JP2007252270A - Solution for lyophilizing mammal-derived cell or sperm - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide such a solution for lyophilizing mammal-derived cells or sperms securing a some extent of time from sampling sperms to lyophilize these sperms when the sperms are to be lyophilized, and meet exceeding 80% blastocyst development percentage after fertilization using the thus lyophilized sperms. <P>SOLUTION: The solution for lyophilizing mammal-derived cells or sperms is provided, being obtained, based on the result of examining an EGTA[ethylene glycol bis(2-aminoethyl ether)tetraacetic acid] tris-hydrochloric acid buffer solution(50 mM NaCl, 50 mM EGTA, 10 mM Tris-HCl)(Non-Patent Literature 2), by increasing the concentration of the buffer solution(Tris-HCl) and either adding no osmotic pressure adjuster(NaCl) or decreasing its concentration. That is, this solution for lyophilizing mammal-derived cells or sperms comprises 10-100 mM of an anti-coagulant, 50-500 mM of a buffering agent and 0-10 mM of an osmotic pressure adjuster. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a solution for lyophilizing mammalian cells and sperm.

  In 1998, it was reported that pups were obtained for the first time from mammalian (mouse) lyophilized sperm. The freeze-dried solution used at this time was a CZB medium (calcium and EDTA-free) and DMEM medium (with 10% fetal calf serum) (Non-patent Document 1). Three years later, the present inventors reported EGTA Tris-HCl buffer as a lyophilization solution for stably increasing the normal rate of chromosomes in mouse lyophilized sperm (Non-patent Document 2). Since then, studies on freeze-dried sperm or dried sperm using this EGTA Tris-HCl buffer have been reported all over the world (Non-patent Documents 3 and 4), rabbits (Non-patent Document 5) and rats (Non-patent Document 6). The pups are obtained from lyophilized sperm.

Nat. Biotechnol. 16: 639-641 (1998) Proc. Natl. Acad. Sci. USA. 98: 13501-13506 (2001) Biol. Reprod. 68: 1779-1786 (2002) Biol. Reprod. 73: 627-633 (2005) Biol. Reprod. 70: 1776-1781 (2004) Zygote 13: 79-85 (2005)

However, when sperm were freeze-dried using a conventional freeze-drying solution, the blastocyst development rate after fertilization did not exceed 80%, and it was at most 70%.
In addition, when a conventional lyophilization solution is used, chromosomal abnormalities accumulate in sperm when left in suspension at room temperature (Theriogenology 62: 897-905 (2004)), so it is frozen immediately after collecting the sperm. Must dry. Therefore, there is a problem that sufficient time cannot be taken for transport of sperm and preparation of a freeze-drying apparatus between sperm collection and freeze-drying.
Therefore, when freeze-drying cells and sperm, the present invention can secure a certain amount of time between collection of cells and sperm and freeze-drying, and after fertilization using freeze-dried sperm. An object of the present invention is to provide a solution for lyophilization of mammalian cells or sperm that satisfies a blastocyst development rate exceeding 80%.

As a result of examining the EGTA Tris-HCl buffer (50 mM NaCl, 50 mM EGTA and 10 mM Tris-HCl) (Non-Patent Document 2) that has already been developed, the present inventors have increased the concentration of the buffer (Tris-HCl), And it discovered that the said subject could be solved by the improvement by not adding the osmotic pressure regulator (NaCl) or making the density | concentration low, and came to complete this invention.
That is, the present invention is a solution for lyophilizing mammalian cells or sperm comprising an anti-coagulant 10 to 100 mM, a buffer 50 to 500 mM, and an osmotic pressure regulator 0 to 10 mM.
The present invention also relates to lyophilization of mammalian sperm, comprising suspending mammalian cells or sperm in this lyophilization solution, holding the suspension at 0 ° C. to 37 ° C. for a certain period of time, and then lyophilizing. Is the method.
Further, the present invention is a fertilization method for a non-human mammal comprising fertilizing a female of the same non-human mammal using a sperm derived from the non-human mammal freeze-dried by this method, and further fertilized by this method. A method for producing a non-human mammal comprising the birth of a female non-human mammal.

The ability to secure a certain amount of time between collection of cells and sperm and freeze-drying, and the improvement of normality after freeze-dried sperm storage at room temperature, are free in the way of transporting and transporting cells and sperm. The sperm can be transported and transported more easily and safely.
Compared with the lyophilized sperm prepared using the conventional lyophilized solution, the lyophilized sperm prepared using the lyophilized solution of the present invention has a normal chromosomal rate, a blastocyst development rate and a refrigerated and room temperature storage sample. Fetal incidence is significantly improved.
The freeze-dried sperm prepared using the freeze-dried solution of the present invention has an expiration date longer than that of a conventional freeze-dried sperm prepared using a freeze-dried solution, and pups are also produced from sperm stored at room temperature for 1 year. It is estimated that there is no significant decrease in the normal fetal incidence after refrigeration for 1 year.

The composition of the lyophilization solution for mammalian cells or sperm of the present invention comprises 10-100 mM anti-coagulant, preferably 10-70 mM, 50-500 mM buffer, preferably 50-200 mM, and osmotic pressure regulator 0. -10 mM, preferably 0 mM.
The “anti-coagulant” may be an organic compound that can prevent blood coagulation and the like by binding to metal ions, and a metal divalent cation chelating agent is preferably used. For example, ethylenediaminetetraacetic acid ( EDTA), diethylenetriaminepentaacetic acid (DTPA), 1,2-diaminocyclohexanetetraacetic acid (DCTA), or ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA), preferably EGTA.
The “buffering agent” may be a solution whose pH is stable at room temperature (25 ° C.), for example, Tris-HCl, HEPES buffer, phosphate buffer, or phosphate buffered physiological salt solution (PBS), preferably Is Tris-HCl.
The “osmotic pressure adjusting agent” may be any water-soluble substance added for the purpose of adjusting the osmotic pressure of the cell preservation solution to be isotonic or hypertonic with respect to the cytoplasm, for example, a salt or a saccharide, A salt is preferred. The salt is preferably a metal salt, more preferably an alkali metal salt, more preferably an alkali metal halide, and examples thereof include sodium chloride and potassium chloride. The saccharide is preferably a monosaccharide or oligosaccharide, more preferably a monosaccharide or a disaccharide, and examples thereof include glucose, sucrose, trehalose and the like.
The lyophilization solution of the present invention may further contain an antioxidant substance such as vitamin E, dimethyl sulfoxide, catechins and the like in addition to the above components.

A cell or sperm derived from a mammal is suspended in such a freeze-drying solution, and this is retained for a certain period, and then freeze-dried.
This period is called pre-incubation, and will be clarified in the examples described later, but is an indispensable time when using the lyophilization solution of the present invention, and the holding time varies depending on the temperature. For example, in the case of 25 ° C., it is 6 hours to 7 days, preferably 1 day to 4 days, and in the case of 4 ° C., it is 2 days to 7 days, preferably 4 days to 7 days. Other temperatures can be inferred from these.
The temperature during this period is 0 ° C. to 37 ° C., preferably 4 ° C. to 25 ° C. If the temperature is higher than this range, sperm chromatin is altered and damage to the sperm DNA tends to occur. May cause mechanical damage to the sperm cell membrane and may affect the normal rate of chromosomes of sperm after lyophilization.
The conditions for lyophilization were as follows: pre-freezing time with liquid nitrogen: 1 minute, degree of vacuum: 22 × 10 ^{−3 to} 42 × 10 ⁻³ mbar, vacuum time: 4 hours to 10 hours, freezing trap temperature: −44 to − 50 ° C.

The lyophilization solution of the present invention is suitable for preservation of mammalian cells or sperm. Such mammals include humans, cows, mice, rats or rabbits.
The period that can be stored after freeze-drying is about 0 to 3650 days. After this period, 0.05-0.1 ml of water is added to the lyophilized sample to resuspend the cells or sperm. The suspended cells or sperm are sucked with an injection pipette by microscopic operation and injected into an unfertilized egg. When injecting cell nuclei other than sperm, or when sperm has lost the ability to activate the egg, the egg is artificially activated by chemical treatment or the like. Activated eggs are generated in vitro from the 2-cell stage embryo to the blastocyst and transplanted to the oviduct or uterus of a pseudopregnant individual.
When cells other than sperm are lyophilized, cells obtained by suspending cells of a target tissue or primary cultured cells derived from the tissue in the lyophilization solution of the present invention are used.

The lyophilization solution and lyophilization method of the present invention can be used when sperm cannot be obtained from a subject individual, for example, when genome storage is performed by lyophilization from a female individual or an azoospermia individual. It is also suitable for the purpose of freeze-drying other cells.

The following examples illustrate the invention but are not intended to limit the invention.

0.5M EGTA (EGTA [ethylene glycol-bis (β-aminoethyl ether) -N, N, N ', N'-tetracetic acid] (Sigma), saturated NaOH solution in water (Sigma, Water for embryo transfer) The pH was adjusted to 8.0.), Tris-HCl (Sigma, Trisma (R) -HCl, 1 M, pH 7.4), NaCl (Sigma, Sodium Chloride 5M solution) and water ( Using Sigma, Water for embryo transfer, various solutions having the compositions shown in Table 1 were prepared.
1.2 ml of the various solutions prepared as described above were collected, added to a 1.5 ml centrifuge tube, and incubated at 37 ° C. One excised piece of the epididymis tail of a 7-12 week old male mouse (B6D2F1: BDF1) was added to each tube containing the various solutions. After further incubation at 37 ° C. for 10 minutes, the sperm was swimmed up or dispersed, and 1 ml of sperm suspension was collected from the upper layer and transferred to another tube. The sperm suspension was dropped on a slide glass, and the recovered sperm was observed. The number of spermatozoa collected was evaluated in four stages: Very good (more than about 500,000 / ml), Good (some), Poor (very little), and Very poor (although almost unidentifiable).
The sperm suspension was incubated at 4 ° C. for 4-7 days and then lyophilized (FD).
Then, after storing at 4 ° C. for 1 day to 3 weeks, sperm was injected into an unfertilized mouse by an intracytoplasmic sperm injection method (ICSI).
After completion of ICSI, the eggs were transferred to m-CZB medium supplemented with mitotic inhibitor vinblastine sulfate. The next day, a chromosome sample was prepared and air-dried after Giemsa staining.
A fertilized egg having a total chromosome number of 40 and having no chromosomal structural abnormality (such as chromatid or chromosomal breakage or exchange) was defined as a fertilized egg having a normal chromosome structure. In this example, the ratio (%) of fertilized eggs having a normal chromosomal composition among fertilized eggs subjected to chromosome analysis was determined and used as the chromosome normal rate.

The results are shown in Table 1.

When EGTA was 10 mM and the concentration of Tris-HCl (pH 7.4) was 100 mM, the lyophilized sperm sample was crushed immediately after ampoule cutting and most of it disappeared. This is probably because the cells were swollen due to the low osmotic pressure of the solution, and the concentration of Tris-HCl (pH 7.4) is considered to be 50 mM or more.
When EGTA was 50 mM and the concentration of Tris-HCl (pH 7.4) was 50 to 200 mM, the chromosome normal rate of fertilized eggs injected with freeze-dried sperm showed 80% or more. Moreover, when the concentration of Tris-HCl (pH 7.4) was 500 mM, the sperm recovery ability was poor.
When EGTA was 100 mM, sperm collection ability was poor, and when the concentration of Tris-HCl (pH 7.4) was 10 mM, the normal chromosome rate of fertilized eggs was 40% or less. The reason for poor sperm recovery is probably due to the toxicity of EGTA.
When NaCl is added to 50 mM EGTA + 100 mM Tris-HCl (pH 7.4) at 10 to 50 mM, it seems that pups can be obtained sufficiently, but the chromosome normal rate of fertilized eggs is 60 to 70%, NaCl. It was low compared to when not adding. In particular, when NaCl was 50 mM, sperm collection ability was poor.

A stock stock solution (hereinafter referred to as “m-ETBS”) having the composition shown in the following table was prepared using each component used in Example 1.

Mouse (B6D2F1) sperm was suspended in m-ETBS and stored at room temperature (25 ° C) or refrigerated (4 ° C) (referred to as "preincubation"), then lyophilized, and mouse (B6D2F1) unfertilized within 2 weeks Injected into eggs. Table 3 shows the normal rate of chromosomes of fertilized eggs in the first cleavage stage.
When mouse (B6D2F1) sperm was suspended in m-ETBS and freeze-dried immediately after sperm injection, the normal chromosome rate of fertilized eggs after sperm injection showed a low value (16%). However, depending on the preincubation time, the normal rate of chromosomes in fertilized eggs increases. When pre-incubation was performed for 6 hours or more at room temperature (25 ° C) and for 2 days or more in refrigeration, freeze-drying increased the normal rate of chromosomes (4 ° C) in fertilized eggs to around 80%.
Although data are not shown here, when preincubation was performed at room temperature (25 ° C.) for 4 to 7 days, sperm after lyophilization lost egg activation ability. Therefore, after injecting sperm into an egg, it is necessary to artificially activate the egg by strontium chloride treatment. When pre-incubated in a refrigerator (4 ° C.), egg activation ability remained for 7 days or less.

  Mouse (B6D2F1) unfertilized eggs were pre-incubated with mouse-BBS prepared in Example 2 (B6D2F1) after lyophilization (4 ° C, 6 days) and refrigerated (4 ° C) for 19 days. Injected into. Of the 13 cases transplanted, the number of implantations was 13, and the number of normal fetuses 14 days after mating was 7. The appearance of the fetus is shown in FIG.

It is a figure which shows the mode of the fetus fertilized using the sperm freeze-dried using the solution for freeze-drying of this invention. (1) shows the uterus of the mouse on the 14th day of gestation into which 13 early embryos have been transplanted, and (2) shows the implanted embryo (normal embryo and resorption embryo) obtained from the uterus.

Claims (10)

A solution for lyophilizing mammalian cells or sperm, comprising an anti-coagulant 10-100 mM, a buffer 50-500 mM, and an osmotic pressure regulator 0-10 mM. The anti-coagulant is ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), 1,2-diaminocyclohexanetetraacetic acid (DCTA), or ethylenebis (oxyethylenenitrilo) tetraacetic acid (EGTA). The solution described in 1. The solution according to claim 1 or 2, wherein the buffer is Tris-HCl or a phosphate buffer solution. The solution according to any one of claims 1 to 3, wherein the osmotic pressure adjusting agent is a salt or a saccharide. The solution according to claim 4, wherein the osmotic pressure adjusting agent is sodium chloride, potassium chloride, glucose, sucrose, or a saccharide. Mammal-derived sperm, comprising suspending cells or sperm derived from mammals in the solution according to any one of claims 1 to 5, holding the cells at 0 ° C to 37 ° C for a certain period of time, and then freeze-drying them. Freeze-drying method. The method according to claim 6, wherein the holding time is 6 hours to 7 days at 25 ° C and 2 to 7 days at 4 ° C. A fertilization method for a non-human mammal comprising fertilizing a female of the same non-human mammal using sperm derived from the non-human mammal freeze-dried by the method according to claim 6 or 7. The method according to claim 8, wherein the non-human mammal is a cow, mouse, rat or rabbit. A method for producing a non-human mammal comprising giving birth to a female non-human mammal fertilized by the method according to claim 8 or 9.