Chemistry: molecular biology and microbiology – Maintaining blood or sperm in a physiologically active state...
Reexamination Certificate
2000-06-23
2002-03-26
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Maintaining blood or sperm in a physiologically active state...
C128S898000, C128SDIG008, C424S561000, C435S003000, C436S008000
Reexamination Certificate
active
06361934
ABSTRACT:
The present invention relates to a method and apparatus for cryopreserving biological material including, for example, cell suspensions, such as embryos, gametes, (spermatozoa, oocytes) cell lines, bone marrow, blood stem cells and the like; solutions of proteins and/or other biologically active substances; and perfused tissues and organs, either engineered or obtained from a natural source.
Cryopreservation is a process where samples such as biological materials are stored at low temperatures. Cryopreservation of biological material substantially as described above is generally effected by freezing in appropriate retaining means, such as flexible bags, glass or plastic ampoules, or plastic tubes (usually referred to as “straws” in the field of cryopreservation) of the type suitable for long term storage at the low temperatures employed.
Although cryopreservation has been widely employed for a range of biological materials, there are certain materials for which it is not suitable. For example, with certain materials, cellular injury can occur on thawing. Furthermore, cryopreservation is not applicable for cell types where recovery on thawing may either be low or very variable, or even non-existent. For example, recovery on thawing of sub-fertile human spermatozoa, human testicular spermatozoa, pig spermatozoa and the like is generally low or variable, and in other cases such as mammalian oocytes, fish eggs, fish embryos, tissues, organs or the like, survival is non-existent.
Furthermore, on thawing cryopreserved material, cell survival can be dependent on the conditions and techniques employed, such as appropriate nucleation techniques, addition of suitable cryoprotective additives and also the control of the cooling rate. Nucleation, for example, is known to be a problem when not externally controlled in cryopreservation techniques, and occurs at a wide range of temperatures below the melting point of individual materials. While some materials nucleate at or just below the melting point, others may not nucleate until the temperature has reached up to 20° C. below the melting point, for example, with cell suspensions contained in straws or ampoules the range of temperature is particularly wide, both because the volume of the suspensions, and hence the number of available heteronuclei, is small and also because the containers are generally sealed before freezing, thus removing the possibility of seeding by airborne ice nuclei in the freezer. Nucleation at temperatures significantly away from melting points of materials requiring cryopreservation has resulted in transient temperature changes and cell death even when cooling rates have apparently been optimised for survival (Whittingham D. G., In The Freezing Of Mammalian Embryos, Ciba Foundation Symposium 52, pp 98-102, 1977).
In order to nucleate biological materials in a reproducible manner, it is common practice firstly to cool the materials to a temperature below the melting point thereof, then after a short period of thermal equilibration, to nucleate ice in the supercooled material. Nucleation can be achieved by any of the following techniques—application of cold forceps to the outside of the material, by cold wires, by devices employing “reverse Peltier effects” or by the application of physical disturbances. An alternative method of ensuring nucleation near to the melting point is to incorporate an ice nucleating agent into the biological suspension before temperature reduction. Examples of ice nucleating agents include the so called ice nucleating bacteria for example Pseudomonas syringae, the active proteins from ice nucleating bacteria and organic compounds such as cholesterol. Following nucleation and initial crystal growth, cooling of the materials is then resumed.
However, monitoring the success of the nucleation procedure and also subsequent crystal growth has proved to be a problem. In conventional controlled rate freezing equipment, it is not possible to monitor subsequent crystal growth following nucleation without removing the straws from the equipment for visual inspection. A high risk of melting is associated with such removal. Furthermore, at present no method is available in such equipment to monitor, in a non-invasive manner, the success of the nucleation procedure.
Problems have also been encountered as a result of the techniques employed to lower the temperature following nucleation. It is common practice that during cryopreservation, biological materials are cooled with, as far as possible, a linear reduction in temperature with time. Cryopreservation equipment is generally designed to control the material environment in a linear manner, and depending on the heat transfer characteristics of the equipment, the materials cool in a more or less linear manner. Whilst this approach is convenient for simple design of equipment and has produced acceptable results with a range of cell types, it has proved unsatisfactory with a number of cellular materials.
WO96/02801 discusses the importance of employing a linear cooling rate over the entire cooling phase employed in cryopreservation, and is concerned with the provision of a cooling assembly capable of obtaining a substantially linear and reproducible rate of cooling samples.
WO96/24018 is concerned with the cryopreservation of mammalian tissue, or living cultured equivalents made by in vitro technology. The temperature control employs linear temperature lowering, but different rates of linear temperature lowering are respectively employed in different stages of the cryopreservation process. More particularly, the temperature is initially lowered at a rate of −10.0° C./minute to the solid-liquid phase equilibrium temperature range for an employed cryoprotectant, and following propagation of ice seed crystals throughout the cryoprotectant cooling is resumed at a rate of between −0.02 to about −0.3° C./minute.
U.S. Pat. No. 4,799,358 describes freezing of biological material, and also employs linear temperature lowering but with different rates of linear temperature lowering being respectively employed in different stages of the freezing process. For example, the temperature is typically initially lowered at a rate of −0.5° C./minute between +20° C. and −7° C. and then −0.3° C./minute between −7° C. and −35° C.
The use of non-linear cooling rates has also been discussed in the prior art. WO91/01635 is concerned with a cooling process and apparatus where the material being frozen is subjected to a greater rate of heat extraction when the latent heat is being given up during nucleation, than when the material is cooled further. WO91/01635 indicates that the heat extraction rates may be non-linear, but does not provide clear guidance as to the parameters of non-linear heat extraction employed.
We have now devised a method and apparatus for cryopreserving biological material, which method and apparatus reduce or alleviate the problems associated with the prior art. In particular, we have devised a method and apparatus to reduce or overcome the problems associated with cellular freezing injury.
According to the present invention, there is provided a method of cryopreserving biological material, which method comprises: providing a sample of the biological material, where a liquid phase of the sample includes at least one solute; lowering the temperature of the sample to a nucleating point at which ice nucleation can occur in the sample; effecting ice nucleation in the sample; and lowering the temperature of the sample from the nucleating point to the solidification point thereof, characterised in that the temperature lowering from the nucleating point to the solidification point is non-linear, whereby the rate of change of solute concentration in the liquid phase decreases for more than 80% of the time taken to lower the temperature from the nucleating point to the solidification point.
Preferably, in the method of the invention the liquid phase is aqueous and, preferably, the biological sample is suspended or diss
Acton Elizabeth
Morris George John
Lankford , Jr. Leon B.
Srivastava Kailash C.
Stevens Davis Miller & Mosher L.L.P.
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