GB2091534A - Method of preservation - Google Patents

Abstract

Cells or other microscopic small biological materials are preserved as follows. A water-in-oil emulsion of the cells is formed from an aqueous suspension of the cells and a non- toxic hydrophobic liquid (the 'oil'), the cells entering the aqueous phase of the emulsion. The temperature of the emulsion is reduced to a value at which the water within the cells is frozen (typically in the range -25 to -35 DEG C). The emulsion is then stored at a temperature of minus 70 DEG C or below. Cells of botanical species (e.g. soya beans), microscopic blood bodies (e.g. red blood cells) or differentiated plant tissue may be preserved without the need to use a conventional cryoprotectant.

Description

SPECIFICATION Method of preservation This invention relates to a method of preservation. In particular, it relates to a method of preserving living cells at low temperatures. The cells may be obtained from, for example, a plant.

It is known that at temperatures below about minus 700C most chemical reactions are halted.

Accordingly, it has been appreciated for a long time that it is theoretically possible to preserve biomaterials over extended periods of time at temperatures of minus 700C or below. However, subjecting cells to a temperature cycle in which the water they contain in vivo is frozen and melted has been found to damage, for example, their membranes causing the cells to die.

It has been discovered that if biological materials are frozen in the presence of certain substances, known as cryoprotectants or cryophylactic agents, subsequent thawing is not so likely to cause cell damage or death. Using such cryopreservants several classes of biological matter have been successfully preserved on a clinical scale, such classes including blood, spermatozoa, cornea and skin.

In general there are two known kinds of cryoprotectants. First, there are those substances used conventionally such as dimethylsulphoxide and glycerol which need to penetrate the cell membrane in order to be effective. It is generally believed that such cryoprotectants help to reduce cell dehydration by acting as substitutes for water.

They also reduce the amount of ice formed during cooling. The other kind of cryoprotectant does not penetrate the membrane of the cell, and less is known about their mode of action and they have not yet been used other than experimentally.

However, they may be used in lower concentrations than penetrative cryoprotectants.

It has been found that red blood cells when frozen and then thawed in the presence of a nonpenetrating cryoprotectant show extensive changes in their internal electrolyte concentrations and it has been postulated that such cryoprotectants increase the permeability of the cell membranes (as do dimethyl sulphoxide and glycerol) and thus allow osmotic equilibration to take place more rapidly during freezing.

Although the choice of a suitable cryoprotectant has made possible the clinical storage at low temperatures of, for example, blood bodies, their use is considered to be by no means entirely satisfactory. For a start, they tend not to eliminate the occurrence of cell damage but merely limit its incidence. Moreover, substances such as dimethylsulphoxide (used in the storage of blood) require rigorous removal from e.g. the blood (involving dialysts, washing, centrifuging etc.) are expensive and have an unpleasant smell, and may induce genetic mutation.

Object of the Invention It is an object of the present invention to provide a method of preserving cells at low temperatures which does not necessarily require the use of a substance that functions as a conventional cryopreservant.

The Invention According to the present invention, there is provided a method of preserving cells (or other microscopic or small biological materials) comprising the steps of forming a water-in-oil emulsion from an aqueous suspension of the cells and a non-toxic hydrophobic liquid (the 'oil'), the cells being contained in the aqueous phase of the emulsion, reducing the temperature of the emulsion to a value at which the water within the cells is frozen, and storing the emulsion at a temperature of minus 700C or below. Individual cells or aggregates of cells may be preserved by this method.

We believe that this method makes possible satisfactory preservation of at least some kinds of cell without the use of a conventional cryoprotectant. Since the non-toxic hydrophobic liquid does not contact the cells, the latter being suspended in droplets of aqueous growth medium, this non-toxic hydrophobic liquid does not in any conventional sense act as a cryoprotectant.

The aqueous growth medium may, for example, include sucrose as a carbon source. Sucrose has been reported to be a cryoprotectant when used in sufficiently large quantities. The proportions of sucrose in a conventional growth medium are not sufficient for the sucrose to have any substantial cryoprotectant effect we believe, and the present invention does not therefore rely on the use of sucrose in the growth medium.

In the method according to the invention the emulsion typically includes a multitude of small drops (or droplets) of the aqueous growth medium. Typically, the size of most drops is such that the aqueous phase forms a thin aqueous film around the cell or cells (which may be present in clusters rather than as single or double cells depending on the nature of the species being preserved. Such drops tend to become supercooled when the emulsion is reduced in temperature to just below OOC and freeze at a temperature well below OOC, for example, at a temperature in the order of minus 25 or minus 350C. We believe that freezing at such temperatures tends to cause less damage than at 000.

The emulsion may, if possible, be formed such that the agglomeration of large clusters of cells is avoided. This is not always possible since many cells grown in culture occur in clusters. If desired, the aqueous suspension of cells may be filtered so as to remove clusters or large clusters (i.e. clusters containing more than ten cells) of cells.

The hydrophobic liquid (or 'oil') is preferably a solvent for oxygen and carbon dioxide. Suitable liquids for use in the invention as 'oils' include silicone oils and glycerides. The viscosity of the 'oil' is not critical to the method according to the invention. The oil may, for example, have a viscosity in the range of about 1 centistoke to about 20 centistokes.

The emulsion typically requires the presence of an emulsifier. An emulsifier having a low hydrophil/lipophil balance and soluble in the oil is used. For example, the emulsifier may be sorbitan tristearate. In preparing the emulsion the emulsifier is added to the oil, and the resulting liquid blended in a homogeniser with an aqueous suspension of the cells (typically in their growth medium) to be preserved. Care must be taken not to destroy the cells.

The relative proportions of the oil to aqueous phase in the emulsion are not critical. Typically, 2 parts by volume of the oil to one of the aqueous phase may be employed.

The rate of the reduction in temperature of the emulsion from +50C to the freezing temperature is typically less than 1 OC per minute. Any refrigerator which is capable of attaining the required freezing rate and temperature may be employed. Typically, the emulsion is loaded into the refrigerator in one or more suitable containers.

Once in vitro freezing of the aqueous medium and the cystoplasm within the cells has occurred, the emulsion containers may be transferred to a storage vessel at a temperature of-700C or below. Typically, the vessel contains liquid nitrogen at a temperature of -1 960C.

The method according to the present invention is particularly suited to the freezing of cells of plants or other botanical species. The cells for preservation may be grown in a conventional aqueous culture medium by conventional methods. Typically, the aqueous culture medium will include sucrose as a source of carbon. The preservation by the method according to the invention of cells of botanical species offers a method of preserving valuable genetic material for the culture of plants, vegetables, cereals and other botanical genera. The method according to the invention may we believe also be used to preserve pieces of differentiated (plant) tissue and micro-organisms.

The method according to the present invention is not limited to the preservation of cells of botanical species. It may also, for example, be used to preserve mammalian cells (generally those cells that can be preserved by conventional cryogenic methods). For example, the method according to the present invention may be employed to preserve whole blood, red blood cells or white cells or other microscopic blood bodies.

Red or white blood cells may be separated from whole blood by conventional techniques and a suitable aqueous fluid added thereto in order to prepare the aqueous phase for the emulsion. It is believed that the method according to the invention is potentially advantageous when applied to red or white blood cells as it eliminates the need to use conventional cryoprotectants such as dimethylsuphoxide.

When it is required to use the cells the emulsion containers may be removed from the storage vessel containing, for example, liquid nitrogen and the aqueous phase is thawed as rapidly as possible.

The method according to the present invention has been used to preserve cultures of soya bean cells and of yeast cells.

On returning the frozen cells to ambient temperature after a prolonged period of storage at -1 960C it has been found that a large proportion of the cells retained their viability giving positive results when FDA, Aniline Blue, cytoplasmic streaming and cell division tests were performed.

The method according to the present invention will now be further illustrated by the following Examples.

EXAMPLE 1 100 ml of a soya bean cell suspension in Miller's growth medium A (C. O. Miller, Ann. N.Y.

Acad, Sci. 144, 251(1967)) was kept in the dark at 250C under constant shaking. The cells were harvested one week after subculturing. 20 ml of the cell suspension was transferred to a sterile tube and centrifuged for 2 minutes. The tube was stored at room temperature.

40 ml of silicone oil (Dow-Corning, viscosity 1 centistoke) was sterilised by exposure to ultraviolet radiation. 2 g of sorbitan tristearate was added and the mixture was warmed gently until a clear solution resulted. The head of Polytron homogeniser was inserted into the oil and with the motor set at setting 4, the supernatant liquid from the cell suspension was added to the oil dropwise over a period of 5 minutes. The homogeniser was turned off and the wet cell pellet was stirred into the emulsion which was then further homogenised (setting 2) to disperse the cells. A sample of the emulsion was subjected to the fluorescin diacetate (FDA) test B (J. M.Widholm, Stain Technol. 47, 189(1972)) to check that the cells were viable after the emulsification treatment. 2 mg FDA was dissolved in 1 ml acetone and kept in a dark bottle. 2-3 drops were added to 5 ml sterile growth medium. One drop of this solution was added to one drop of cell suspension on a microscope slide and left for 10 minutes after which the slide was examined under the fluoresence microscope at 380 and 430 nm. A bright green fluorescence of the plasma membrane and the organelles was taken as evidence of cell viability.

3 ml cell emulsion samples were transferred to sterile plastic tubes and immersed in a thermostated bath at 50 below the ambient temperature. The samples were cooled at a rate of less than 1 OC/min to --280C. A sample was removed, thawed and tested for viability with FDA; the remaining tubes were placed in a deep freeze at -9O0C for three days, after which they were transferred to liquid nitrogen for storage.

After one week a sample was thawed by immersing the tube in tepid water for 3 minutes, and the cell viability checked with FDA immediately.

To break the emulsion and recover the cells, the contents of the tube were transferred to a sterile beaker, an equal volume of sterile medium was added and the mixture shaken until phase separation occurred. The aqueous phase was removed and centrifuged. The supernatant fluid was removed, the cells resuspended in fresh medium and a final FDA viability test and cell count performed. According to the fluorescence criterion more than 60% of the cells survived the freeze/thaw treatment. When the emulsification step was omitted but otherwise the experimental procedure was followed, no cells survived.

EXAMPLE 2 100 ml of a soya bean cell suspension in Miller's growth medium A (C. O. Miller, Ann. N.Y.

Acad, Sci. 144, 251 (1967)) was kept in the dark at 250C under constant shaking. The cells were harvested one week after subculturing. 4 ml of the cell suspension was transferred to a sterile tube and centrifuged for 2 minutes. The tube was stored at room temperature.

4 ml of silicone oil (Dow-Corning, viscosity 1 centistoke) was sterilised by exposure to ultravoilet radiation. 0.2 g of sorbitan tristearate was added and the mixture was warmed gently until a clear solution resulted. The head of a Polytron homogeniser was inserted into the oil and with the motor set at setting 3, 2 ml of supernatant liquid from the cell suspension was added to the oil dropwise over a period of 5 minutes. The homogeniser was turned off and the concentrated cell suspension after centrifuging (2 ml) was added dropwise to the emulstion which was then further homogenised (setting 23) to disperse the cells. This was completed within one minute of starting to add the concentrated cell suspension. A sample of the emulsion was subjected to a asiline blue fluorescence test to check that the cells were viable after the emulsification treatment.

Cell emulsion samples were transferred to sterile plastic tubes and immersed in a thermostated bath at 1 OO.The samples were cooled at a rate of less than 1 C/min to -280C. A sample was removed, thawed and tested for viability by an analine blue-fluorescence technique; the remaining tubes were placed in a deep freeze at -900C for three days, after which they were transferred to liquid nitrogen for storage.

After one week a sample was thawed by immersing the tube in tepid water for 3 minutes, and the cell viability checked with aniline blue immediately.

To break the emulsion and recover the cells, an excess of the growth medium was added and the homogenic was used to agitate the resulting liquid for two minutes at setting three. A final assay with aniline blue was then performed. According to the fluorescence criterion more than 60% of the cells survived the freeze/thaw treatment. When the emulsification step was omitted but otherwise the experimental procedure was followed, no cells survived.

Each test with aniline blue showed that a substantial proportion (at least 60%) of the cells were viable and in the stage of division. Each test involved adding one drop of aniline blue solution (0.005% in M/15 K2H PO4 with pH adjusted to between 8 and 10) to one drop of the cell extract.

In each case, when the cell extract was examined under a fluorescent microscope, a blue-green fluorescence was observed indicating that the cells were in the stage of division.

In a further experiment another water-in-oil suspension of soya bean cells was prepared and preserved by the method described above and under sterile conditions. After storage over a period of days at -20 degrees C in liquid nitrogen, a sample was thawed (as described above) and plated out on agar. The cell culture was observed to grow. When the emulsification stage was omitted, no cells survived.

Red blood cells have emulsified by a technique analogous to that described in Example 2 and it is believed that a substantial proportion of the cells will survive a freeze-thaw cycle analogous to that described in Figure 2. It is further believed that the ratio of aqueous to oil phase used in Example 2 should be reduced when red blood cells are being emulsified.

Claims (12)

1. A method of preserving cells (or other microscopic or small biological materials) comprising the steps of forming a water-in-oil emulsion from an aqueous suspension of the cells and a non-toxic hydrophobic liquid (the 'oil'), the cells being contained in the aqueous phase of the emulsion, reducing the temperature of the emulsion to a value at which the water within the cells is frozen, and storing the emulsion at a temperature of minus 700 or below.

2. A method according to claim 1, in which the temperature of the emulsion is reduced to a value within the range of -25 to -350C.

3. A method according to claim 1, or claim 2, in which the oil is a solvent for oxygen and carbon dioxide.

4. A method according to any one of the preceding claims, in which the oil has a viscosity in the range 1 centistoke to 20 centistokes.

5. A method according to any one of the preceding claims, in which the oil is a silicone oil or glyceride.

6. A method according to any one of the preceding claims, in which sorbitan tristearate is employed as an emulsifier in the formation of the emulsion.

7. A method according to any one of the preceding claims, in which the temperature of the emulsion is reduced to the freezing temperature at a rate of less than 1 0C per minute.

8. A method according to any one of the preceding claims, in which the cells are of a botanical species.

9. A method as claimed in claim 8, in which the said suspension is formed in an aqueous growth medium.

10. A method according to any one of the preceding claims in which soya bean cells are preserved.

11. A method according to any one of claims 1 to 8 in which whole blood or microscopic blood bodies are preserved.

12. A method according to claim 11 in which the blood bodies are red blood cells or white blood cells.

1 3. A method of preserving cells substantially as described in Example 1 or Example 2.