DE3148551C2 - - Google Patents

Description

The invention relates to the preservation method specified in claim 1 of cells or other microscopic or small biological materials. Claims 2 to 3 relate to configurations of this method.

It is known that at temperatures of less than about -70 ° C most chemical reactions come to a standstill. Man has therefore long known that it is theoretically possible to organic materials over long periods of time at temperatures of -70 ° C or lower to preserve. However, it was found that by exposing the cells to a temperature cycle, in which the water contained in them frozen and is then melted, e.g. B. the cell membranes are damaged, so that the cells die.

It is also a procedure known (Chemical Abstracts Vol. 73, 1970, 74931), after which tumor cells quickly in glycerin and in silicone oil were frozen. It was found that when freezing bio logical materials in the presence of such substances that are known as cryoprotectants, later  Thawing the risk of cell damage or cell defrost can be reduced. Unuse such a low Temperature protection agents have been different classes of bio logical substances successfully clinically preserved, e.g. B. blood, Spermatozoa, cornea (of the eye) and skin.  

In general there are two known types of low temperature anti-aging agents:

First, these are substances that be used conventionally, e.g. B. dimethyl sulfoxide and glycerin, which have to penetrate the cell membrane, to take effect. Furthermore, it is generally assumed that such cryoprotectants help reduce cell dehydration by acting as Water set act. Furthermore, they reduce the during the Amount of ice formed.

The second type of low temperature Anti-saturants do not penetrate the cell membrane, and less is known about how they work; they were previously only used experimentally. However, you can in lower concentrations than the cell membrane urgent cryoprotectants are used. It was found that red blood cells after they in the presence of a non-penetrating low temperature preservatives had been frozen and thawed, significant changes in their internal electrolyte concentration shows. It was believed that such low temperature protection agents the permeability of Increase cell membranes (like this also with dimethyl sulfoxide and glycerin is the case) and thus the production an osmotic equilibrium state during Ge Freeze faster.

The choice between various suitable low temperatures The clinical storage of e.g. B. basically allows blood cells, the use of these funds is by no means completely satisfactory. First, there is a tendency for the occurrence cell damage is not eliminated, but only is reduced. Second, substances like dimethyl sulfoxide (which is used for the storage of blood will), very thoroughly z. B. removed from the blood (what  by dialysis, washing, centrifuging, etc.). you are expensive and have an unpleasant smell; you can too induce genetic mutation.

The object of the invention is an improved con serve cells with simple means at risk cell damage from storage even at very low temperatures temperatures is further reduced.

The method according to the invention for preserving cells or other microscopic or small biological Materials is characterized in claim 1. The watery Flushing of the cells with the "oil" does not form toxic hydrophobic liquid a water-in-oil (W / O) emulsion. This will change the temperature at which the water in the cells freezes to a low level. The emulsion is stored at a temperature of -70 ° C or lower maintains. With this method it is possible to use single cells or Preserve cell clusters.

This method is believed to be the most satisfactory Preservation of at least some types of cells without the use a conventional low-temperature protection agent. Since the non-toxic, hydrophobic liquid, the cells even not contacted significantly, as the latter in droplets of aqueous nutrient medium are suspended, this does not work toxic hydrophobic liquid also not as low temperature protective means in the conventional sense. In the procedure after In the invention, the emulsion typically contains a variety of small drops or droplets of the aqueous nutrient medium. Typically the droplets are of such a size that the aqueous phase a thin aqueous film around the cell or forms the cells (the cells can be in the form of  Grapes instead of in the form of single or double cells depending on the type of species to be preserved). Such Droplets tend to become hypothermic when the Temperature of the emulsion is reduced to just below 0 ° C; they only freeze at a level well below zero Temperature, e.g. B. at -25 ° or -35 ° C. It is believed that this slower freezing at such lower temperatures leads to less damage than freezing at 0 ° V.

The aqueous nutrient medium can e.g. B. sucrose as carbon source included. While it has been reported that sucrose, if used in sufficient quantities, a Low temperature protection agent is. The percentage of sucrose in a conventional nutrient medium is not so high that the sucrose has some significant low-temperature May have protective effect, and the invention is therefore not based on the use of sucrose in the nutrient medium.

If possible, the emulsion can be formed so that the Agglomeration of large cell clusters is avoided. This is however not always possible, since many cells in cultures be grown in grapes. If desired, can the aqueous suspension of cells are filtered to Grapes or large grapes (i.e. grapes with more than ten Cells) from cells.

The hydrophobic liquid (or oil) is preferably a Solvent for oxygen and carbon dioxide. Suitable Liquids for the procedure are silicone oil and glycerides.  

The viscosity of this "oil" is not for the process critical, but is preferably between 1 mm² / s and 20 mm² / s.

Typically, an emulsifier must be present in the emulsion It will be a low HLB emulsifier that is soluble in the oil. For example, the emulsifier Be sorbitan tristearate. When preparing the emulsion the emulsifier is added to the oil and the resultant Liquid is mixed with an aqueous in a homogenizer Suspension of cells (typically in their nutrient medium), that have to be preserved, mixed. It is careful make sure that the cells are not destroyed.

The relative proportions of the oil and the aqueous phase in the Emulsions are not critical. Typically 2 parts by volume of oil to one part by volume of aqueous phase used.

The rate of temperature decrease of the emulsion from + 5 ° C to the freezing temperature is typically less than 1 ° C / min. A cooling device, that reaches the required freezing rate and temperature, be used. Typically the emulsion is in one or more suitable containers in the freezer device introduced. After the aqueous medium and Cell plasma can be made in the cells in the container the emulsion container is transferred to a storage container be in a temperature of -70 ° C or lower prevails. Typically this container contains liquid Nitrogen with a temperature of -196 ° C.

The specified method is particularly suitable for Freeze the cells of plants or other botanical Genera. The cells to be preserved can be in one  conventional aqueous culture medium with conventional Methods are bred. Typically that contains aqueous nutrient medium sucrose as a carbon source. The conservation of cells of botanical genera with the specified procedure offers a possibility for Preservation of valuable genetic material for the Growing plants, vegetables, cereals and others botanical genera. It is believed that this specified method also for the preservation of pieces of differentiated (vegetable) tissue and of micro organisms.

The procedure is not based on cell preservation botanical genres limited. It can e.g. B. also for the conservation of mammalian cells can be used (These are generally cells that can be preserved by conventional low-temperature processes are). For example, the procedure for preservation of whole blood, red or white blood cells or others microscopic blood cells are applied. Red or White blood cells can be obtained from conventional whole blood Methods are separated, after which they can be used with a suitable aqueous liquid are added to the aqueous phase prepare for the emulsion. It is believed that specified method is very advantageous if it is in Applied with red or white blood cells then because of the need for conventional lows temperature protection agents such as dimethyl sulfoxide are not required.

When the cells are to be used again, the Emulsion container from the storage container, the z. B. liquid Contains nitrogen, removed, and the aqueous phase thawed as soon as possible.  

The specified procedure was for the preservation of Cultures of soybean cells and yeasts are applied.

When the freezer cells are stored at -196 ° C for a long time were brought back to ambient temperature found that the earning power of a large part of the cells had been preserved so that positive results were obtained were performing fluorescent diacetate, Aniline blue, cytoplasmic flow and cell division Testing.

The following examples serve to illustrate the invention.

Example 1

100 ml soybean cell suspension in Miller's nutrient medium A (C.O. Miller, Ann. N.Y. Acad, Sci 144, 251 (1967)) kept at 25 ° C in the dark with constant shaking. The cells were harvested one week after inoculation. 20 ml of the cell suspension was placed in a sterile Glass is placed and centrifuged for 2 min. The glass was kept at room temperature.

40 ml of silicone oil (Dow-Corning, viscosity 1 mm² / s) were sterilized by exposure to UV rays. 2 g sorbitan tristearate was added and the mixture became slow warmed until a clear solution results. The head of one Polytron homogenizer was placed in the oil, and at engine setting 4 the excess liquid became drop of oil from the cell Added 5 min. The homogenizer was turned off, and the wet cell pellet was stirred into the emulsion which was then further homogenized (engine setting 2) to disperse the cells. A sample of the emulsion was made  the fluorescein diacetate test (FDA test) B (J.M. Widholm, Stain Technol. 47, 189/1972)) to check that the cells yield after the emulsification treatment were capable. 2 mg FDA was dissolved in 1 ml acetone and in kept in a dark bottle. 2-3 drops were made 5 ml of steep nutrient medium was added. A drop of this Solution was dropped into a drop of cell suspension Microscope slide added and left for 10 min, after which the slide under the fluorescence microscope was examined at 380 and 430 nm. A light green Fluorescence of the phase membrane and the organelles was observed regarded as proof of the profitability of the cells.

3 ml cell emulsion samples were placed in sterile plastic tubes spent and in a thermostatically controlled bath immersed, the temperature of which is 5 ° C lower than that Ambient temperature was. The samples were with a Ge speed reduced from less than 1 ° C / min to -28 ° C cools. A sample was taken, thawed and with FDA examined for profitability; the remaining tubes were placed in a freezer at -90 ° C for three days, after which they are stored in liquid nitrogen was brought.

After a week a sample was made by immersing the Tube thawed in lukewarm water for 3 min, and the Cellular yield was immediately examined with the FDA.

To break the emulsion and recover the cells the contents of the tube were placed in a sterile beaker spent an equal volume of sterile medium added and the mixture was shaken until the Phase separation took place. The aqueous phase was removed and centrifuged. The supernatant liquid was removed  the cells were re-opened in fresh medium washed up, and a recent FDA profitability study and the cells were counted. To the fluorescence criterion survived more than 60% of the Cells freeze / thaw treatment. If the Emulsification step was omitted and the other Course of the experiment was the same, none survived of the cells.

Example 2

100 ml soybean cell slurry in miller Culture medium A (C.O. Miller, Ann. N.Y. Acad, Sci. 144, 251 (1967)) were at 25 ° C with constant shaking kept in the dark. The cells became one week after harvested from vaccination. 4 ml of the cell suspension were placed in a sterile tube and centrifuged for 2 min fugated. The tube was stored at room temperature.

4 ml of silicone oil (Dow-Corning, viscosity 1 mm² / s) were sterilized by exposure to UV radiation. 0.2 g Sorbitan tristearate was added and the mixture was warmed slowly until a clear solution was obtained. The head of a Polytron homogenizer was placed in the oil immersed, and with engine setting 3, 2 ml of the supernatant liquid from the cell suspension added dropwise to the oil over 5 minutes. The homo genizer was turned off and after centrifugation the concentrated cell suspension (2 ml) added dropwise to the emulsion, which then continues was homogenized (motor setting 2.5) to the cells to disperse. This was done within one Minute after the start of adding the concentrated  Flooding of cells ended. A sample of the emulsion was made subjected to an aniline blue fluorescence test to determine deliver the cells after the emulsification treatment were profitable.

Samples of the cell emulsion were placed in sterile plastic transferred to a tube and placed in a thermostatically controlled bath submerged at 10 ° C. The samples were taken at a speed cooled from less than 1 ° C / min to -28 ° C. A sample was taken, thawed and found to be profitable examined with an aniline blue fluorescence method; the remaining tubes were placed in a freezer for 3 days spent at -90 ° C, after which they are in liquid nitrogen were stored.

After a week, a sample was made by immersing the tube chens in lukewarm water for 3 min, and the Cell yield was immediately examined with aniline blue.

To break the emulsion and recover the cells an excess of nutrient medium was added and the homogeni sator was used to stir the resulting liquid used in position 3 for 2 min. An end sub Search with aniline blue was then carried out. According to the fluorescence criterion survived more than 60% of the Cells the freeze / thaw treatment. If the emulgy omitted and the rest of the experiment as described, none of the survived Cells.

Every attempt with aniline blue showed that a considerable Proportion (at least 60%) of the cells profitable and was in the division phase. With every attempt, a Drop of aniline blue solution (0.005% in M / 15 K₂H PO₄, where  the pH is adjusted to a value between 8 and 10 was) added to a drop of cell extract. If the Cell extract under a fluorescence microscope in each case there was a blue-green fluorescence observed what was a sign that the Cells were in the state of cell division.

In another attempt, there was another flood made from soybean cells and with the specified Procedure preserved under sterile conditions. To Storage for several days at -20 ° C in liquid stick a sample was thawed as explained and distributed on agar. Cell culture growth was observed takes care. If the emulsification step has been omitted, none of the cells survived.

Red blood cells were analyzed using a method similar to that emulsified according to example 2, and it is believed that a significant proportion of blood cells have a Freeze / thaw cycle according to example 2 survived. It is also believed that red emulsification Blood cells the ratio of the aqueous phase to Oil phase, as used in Example 2, reduced should be.

Claims (3)

1. A method for preserving cells or other microscopic or small biological materials, characterized in that
that a water-in-oil emulsion is formed from an aqueous suspension of the cells and a non-toxic hydrophobic liquid (the "oil") using an emulsifier, the cells being contained in the aqueous phase of the emulsion,
that the temperature of the emulsion is reduced to a value in the range of -25 to -35 ° C at which the water in the cells freezes, and
that the emulsion is stored at a temperature of -70 ° C or lower. 2. The method according to claim 1, characterized, that the temperature of the emulsion at a rate reduced to below freezing temperature by less than 1 ° C / min becomes.   3. The method according to claim 1 or 2, characterized, that an emulsion with a ratio of 2 parts by volume Oil is made into a volume of aqueous phase.