WO2002009514A1

WO2002009514A1 - Method of preserving cells and uses thereof

Info

Publication number: WO2002009514A1
Application number: PCT/AU2001/000921
Authority: WO
Inventors: Jillian Marianne Shaw; Mulyoto Pangestu
Original assignee: Monash University
Priority date: 2000-07-28
Filing date: 2001-07-27
Publication date: 2002-02-07
Also published as: AUPQ906300A0

Abstract

The present invention relates to a method of preserving and storing cells and the cells prepared by the method. The invention also relates to the use of the preserved cells. In a first aspect of the present invention, there is provided a method of preserving cells in a cell sample, said method comprising: evaporatively drying a cell sample in the presence of an inert gas to substantially dry the cell sample; and storing the substantially dried cell sample in an environment that is substantially oxygen depleted.

Description

METHOD OF PRESERVING CELLS AND USES THEREOF

The present invention relates to a method of preserving and storing cells and the cells prepared by the method. The invention also relates to the use of the preserved cells.

INTRODUCTION

Strategies which allow cells or their genetic material to be recovered in a functional state after short, medium or long term storage are important in many areas of science, agriculture and medicine. For cells or tissues this is currently most commonly achieved by cooling the material to low sub zero temperatures and maintaining them at such temperatures until further use. While the current protocols allow cells to be recovered after an indefinite period of time, low temperature storage is neither cheap nor convenient.

Biological material is most commonly suspended in solutions especially formulated for the mode of storage. For instance, for freezing, a suitable cryoprotectant formulation is added to the cell suspension so that when the cells are cooled to the desired storage temperature and then returned to physiologic temperatures when needed, they retain their functionality. Additionally, selection of the appropriate protocol for supporting the biological material during the storage process can be a hit and miss affair and may require a precise combination of cryoprotectant type and concentration, additives such as sugars and, cooling and warming rates. This will be dependent upon the cells being stored and the conditions under which they are stored.

Drying is an alternative storage method. Dried mammalian sperm can fertilise oocytes. However, the drying methods used included the use of ethanol, methanol, acetone, chloroform methanol and freeze drying. Whilst sperm reconstituted after freeze drying has shown the ability to give rise to young following intracytoplasmic injection (ICSI) into oocytes, this procedure to preserve the cells relies heavily on costly equipment and gas supplies and is often not suitable for routine use in cell storage.

Accordingly, it is an object of the present invention to provide a method of preserving cells which is simple and economical and is generally not reliant on costly equipment.

SUMMARY OF THE INVENTION

In a first aspect of the present invention, there is provided a method of preserving cells in a cell sample, said method comprising: evaporatively drying a cell sample in the presence of an inert gas to provide a substantially dry cell sample; and storing the substantially dried cell sample in an environment that is substantially oxygen depleted.

In another aspect of the present invention there is provided a preserved cell sample prepared by the methods described above.

In yet another aspect of the present invention, there is provided a method of reviving a cell sample, said method comprising: obtaining a preserved cell sample prepared by the methods described above; rehydrating the cell sample in a suitable medium compatible to the cell sample; and optionally culturing the cell sample.

Accordingly, in another aspect of the present invention, there is provided a rehydrated cell sample preferably one which has been rehydrated from a preserved cell sample of the present invention.

In another aspect of the present invention, there is provided a method of fertilising an oocyte or egg, said method comprising: obtaining an oocyte or egg; obtaining a preserved cell sample prepared by the process described above; rehydrating the cell sample in a suitable medium compatible to the cell sample; and introducing a cell from the cell sample into the oocyte or egg to fertilise the oocyte or egg.

In yet another aspect of the present invention, there is provided a fertilised oocyte or egg prepared by injection of a rehydrated cell sample or a rehydrated sperm into an oocyte or egg. The sperm, oocyte or egg may be the sperm, oocyte or egg from a plant or animal or the equivalent cells from any species.

Accordingly, in another aspect of the present invention, there is provided a method of producing an offspring, said method comprising: obtaining an oocyte or egg; obtaining a preserved cell sample prepared by the process described above; rehydrating the cell sample in a suitable medium compatible to the cell sample; introducing a cell from the cell sample into the oocyte or egg to fertilise the oocyte or egg to create an embryo; and allowing the embryo to develop to the offspring.

In another aspect of the present invention, there is provided an offspring prepared by the processes described above.

FIGURES

Figure 1A shows a plastic straw containing a small sample of fluid containing viable cells (arrow).

Figure 1 B shows how this sample is aspirated into the straw to spread its contents thinly along the straw walls. Figure 1 C shows the sample is then dried by letting inert gas flow through the straw (dark arrows).

Figure 1 D shows the straw is sealed (preferably both ends) once the sample is deemed dry and then stored in an oxygen free dry atmosphere.

Figure 2A shows the alternative to using a straw by drying cells onto a surface. This figure shows a dish containing a small sample of fluid containing viable cells.

Figure 2B shows the sample is then dried by letting inert gas flow across the dish/ surface

DESCRIPTION OF THE INVENTION

A cell sample may comprise a cell suspension, clumped or aggregated cells or even a tissue sample. A tissue sample should be of a small size capable of being evaporatively dried. Clumped cells may come in the form of cell aggregates possibly resulting from inadequate cell suspension. Clumped cells may also come in the form of monolayers or cell colonies grown on a tissue culture plate or any surface that can support cell growth. Additionally, the cell sample may come as unattached monolayers of cells which have been detached from a cell surface.

A cell suspension is mostly preferred when the cells are individually separated from each other. The cell sample may be of any origin including plant, animal or bacterial. Preferably the cells are animal cells which may include sperm. However, sperm may also derive from humans or plants.

The cell samples may comprise cells suspended in their normal media used for growing or suspending the cells. However, other additions selected from the group including ions, carbohydrates, amino acids, antioxidants, free radical scavengers and cryoprotectants and/or lyoprotectants such as trehalose and raffinose may be added. However, it is preferred that the solution is isotonic with the cells to be preserved. The concentrations of the components may contribute to the overall osmolarity of around but not limited to 270 - 330 mOsmol.

Where the cell is a sperm, the sperm may be suspended and dried in any medium known to the skilled addressee which is suitable for sperm, but preferably it is dried in a composition comparable to that of the intracellular environment of the sperm.

Evaporative drying is used to dry the cells. Unlike freeze drying, the cells are not subjected to low temperatures where crystal formation may result. Evaporative drying simply removes moisture from the cells and may be performed at room temperature. This is achieved in the presence of an inert gas generally by passing a stream of gas over the cell sample. Preferably a high gas flow rate is applied. Rates of gas flow in the order of one litre per minute may be used. However this is merely illustrative. Any rate may be used to achieve removal of moisture from the cells.

Evaporative drying is best achieved from a small volume. This may be achieved from any surface on which the cell sample is applied. Conveniently, as shown in Figure 1 , the cell sample is dried in a straw for subsequent rehydration and delivery (Figures 1A). The contents may be aspirated into the straw and its contents spread thinly along the straw walls (Figures 1B). The sample may be dried by passing inert gas through the straw (Figure 1C). However, the invention is not limited to this example and the cell sample may be dried directly from the culture dish or cover slip to which the sample is applied. This is particularly useful for monolayers of cells. The sample should be kept small. For example, a sample of approximately 10 micro litres would be ideal.

The inert gas may be any inert gas known to the skilled addressee. Preferably, the gas is nitrogen. Oxygen should be avoided. However, since the sample will be stored in an oxygen depleted environment, oxygen exposure should be limited.

The samples are "substantially" dried. This means that most of the moisture is removed.

The term "substantially dry" means that at least 95% of the moisture is lost, preferably 97% of the moisture is lost. However it would be understood that 100% of the moisture is unlikely to be removed. In fact, it may be best that at least 2% of the moisture remains.

In order for the dried cell sample to be stored, the sample must be stored in an oxygen free environment. This may be achieved by any means available to the addressee.

The dried samples may be protected from atmospheric oxygen and moisture by any method available to the addressee but preferably they may be protected by placing them in a bag or chamber filled with dry inert gas. The bag or chamber is preferably impervious to both oxygen and moisture, or may contain oxygen and moisture scavengers, or be placed within a dry, oxygen free environment. The specimens may be stored at a range of temperatures including but not limited to room temperature and temperatures below 0°C.

However, in a preferred embodiment of the present invention, dried samples in straws may be prepared for storage by sealing the straw containing the cell sample while nitrogen gas is flowing through it (Figure 1 D), and then placing the straw in a storage container such as but not limited to an oxygen impermeant bag such as 4 ply or greater than or equal to 15μm aluminium foil bags. Preferably, the storage container is then flushed with dry inert gas (e.g. nitrogen) to remove the oxygen and then sealed to retain the dry inert gas.

Alternatively, the sample may be dried on a surface such as a tissue culture surface (Figures 2A). A small sample of the cells, tissue or a monolayer of cells may be placed on the surface and dried by letting inert gas flow across the dish or surface (Figure 2B).

In another aspect of the present invention there is provided a preserved cell sample prepared by the methods described above. Preferably, the cell sample is a sperm sample.

This process is particularly useful where a cell sample which has previously been used is lost, or contaminated and a new source of that same cell sample is required. Effective preservation and storage of the same cell sample would allow for continued cell and sample propagation. Also, sperm samples may be stored for future use and rehydrated for further fertilization of oocytes.

The cells may be rehydrated by any solutions or media deemed appropriate by the addressee, including but not limited to water, saline, tissue culture media, buffered physiological solutions, solutions in which the ionic composition mimics that of the intracellular environment (high potassium, low sodium, low calcium) or other compounds and media which are compatible with cell survival or which can be injected into cells, including but not limited to antioxidants such as vitamin E (alpha tocopherol). The solutions may also include cryo and lyoprotectants.

Where the cells are sperm, the sperm may be rehydrated in any medium known to the addressee but in a preferred embodiment of the present invention, the sperm is rehydrated in a high potassium and low sodium solution. Concentrations of potassium and sodium may be in the order of 120mM potassium and 20mM sodium. After thawing of the sperm sample, the sperm may be used to fertilise an egg, preferably using intracytoplasmic sperm injection. However, any suitable method known to the addressee may be used. This may be achieved by collecting the sperm or cell singly into a micropipette and injecting it through the membrane of the egg.

Cells other than sperm can be cultured after re-hydration under standard culture conditions.

Accordingly, in another aspect of the present invention, there is provided a rehydrated cell sample preferably one which has been rehydrated from a preserved cell sample of the present invention. Preferably the rehydrated cell sample is a sperm sample.

In yet another aspect of the present invention, there is provided a fertilised oocyte or egg prepared by injection of a rehydrated cell sample or a rehydrated sperm into an oocyte or egg. The sperm, oocyte or egg as used herein may be the sperm, oocyte or egg from a plant or animal or the equivalent cells from any species.

The cell sample may be a somatic cell line or sperm which has undergone genetic manipulation either before or after drying. It is also possible the oocyte or egg has been modified either by enucleation or genetically modified for the production of a transgenic animal or plant

The offspring may be any descendent resulting from the fertilization of an oocyte or egg of a plant or animal. Hence the offspring may be a plant or animal offspring.

The oocyte or egg may be obtained from any species including but not limited to the group including human, bovine, ovine, porcine, caprine, murine, and feline. It may be genetically modified as described above. In the case of producing a plant, the egg may be obtained from any plant particularly a flowering plant.

The cell sample may be a cell or a sperm or a somatic cell capable of fertilising an oocyte or egg. However, the cell sample will be a sample as prepared by rehydrating a preserved cell sample.

Methods of introducing the cell into the oocyte or egg may include but is not limited to, injecting the cell or sperm by intracytoplasmic sperm injection, microinjection or micromanipulation, or any in vitro fertilization methods providing the oocyte or egg becomes fertilised by the cell sample. However, any methods available to the skilled addressee for introducing a sperm or cell into an oocyte or egg can be used.

Allowing the embryo to develop to the offspring may involve transfer of the embryo to a receptive animal. The receptive animal is an animal capable of carrying a foetus to term and may be a female animal in a breeding cycle or artificially induced to accept an embryo and to carry the foetus to term. By "artificially induced" it is meant that pharmaceutical grade synthetic hormones such as follicle stimulating hormone (FSH) in conjunction with luteinizing hormone (LH), using prescribed stimulation protocols for a given species, be injected in to the animal to prepare the womb for receiving the blastocyst.

In another aspect of the present invention, there is provided an animal or plant or any species prepared using cells previously stored by the processes described above.

The procedures described herein are designed to allow the long term storage of cells. The cells may be any cell derived from any species, plant or animal. The procedures have the advantage over previous methods of being simple, cost effective and not requiring the expensive equipment associated with freezing approaches to ceil storage. The procedure described allows cells to be stored and transported at ambient temperature (or below).

Once the cells are dried and stored they may be rehydrated at the convenience of the user for any method which utilizes cells. For instance, the production of plants and animals is a suggested use of the process described herein. However, the present invention is not limited to this use. The injection of cells into blastocysts to produce chimeras or the isolation of DNA from stored cells are other such suggested uses of the stored cells. Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

Examples of the procedures used in the present invention will now be further described. It should be understood, however, that the following description is illustrative only and should not be taken in any way as a restriction on the generality of the invention described above.

Examples

Example 1 : Viable sperm following the use of a novel method for drying and storing cells.

(a) Source of material: Sperm and oocytes were collected from C57BL/6J x CBA mice. The females were superovulated with PMSG and hCG using standard protocols (Hogan et al,1986) with oocytes collected 13 h after the hCG injection. Sperm were collected by removing the cauda from mature male mice. These cauda were minced into a modified intracellular medium (mHBS Eroglu et al, 2000) for 15 minutes at room temperature (RT) to allow sperm to swim into the solution.A 10μl drop of the sperm suspension was then aspirated into a 250μl plastic insemination straw (IMV, France). The drop was aspirated up the straw to form a thin layer along the inner wall of the straw. To dry this layer a steady flow of dry nitrogen gas was then blown through the straw (approx flow rate of 1 liter/min) for five minutes to dry the specimen.

In the earliest experiments the 0.25 μl straws were dried while being held within a surrounding outer 500μl straw (also gassed with nitrogen). The two straws were heat sealed while still being gassed to trap the nitrogen inside the straws. This straw-in - straw pair were then wrapped in aluminum foil and stored at room temperature in a modular incubator filled with dry nitrogen gas.

In later experiments the 0.25 μl straws were dried with dry nitrogen gas, and heat sealed while still being gassed. The straws were then inserted into A6 size heat sealable foil bags of the type currently used for submarine embryo culture. The oxygen permeability of the bags used in this study was 0.02cc/m²/24 h, which is adequate for short term storage. For longer-term storage bags with the lowest possible oxygen permeability rating should be used. The foil bags were heat sealed and dry nitrogen introduced by piercing one corner of the bag with a hypodermic needle attached to a dry nitrogen gas supply. The bag was repeatedly (2 or more times) filled and then evacuated (by manually squashing the bag), before removing the needle and sealing the bag so that the puncture was isolated from the gas filled portion of the bag.

(b) Utilization of the stored sperm: The sperm were prepared for microinjection by removing a straw from storage (the modular chamber or a foil bag) removing the heat sealed ends of the straw and then gently flushing the straw with 300μl mHBS, to expel the sperm. Single sperm were transferred in to the cytoplasm of C57xCBA F1 Mil oocytes by ICSI procedures using two micromanipulators and an inverted compound microscope. The oocytes were held in M2 media for the duration of the injection. Those which survived the injection were cultured in KSOM (Specialty Media, Cat. MR-041-D) at 37°C in humidified 5%CO₂. The viability of the resulting embryos was assessed by culturing the embryos for up to 72 hours in vitro, or by transferring the embryos to day 1 pseudopregnant recipients.

Mouse sperm dried under and stored in a dry nitrogen atmosphere have been stored at room temperature for 1 month or more before being injected into mouse oocytes. A proportion of these develop into blastocysts in culture, and fetuses and live young after transfer to pseudopregnant recipient mice.

A total of 344 oocytes have been injected with evaporative dried sperm. As a control 75 oocytes were injected with control fresh sperm. Following ICSI with control (fresh) sperm 37% of injected oocytes survived the injection; 18% formed 2PN, and 8% formed blastocysts. The corresponding results following the use of dried sperm was 39%, 29% and 3% respectively.

In separate experiments with sperm stored for 1 month or more, in vivo development was shown. In the first test 6 embryos derived from oocytes injected with fresh sperm were transferred to one uterine horn of a day 1 pseudopregnant recipient, and 6 embryos derived from oocytes injected with dry-stored sperm were transferred to the opposite uterine horn. On the 15th day of gestation the control embryos had developed into 3 fetuses and 3 resorptions, while the embryos derived from dried sperm had developed into 4 fetuses and 2 resorptions. All fetuses were morphologically normal and of normal weight.

A further 10 embryos derived solely from dried sperm were transferred to both uterine horns of a pseudopregnant recipient. She gave birth to 5 pups. These are now 3 weeks old and have exhibited normal development to date.

Finally, it is to be understood that various other modifications and/or alterations may be made without departing from the spirit of the present invention as outlined herein.

Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method of preserving cells in a cell sample, said method comprising: evaporatively drying a cell sample in the presence of an inert gas to provide a substantially dry cell sample; and storing the substantially dried cell sample in an environment that is substantially oxygen depleted.

2. A method according to claim 1 wherein the cell sample is selected from the group including a cell suspension, tissue sample, monolayer or cell colony from tissue culture.

3. A method according to claim 1 or 2 wherein the cell sample is a sperm sample.

4. A method according to any one of claims 1 to 3 wherein the evaporative drying is conducted at room temperature.

5. A method according to any one of claims 1 to 4 wherein the evaporative drying is conducted by passing a stream of inert gas over the cell sample.

6. A method according to any one of claims 1 to 5 wherein the inert gas is nitrogen.

7. A method according to any one of claims 1 to 6 wherein the sample is at least 95% dry.

8. A method according to any one of claims 1 to 7 wherein the sample is at least 97% dry.

9. A preserved cell sample prepared by a method according to any one of claims 1 to 8.

10. A preserved cell sample according to claim 3 wherein the cell sample is sperm.

11. A method of reviving a cell sample, said method comprising: obtaining a preserved cell sample according to claim 9 or 10; and rehydrating the cell sample in a suitable medium compatible to the cell sample.

12. A method according to claim 11 wherein the cell sample is rehydrated in a solution or media selected from the group including water, saline, tissue culture media, buffered physiological solutions and ionic compositions compatible with intracellular environments of the cell.

13. A method according to claim 11 or 12 wherein the cell is rehydrated in the presence of high potassium and low sodium.

14. A method according to claim 13 wherein the solution includes 120mM potassium and 20mM sodium.

15. A method according to any one of claims 11 to 14 wherein the cell sample is a sperm sample.

16. A rehydrated cell sample prepared by the method according to any one of claims 11 to 15.

17. A rehydrated cell sample according to claim 16 wherein the cell sample is sperm.

18. A method of fertilising an oocyte or egg, said method comprising: obtaining an oocyte or egg; obtaining a preserved cell sample prepared by the method according to any one of claims 1 to 8; rehydrating the cell sample in a suitable medium compatible to the cell sample; and introducing a cell from the cell sample into the oocyte or egg to fertilise the oocyte or egg.

19. A method according to claim 18 wherein the cell line has been genetically modified.

20. A method according to claim 18 or 19 wherein the cell sample is a sperm or a somatic cell.

21. A method according to any one of claims 18 to 20 wherein the cell is introduced into the oocyte or egg by a process selected from the group including injecting the cell or sperm by intracytoplasmic sperm injection, microinjection or micromanipulation.

22. A fertilised oocyte or egg prepared by the method according to any one of claims 18 to 21.

23. A method of producing an offspring, said method comprising: obtaining an oocyte or egg; obtaining a preserved cell sample prepared by the method according to any one of claims 1 to 8; rehydrating the cell sample in a suitable medium compatible to the cell sample; introducing a cell from the cell sample into the oocyte or egg to fertilise the oocyte or egg; and allowing the embryo to develop to the offspring.

24. A method according to claim 23 further including transferring the embryo to a receptive animal.

25. A method according to claim 24 wherein the receptive animal has been artificially induced.

26. An offspring prepared by the method according to any one of claims 23 to 25.

27. A method according to claim 1 substantially as hereinbefore described with reference to the example.