either 5% v/v goat serum or 5% v/v donkey serum, 1% w/v bovine serum albumin, and 0.1% v/v Triton X-100 (Fisher) in PBS for 30 min. The fixed specimens were then incubated with primary antibodies at 4° C. overnight. After washing, they were exposed to secondary antibodies. Colonies exposed only to secondary antibody served as controls. VECTASHIELD mounting medium with DAPI (Vector Laboratories) was used to mount the coverslips. Primary antibodies were: POU5F1 (1:100, Santa Cruz Biotechnology), SOX2 (1:1000; Millipore), NANOG (1:200; Abcam), SSEA1 (1:50; Developmental Studies Hybridoma Bank [DSHB]). As shown in
The morphology of ID6 porcine iPSC during culture is shown in
To overcome the above technical issue, ID6 cells were dispersed into smaller cell aggregates prior to freezing by using a “gentle dissociation reagent” (Stem Cell Technologies) for 6 minutes and supplemented with 10 uM of ROCK inhibitor prior to freezing. Cells separated in this manner typically provided clumps of 6-8 cells, as shown in
For maintenance, ID6 piPSC were cultured on irradiated mouse embryonic fibroblasts (iMEF) feeder layers in six-well culture plates in standard hESC medium (hESCM) supplemented with 20% knockout serum replacement (KOSR, Gibco) and 4 ng/ml human FGF2. The procedure for cooling, storage, and thawing, were the same as those described in Example 4. Samples were thawed after 5 and 15 weeks of storage. Thawed cells from three samples in each treatment group were transferred to 6-well plates coated with iMEF, with cells from one vial divided equally between two wells. On day 4 after thawing and plating, five images of different areas of each culture well were captured at 40× magnification to determine colony areas relative to the control group that had been stored in LN2. Cells were then fixed in 4% v/v paraformaldehyde in phosphate-buffered saline (PBS, Hyclone) for 2 min and stained for alkaline phosphatase activity to increase contrast. Nine images were taken at 8× magnifications to cover the entire area of the well and used to measure the total number of colonies present. All images were analyzed by the Image J software. The results are shown in
The medium of the present invention was also able to provide effective cryopreservation for human iPSC. The human iPSC line was derived from human umbilical cord fibroblasts reprogrammed with five factors (POU4F1, SOX2, KLF4, LIN28, and MYCL) and TP53 shRNA by using episomal plasmid transfection. Cells were cultured on Matrigel (BD Bioscience) coated six-well culture plates (Nunc) in defined mTeSR1 medium (STEMCELL Technologies). The morphology of cell colonies of human iPSC lines is similar to ID6 cells in Example 5. Therefore, before freezing, the cell colonies were also dispersed into smaller cell aggregates as described in Example 5. The procedure for cooling, storage, thawing, post-thaw viability tests, were almost the same as those described in Example 5, except that thawed cells were transferred to 6-well plates coated with Matrigel. As shown in
H1 hESC (WA01) were obtained from the WiCell Research Institute, Madison Wis. in 2002. The procedure for culturing, maintaining, dispersing, cooling, storage, thawing, and post-thaw viability tests, were the same as those described in Example 6. The results are shown in
Alternatively, we next used TrypLE dispersion of the H1 hESC colonies in presence of a RHO-kinase inhibitor (ROCKi, Y-27632), which protects hESC from cell death, to achieve a suspension of single cells (
First, use of the different base media (FBS in the left panel and DMEM in the right panel) for making the medium of the present invention doesn't cause any noticeable difference in post-thaw cell survival and plating efficiency. Therefore, the medium of the present invention can be completely serum free; second, the use of the medium of the present invention did slightly improve the cryopreservation efficiency in liquid nitrogen, comparing the data demonstrated on the center left bars (using the medium of present invention for liquid nitrogen storage) and left bars (using the solely 10% DMSO for liquid nitrogen storage), without any negative effects; third, −80° C. storage using the medium of present invention (right bars) yielded almost identical cell survival and post-thaw plating efficiency as those from liquid nitrogen storage (left bars); and at last, −80° C. storage without using the medium of present invention resulted in significantly lowered post-thaw plating efficiency and viability (center right bars) than other treatments.
For the post-thaw H1 hESC cells from −80° C. storage when the medium of the present invention was used,
Experiments using the medium of the present invention were also performed with ID6 porcine iPSC epiblast-type cells separated using different methods. Colonies (
Spermatozoa, peripheral blood mononuclear cells (PBMC), and E. coli are typical cell types that can be successfully cryopreserved in liquid nitrogen without gradual loss of cell viability during cryostorage even when storage period extends to years. However, their storage at −80° C. using homemade or commercially available cryopreservation media results in remarkable loss of viability and functionalities even after mid-term periods of storage (e.g., several months), and the rate and degree of such losses depend on cell types. Using the examples described below, we are demonstrating that those losses were prevented when cryopreservation of the invention was adopted for these cell types.
Improvement of the efficiency of preservation of porcine semen is critical for pig breeding improvement and is highly valuable for food industry. Cryopreservation or transportation of porcine semen in liquid nitrogen or its vapor, using liquid nitrogen dewars, dry shippers or much more expensive cryogenic freezers, is highly expensive and impractical for use in most farm operations. As a result, freshly collected semen is widely mixed with commercially available extender solutions (no cryoprotectants) at around 4° C., but that method can only maintain sperm viability for approximately one week.
To overcome that limitation, a medium of the invention was used for pig semen cryopreservation at −80° C., which enables collectors to efficiently cool and ship the sperm suspension on dry ice (available in many supermarkets) and store in regular −80° C. deep freezers. The sperm rich fraction of boar semen (˜100 ml) was collected and filtered twice through a sperm filter and placed at room temperature for 1.5 hr. Filtered semen samples (25 ml for each sample) were transferred to 50 ml conical tubes and washed by gently mixed by 1:1 (v/v) with 25 ml sperm wash medium and then centrifuged 1000×g for 7 minutes. Supernant of each sample was removed and then 5 ml of commercially available sperm extender (BF5, whose major components include egg yolk and glucose, etc.) was gently mixed with the centrifuged spermatozoa as a standard extension or suspension procedure. Each suspension sample was precooled to 4° C. in a refrigerator before freezing. For the Control the commercially available boar semen cryopreservation medium (BF5 plus 4% v/v cell culture grade glycerol), which is only valid for storage of porcine semen in liquid nitrogen, was added dropwise to the suspension with final volume ratio as 1:1, and resulted in a final volume approximately as 10 ml. The new suspension was aliquoted into 0.5 ml straws (10-20 straws) and the straws were put on dry ice for one hour and then stored in a −80° C. freezer. For the treatment groups, the BF5 was used as the base medium of the new freezing media (just as using DMEM as the base medium for stem cells in previous examples). Two treatment groups were prepared: the cryopreservation medium for Treatment A was BF5 mixed with 4% v/v cell culture grade glycerol and 20% w/v Ficoll 70; Treatment B was BF5 mixed with 4% v/v cell culture grade glycerol and 10% w/v Ficoll 70. For both treatments, the sperm suspensions in BF5 were mixed with the new Ficoll containing freezing media (A and B, respectively) with a 1:1 ratio, then aliquoted into 0.5 ml straws, put on dry ice for freezing and stored in the deep freezer (same as the Control).
After two months of storage, straws from three groups were thawed by directly plunging the straws into room temperature water. For each group, suspensions from two of the straws were diluted by 10× and cultured for 2 hrs. to evaluate post-thaw motility, and cells from other straws were collected and co-cultured with approximately 100 porcine oocytes in IVF medium for IVF efficiency evaluation. The results are listed in Table 2, below.
It is straightforward to conclude that the Treatment A, a typical embodiment of the invention, e.g. the use of the base medium with the addition of 20% w/v Ficoll 70 and 4% v/v glycerol as the permeating cryoprotectant and mixing it with cell suspension with a 1:1 ratio, significantly improved the post-thaw motility of the porcine sperm after two months of storage, whose viability and IVF efficiency is comparable to the outcome from liquid nitrogen storage. In contrast, if the medium contained either no Ficoll or an insufficient amount of Ficoll, then the post-thaw motility and functionality was severely impaired after two months of storage in a −80° C. freezer.
All the cryopreservation media were mixed with the cell suspensions with a 1:1 volume ratio.
PBMCs are highly valuable for blood banking and widely used in research or biomedical applications related to immunology (including auto-immune disorders), infectious diseases, hematological malignancies, vaccine development, etc Using the combination of DMSO, FBS or BSA, and base medium (e.g. DMEM), these cells can be successfully cryopreserved in liquid nitrogen or its vapor for many years without loss of cell viabilities. However, when they are stored in −80° C. freezers, gradual cell loss has been observed, and after slightly more than one year of storage, the recovery is minimal. And using dry ice boxes for transportation of those samples previously preserved in liquid nitrogen or its vapor results in inevitable cell loss due to recrystallization during transition (warming from −120° C. or lower to −78° C. or higher). For many small clinics or hospitals, establishing cell stocks of PBMC without using expensive liquid nitrogen facilities is technically impossible. In addition, the high concentrations of FBS (generally 40% v/v) used in PBMC cryopreservation medium also significantly increase the associated cost, because the price of FBS (˜$800 to 1,000 per liter) is much higher than DMEM or other simple base medium (˜$20 per liter), and more importantly, FBS as an animal product generates contamination and regulatory issues.
To solve these practical issues, a medium of the invention was used for porcine PBMC cryopreservation in −80° C. freezers without using any FBS. Approximately 10 ml pig blood was first mixed with equal volume of PBS+2% FBS, and the cells were collected through standard density gradient centrifugation (1200 g for 10 minutes) by using the top layer. The enriched cells were washed and centrifuged again (450 g for 10 minutes) and cultured in commercially available culture medium (RPMI) supplied with diluted GM-CSF (human granulocyte-macrophage colony-stimulating factor) at 37° C. and with 5% CO2 in an incubator for 6 days. Cells were then collected and concentrated by centrifugation and resuspended in RPMI. The new suspensions were aliquoted as 0.5 ml in each 1 ml cryovial (˜105 cells per vial). The Control group was treated with traditional cryopreservation medium, which contains 20% v/v DMSO, 40% v/v FBS and 40% v/v DMEM, by adding 0.5 ml of the medium into the cell suspension in the cryovial dropwise so that the final volume ratio between the cell suspension and cryopreservation medium is 1:1. The Treatment group was treated with the medium of invention without any FBS, which is based on DMEM with the addition of 20% v/v DMSO and 20% w/v Ficoll 70 and also mixed with the cell suspension dropwise in the cryovial with a final 1:1 volume ratio.
After mixture, the cryovials were then mounted in commercially available freezing box, Mr. Frosty, and the box was cooled in a −80° C. lab freezer overnight and the cryovials were then placed in storage boxes in the same freezer for storage. After two months of storage, the cryovials were thawed in a 37° C. water bath. The cell viability of all samples prior to freezing and post-thaw was determined using TC20™ automated cell counter. The ratios between the post-thaw viability and the viability prior to freezing for both groups are listed in Table 3, below. The results clearly demonstrated that the medium of the invention resulted in high recovery of cells, comparable to published data when PBMC are preserved in liquid nitrogen, and more importantly, efficiently prevented the cell loss during the two month storage period shown in the results of the group when traditional DMSO+ FBS medium was used. The medium of invention is also serum free (without any FBS).
Both of the cryopreservation media were mixed with the cell suspensions with a 1:1 volume ratio.
E. coli competent cells are most commonly used bacterial cell types for transformation of DNA in molecular biology research and technological development. Cryopreservation of E. coli competent cells in liquid nitrogen using DMSO is a widely used protocol for long-term storage. Although many labs use high concentrations of glycerol for temporary storage in −80° C. deep freezers, the preserved cells stocks will expire after several months and using high concentration of glycerol (highly viscous) is problematic in operation.
To improve the efficiency of long-term storage of E. coli competent cells in deep freezers, a medium of the invention was tested in comparison with the treatments of using low concentration of DMSO and high concentration of glycerol when the cells were stored at −80° C. for two months. The pre-cultures of NEB® 5-alpha F'Iq competent E. coli were diluted (1:50 in LB medium at −37° C.), grown till the OD reached 0.6, and then cooled on ice. The samples were transferred to centrifuge tubes and centrifuged for 5 minutes at 3000 rpm and the pellets were then re-suspended in 25 ml DI water. This washing step was repeated three for all samples. The final pool of pellets was resuspended in 0.1M CaCl2 water solution and aliquoted as 0.1 ml in each 0.5 ml cryovials and cooled on ice. Three cryopreservation media were prepared for three different treatments: Treatment A as 14% v/v DMSO and 0.1M CaCl2 in DI water; Treatment B as 40% v/v glycerol and 0.1M CaCl2 in DI water, Treatment C (a medium of the invention) as 14% v/v DMSO, 20% w/v Ficoll 70 and 0.1M CaCl2 in DI water. For all the samples (0.1 ml cell suspension precooled on ice, as stated above) for each treatment, 0.1 ml of the corresponding cryopreservation medium was added to the cell suspension directly (i.e. volume ratio is also 1:1), and then mixing was completed by gentle shaking and samples were kept on ice for 20 mins. The cryovials were then mounted in sample storage boxes (10×5×2 cm) and directly mounted in −80° C. freezer for cooling and storage (cooling rate approximates 15-20° C./min).
After the storage period for one day, one month, and two months, respectively, cryovials from each treatment group were thawed to compare the influence of the storage periods on cell viability loss across different treatments. The value of colony forming units (CFU) for each sample was determined after performing standard culture and counting protocols for E. coli competent cells. The CFU values after one day, one and two months of storage for these treatments are listed in Table 4, below. As demonstrated in Table 4, the post-thaw colony formation efficiency for both Treatments A and B gradually decreased. In contrast, Treatment C still satisfactorily maintained colony formation efficiency, which will be beneficial for a large number of molecular biology labs to maintain their E. coli stocks in deep freezers. The CFU value resulting from Treatment A is much lower than the other two (one order of magnitude lower), because Treatment A is generally used for liquid nitrogen storage of E. coli but not for their −80° C. storage. The one day storage using Treatment C resulted in a CFU value lower than the Treatment B, but from the point of view regarding long storage periods (esp. longer than two months), its advantage is obvious. It also establishes the usefulness of the media of the invention for prokaryotic cells.
E. coli at −80° C. for one and two months.
All the cryopreservation media were mixed with the cell suspensions with a 1:1 volume ratio.
In summary of the results shown in above examples, the present invention is a simple and reliable method for long term storage of human and porcine pluripotent stem cells at −80° C., based on the use of the medium of the present invention that contains high concentration of Ficoll 70, a synthetic polymer of sucrose, which, it is believed, has not previously been used for this or comparable purposes. It is believed the success of the method is attributable to the ability of the Ficoll polymer to improve the thermal stability of the permeating cryoprotectant at non-cryogenic temperatures and prevent corresponding ice recrystallization that generally causes cell loss during long-term storage at non-cryogenic temperatures. The molecular mechanism is probably due to the physical properties of Ficoll 70, which is comprised of small spheres approximately 5 nm in radius. Slow cooling will lead to macromolecular crowding in the solution that remains unfrozen at −80° C., so that the packed Ficoll 70 spheres form a mechanical barrier that hinders enlargement of small ice crystals. Additionally, Ficoll 70 can avoid FBS in the cryopreservation solution, hence avoiding exposure of cells to animal products. From the foregoing it will be seen that this invention is one well adapted to attain all ends and objectives herein-above set forth, together with the other advantages which are obvious and which are inherent to the invention.
Since many possible embodiments may be made of the invention without departing from the scope thereof, it is to be understood that all matters herein set forth or shown in the accompanying drawings are to be interpreted as illustrative, and not in a limiting sense.
While specific embodiments have been shown and discussed, various modifications may of course be made, and the invention is not limited to the specific forms or arrangement of parts and steps described herein, except insofar as such limitations are included in the following claims. Further, it will be understood that certain features and subcombinations are of utility and may be employed without reference to other features and subcombinations. This is contemplated by and is within the scope of the claims.
This application is based on and claims priority to U.S. Provisional Application Ser. No. 62/336,142 filed on May 13, 2016, which is hereby incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US17/32606 | 5/15/2017 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 62336142 | May 2016 | US |
