JP2010094076A - Method for freezing and preserving mammal tissue, nucleus transplantation embryo of non-human mammal and cloned non-human mammal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for freezing and preserving a mammal tissue, which can simply preserve the mammal tissue at a low cost in order to temporarily preserve the mammal tissue, and the like. <P>SOLUTION: The method for freezing and preserving the mammal tissue include: a picking process for picking the tissue from a mammal and a freezing process for freezing the picked tissue at a temperature of ≤-60°C without performing a cell-dispersing treatment and a cell culture and without using an anti-freezing agent. The nucleus transplantation embryo of a non-human mammal uses a non-human cell contained in the tissue frozen and preserved by the method for freezing and preserving the mammal tissue, as a donor cell. In addition, the cloned non-human is produced from the nucleus transplantation embryo of the non-human mammal. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for cryopreserving mammalian tissue, a nuclear transfer embryo of a non-human mammal using cells contained in the cryopreserved mammalian tissue as donor cells, and a non-transplanted embryo produced from the nuclear transfer embryo. It relates to human mammals.

  Included in somatic tissues such as muscle tissue and adipose tissue that can be used for animal replication by somatic cell clones as well as germ cells of individuals with excellent traits to protect useful genetic resources The stored cells are also cryopreserved. Moreover, the cells contained in the cryopreserved somatic tissue have already been used as donor cells when producing somatic cell cloned animals (see Patent Documents 1 and 2).

  In the conventional somatic cell cryopreservation method, for example, (1) a tissue is collected from an individual having an excellent trait, and (2) the collected tissue is minced and digested with trypsin to obtain cells in a culture solution. In general, (3) the cell suspension is first cultured in an appropriate environment, (4) the cells are subcultured in the second and third generations, and (5) the cells are cryopreserved. is there.

  (5) When cells are cryopreserved, if ice crystals are formed in the cells, the cells are killed. Therefore, the collected tissue is not frozen as it is, and no ice crystals are formed in the cells. As described above, various ideas such as vitrification preservation method by rapid freezing and slow cooling method have been studied (see Non-Patent Document 1).

  Here, the vitrification preservation method means that the grown cells are suspended in a cryoprotectant containing a high concentration of glycerol and then rapidly frozen with liquid nitrogen, so that ice crystals do not form inside or outside the cells. This is a method of freezing in a vitrified state. The slow cooling method is a method in which proliferated cells are suspended in a cryoprotectant containing glycerol or the like at a low concentration, and then frozen by gradually lowering the temperature with a program freezer or the like.

  However, these cell cryopreservation methods have the following problems. First, cells must be proliferated before storing the tissue, and the proliferation of cells required a great deal of cost and effort. In order to freeze the proliferated cells, special operations such as rapid cooling with liquid nitrogen, a program freezer, and special equipment were required.

  In order to solve such problems, a method of freezing the collected tissue without suspending it in the vitrification solution without culturing cells, or suspending the collected tissue in a vitrification solution having a special composition A method of cryopreserving after the treatment is also being studied. However, the method that does not use the vitrification liquid has not been confirmed to be realistic. In addition, the method of using the vitrification solution requires operations such as tissue cutting, washing, preparation of the vitrification solution, quick freezing with liquid nitrogen, removal of the vitrification solution after thawing, etc. Required a certain level of proficiency (see Patent Document 3).

Therefore, in these methods, a large number of samples, such as samples collected from cattle shipped from the market, are not selected based on specific traits and stored in advance, but as many as possible without defining the purpose. Since it would take a lot of labor and cost to store it, it was not suitable for such a use.
JP 2002-262716 A Special table 2003-503046 gazette JP 2002-253207 A Eizo Asahina, Yoko Hisada, Makito Emura, Survival of rapidly frozen and thawed tumor cells, Low temperature science. Ser. B, Biological science, 25: 81-96

  Accordingly, an object of the present invention is to provide a tissue cryopreservation method that can be stored easily and at low cost because a large number of mammalian tissues are stored. The present invention also relates to a nuclear transfer embryo of a non-human mammal that uses a somatic cell contained in the tissue preserved by the cryopreservation method of the tissue as a donor cell, and a clone non-human mammal produced from the nuclear transfer embryo. The purpose is to provide animals.

  As a result of intensive studies, the inventors put a tissue collected from a mammal into a cryoprotectant or a vitrification solution, and then freeze the collected tissue as it is, instead of quick freezing or slow freezing. And the somatic cells contained in the tissue thus cryopreserved can be used as donor cells in nuclear transfer and somatic cell cloning techniques, thereby completing the present invention.

  That is, the method for cryopreserving a mammalian tissue according to claim 1 of the present invention includes a collection step of collecting a tissue from the mammal, and the collected tissue is subjected to cell dispersion treatment and cell culture, and freezing prevention is performed. And a freezing step of freezing at a temperature of −60 ° C. or lower without using an agent.

  A nuclear transfer embryo of a non-human mammal according to claim 2 of the present invention is a non-human animal cell contained in a tissue cryopreserved by the cryopreservation method of a mammal tissue according to claim 1 as donor cells. It is intended to be used as

  The cloned non-human mammal according to claim 3 of the present invention is produced from the nuclear transfer embryo of the non-human animal according to claim 2.

  The method for cryopreserving mammalian tissue of the present invention allows cryopreservation of tissue at a simple and low cost, and the frozen tissue can also be used for donor cells of somatic cell clones. Therefore, instead of selecting individuals with excellent traits in advance and freezing and preserving their tissues, after securing a large number of mammalian tissues, later, individuals with excellent traits as needed Individuals with excellent traits can be replicated using the tissue.

1. A method for cryopreserving a tissue of a mammal according to the present invention is a method comprising (1) a sampling step for collecting a tissue from a mammal, and (2) a freezing step for freezing the collected tissue as it is. is there. Therefore, details of each process will be described below.

(1) Collection process The collection process is a process of collecting tissue from a mammal. Here, examples of the mammal include domestic animals such as cattle, pigs, sheep, and goats, pets such as dogs and cats, laboratory animals such as rats, mice, and guinea pigs, and humans. Among these, cattle are preferable from an economical viewpoint.

  Moreover, as said structure | tissue, if it can obtain easily at low cost, it can be used without specifically limiting. Examples thereof include muscle tissue, adipose tissue, skin tissue, visceral tissue, reproductive tissue such as testis and ovary, nerve tissue such as brain spinal cord and spinal column, and blood. However, when collecting tissues from cattle slaughtered at slaughterhouses, muscle tissue, skin tissue, and visceral tissue are often used as products, and nerve tissues such as the brain and spine are discarded by law. These tissues are difficult to collect because blood is discarded during slaughter. On the other hand, adipose tissues such as internal organs and subcutaneous tissues are often not used as commercial products, are easy to collect, and can be collected regardless of gender, and thus are suitable for use in the present invention.

  Tissue collection can be used without particular limitation as long as it is a known method. Specifically, a method of sucking tissue from a living body with a syringe, a method of locally anesthetizing a living body and collecting it by a surgical operation or a liposuction method, a female adipose tissue around a viscera of a slaughtered mammal or subcutaneous The method of excision is mentioned. Among these, the method of collecting adipose tissue of a slaughtered mammal is preferable because it is easy to collect. The liposuction method is a method generally performed in cosmetic plastic surgery and the like, and specifically includes a method using ultrasonic liposuction, powered liposuction using a cannula, syringe suction, and the like. It is done.

(2) Freezing step The freezing step is a step of freezing the collected tissue as it is. More specifically, it is a step of freezing the collected tissue just by putting it in a freezing container or the like without cell dispersion treatment and cell culture and without using a cryoprotectant. Here, examples of the cryocontainer include containers that can be used for storage of biological samples such as cryotubes, microtubes, test tubes, etc. However, the tissue may be frozen with a resin film.

  In addition, as a tissue freezing method, any conventional method for freezing cells, for example, a method in which a cryocontainer is placed in a refrigerator or liquid nitrogen can be used without particular limitation. In order to prevent the formation of ice crystals, the freezing temperature is -60 ° C or lower, and the deep freezer set temperature of -80 ° C, which is generally used for storage of biological samples, is easy to use. Therefore, it is preferable.

2. Non-human mammalian nuclear transfer embryos Non-human mammalian nuclear transfer embryos can be prepared by conventional nuclear transfer embryo preparation procedures, such as (1) preparation of donor cells, (2) preparation of enucleated unfertilized eggs, ( 3) It may be performed along the nuclear transfer. More specifically, (1) preparation of donor cells may be performed, for example, by thawing cryopreserved tissue, cutting and washing, suspending the cells in an appropriate culture medium, and culturing the cells. In addition, (2) preparation of an enucleated unfertilized ovum is, for example, taking a cumulus ovum complex from the ovary and allowing it to mature in vitro, and then removing and removing the cumulus cells from the cumulus ovum complex to obtain an unfertilized ovum. Thus, the nucleus may be removed from the unfertilized egg. Furthermore, (3) nuclear transfer is performed by, for example, injecting donor cells into an enucleated unfertilized ovum and fusing the cells with an electric pulse to obtain a nuclear transfer ovum, and then culturing the nuclear transfer ovum to generate a blastocyst Incubate until it is done.

3. Cloned Non-Human Mammals Cloned non-human mammals can be produced by basically the same method as the conventional method using a nuclear transfer embryo. Specifically, the non-human mammal nuclear transfer embryo of the present invention may be transplanted into a temporary parent uterus to give birth and birth to a cloned non-human mammal.

4). Others Artificial multipotency that collects cells contained in cryopreserved tissues and introduces several types of genes into these cells to give them pluripotency similar to ES cells (embryonic stem cells) Inducible pluripotent stem cells (hereinafter abbreviated as iPS cells) may be prepared. Since these iPS cells can be induced to differentiate into all tissues and organs that make up the body, for example, if the technology to establish iPS cells from human patients themselves is established, Tissues and organs can be created.

  Hereinafter, the present invention will be described in more detail based on examples. However, the scope of claims of the present invention is not limited in any way by the following examples.

1. Proliferation test I
The tissue was cryopreserved and thawed by the cryopreservation method of the present invention, and the proportion of cells having proliferative ability among the cells contained in the thawed tissue was examined. Specifically, it investigated as follows.

(1) Tissue collection and cryopreservation At the slaughterhouse, a piece of tissue (A to H) is directly collected from the adipose tissue around the uterus of 8 cows with a scalpel, and this sample is about 20 ° C physiological saline. And transported to the laboratory for 3-4 hours. In the laboratory, adipose tissue was divided into approximately 1 g portions, placed in 2 ml cryotubes, and stored frozen in a -80 ° C. freezer for 5 days.

(2) Thawing and tissue decomposition The adipose tissue contained in the cryotube is fixed in a water bath (hereinafter referred to as the thawing temperature. In this case, the thawing temperature is 25, 30, 35, 38, 39, 40, 41) , 42, 45, and 50 ° C.) and then thawed completely. The thawed adipose tissue was taken out from the phosphate buffer, placed in another container, and chopped with scissors or a stick.

  Dulbecco's modified Eagle medium (SIGMA) containing 0.1 wt% collagenase and 0.2 wt% dispase was added to the cut adipose tissue to 3-5 times its volume. This tissue suspension was cultured for 1-2 hours while shaking in a 39 ° C. water bath to obtain an adipose tissue lysate. The resulting adipose tissue lysate was filtered through a 250 μm nylon mesh to remove tissue that could not be enzymatically digested.

(3) Cell collection and cell culture The adipose tissue lysate was diluted with a phosphate buffer, centrifuged, and the supernatant was discarded. The remaining precipitate was suspended in 500 μl of a primary culture medium (MF-start, registered trademark, Toyobo) to which the antibiotic Primocin (registered trademark, INVIVOGEN) was added to a final concentration of 100 μg / ml. This suspension was placed in a 4-well dish so that the concentration was 1 well / sample for each thawing temperature. The 4-well dish was placed in a CO ₂ incubator and cultured for 10 days (5% CO ₂ , 95% air, 39 ° C., saturated humidity).

(4) Cell staining and calculation of growth rate With the cells growing on the bottom, the medium was removed from the 4-well dish, and the cells were fixedly stained with acetoorcein. Each well was washed with 100% ethanol to remove excess staining solution. As a result, only cells capable of proliferation were stained red.

  After the 4-well dish was placed in a dryer to dry the cells in each well, each well was covered with a white poster color. Next, the image was captured with a scanner from the back of the 4-well dish. The result is shown in FIG. 1A shows the results when samples A to D (shown as No. 9 to No. 12 in FIG. 1) are used, and FIG. The results when H (shown as No. 17 to No. 20 in FIG. 1) are used are shown.

  Next, using the image processing software (Adobe Photoshop CS Ver.8.0.1), the captured image was binarized into black and white and stained with the number of pixels of the whole well (hereinafter abbreviated as the total number of pixels). The number of pixels in the cell portion (hereinafter abbreviated as the number of cell pixels) was measured, and the ratio of the number of cell pixels to the total number of pixels was calculated as the cell proliferation rate. The results are shown in Tables 1 and 2.

  From Table 1 and Table 2, it was found that there was not much difference in the cell growth rate depending on the thawing temperature of the cryopreserved tissue, and the optimum thawing temperature could not be determined statistically. On the other hand, it was found that the cell growth rate differs depending on the cow from which the sample and tissue were collected.

2. Proliferation test II
Twelve samples from AA to LL different from Example 1 were used, and by the same method as in Example 1, the proportion of cells having proliferation ability among the cells contained in the cryopreserved tissue was examined. The difference from Example 1 is that the cryopreservation period of the cells is 5 days or 20 days, and the thawing temperature is any of 35, 36, 37, 38, 39, 40, 41, 42 ° C. The cell culture period after thawing is 7 days, and a 24-well dish was used.

  The results are shown in FIG. 2 and Tables 3 to 6. FIG. 2 (a) shows the result when frozen for 5 days, and FIG. 2 (b) shows the result when frozen for 20 days. From these results, it was found that, as in Example 1, there was not much difference in the cell growth rate depending on the thawing temperature of the cryopreserved tissue, and the cell growth rate was different depending on the sample.

3. Proliferation test III
Using 8 samples from M to P different from those in Example 1 and Example 2, the proportion of cells having proliferation ability among the cells contained in the cryopreserved tissue was examined by the same method as in Example 1. . The difference from Example 1 is that the sample is adipose tissue around the kidney or subcutaneously, collected at the slaughterhouse and stored as a lump at 4 ° C for 24 hours, then divided into 1g and frozen in a -80 ° C freezer The freezing period is 7 days, the thawing temperature is 25, 30, 35, 38, 40, 42, 45, 50 ° C, the cell culture period after thawing is 12 Is a day.

  The results are shown in FIG. 3 and Tables 7 and 8. FIG. 3 (a) shows the case where samples M and P (shown as No. 5 and No. 12 in FIG. 3) are used, FIG. 3 (b) shows samples N and O (No. in FIG. 3). .7, No. 9)) The results when stored frozen for 20 days are shown. From these results, it was found that, as in Example 1, there was not much difference in the cell growth rate depending on the thawing temperature of the cryopreserved tissue, and the cell growth rate was different depending on the sample.

4. Quality of frozen tissue-derived cells The quality of cells contained in the tissue preserved by the cryopreservation method of the present invention was examined.

(1) Effect of subculture The effect of subculture of cells contained in cryopreserved tissues on the morphology and proliferation ability of the cells was examined. Specifically, it was performed as follows.

  Samples F and G (denoted as No. 18 and No. 19 in FIG. 1) frozen and thawed according to the procedure described in Example 1 were cultured with MF-start (Toyobo), and the cells were dished. When adhesion to the bottom surface and proliferation were confirmed, the medium was changed to MF-medium (Toyobo) and subcultured 3 times. Cells at passage number 3 (hereinafter abbreviated as p3) were cryopreserved with a cell cryoprotectant (Cell Banker 1, Nippon Zenyaku Kogyo Co., Ltd.) for over 1 year.

Thaw the cryopreserved cells and seed them on a collagen-coated dish (BD Biocoat dish, Becton Dickinson) containing MF-start (Toyobo) and culture (5% CO ₂ , 95% air, 37 ° C, saturation humidity) ) Started. Thereafter, when attachment to the bottom of the dish and proliferation were confirmed, the medium was changed to MF-medium (Toyobo), and subculture was repeated while observing cell morphology and proliferation with an optical microscope.

  As a result, F (No. 18) proliferated remarkably at p7 and could not be passaged after p8. In contrast, G (No. 19) showed good cell growth even after repeated passages. Moreover, the result of having observed the form of the cell in p6 with the optical microscope (100 times) is shown in FIG. 4A shows an observation result of F (No. 18), and FIG. 4B shows an observation result of G (No. 19). As shown in this figure, the cell morphology of F (No. 18) was poor, whereas the cell morphology of G (No. 19) was good.

(2) Chromosome test Using F (No. 18) and G (No. 19), the effect of subculture of cells contained in cryopreserved tissues on the number of chromosomes was examined. Specifically, it investigated as follows.

After adding demecorsin (manufactured by SIGMA) to 20 ng / ml in the medium of growing cells (p6), culture for 3 hours (5% CO ₂ , 95% air, 37 ° C, saturation humidity) Synchronized with the mitotic phase. The synchronized cells were detached from the culture dish by trypsin treatment, and centrifuged (200 × g, 5 min) with phosphate buffered saline (hereinafter abbreviated as PBS, GIBCO). A 0.56% KCl solution was slowly added to the obtained cell pellet, and then allowed to stand for 15 minutes to expand the cells. After the swelling treatment, the cell suspension was centrifuged (125 × g, 5 min), and then the supernatant was removed.

  Carnoy's solution (acetic acid: methanol = 1: 3) was slowly added to the obtained cell pellet, and then allowed to stand for 10 to 15 minutes to fix the cells. After the fixed cell suspension was centrifuged (125 × g, 5 min), the supernatant was removed. 100-200 μl of Carnoy's solution was added to the obtained cell pellet, suspended, and then dropped onto a slide glass. The glass slide was air-dried and then encapsulated with VECTASHIELD mounting medium (registered trademark, Vector Laboratories) containing 5 μg / ml DAPI (SIGMA).

  The slide glass in which the cells were encapsulated was observed with a fluorescence microscope, and the number of chromosomes contained in the nucleus of the cell whose cell cycle was synchronized with the division phase was counted. The results are shown in Table 9. Moreover, the result of having observed the cell with the normal number of chromosomes with the fluorescence microscope is shown in FIG. 5A shows the observation result of F (No. 18), and FIG. 5B shows the observation result of G (No. 19).

  As shown in Table 9, in F (No. 18) and G (No. 19), the percentage of cells having normal (2n = 60) chromosome numbers was 64% and 52%, respectively. . From this result, it was confirmed that cultured cells having a normal number of chromosomes were obtained from adipose tissue cryopreserved by the cryopreservation method of the present invention.

(3) Apoptosis detection Using the above F (No. 18) and G (No. 19), the ratio of cells contained in cryopreserved tissues to undergo apoptosis during subculture was examined by the TUNEL method. Specifically, it investigated as follows.

  The proliferating cells (p6) were detached from the culture dish by trypsin treatment, and washed twice by centrifugation (200 × g, 5 min) using PBS. About 20 μl of PBS was added to the obtained cell pellet and suspended, and then dropped onto a slide glass.

  After air-drying the slide glass, the cells were fixed with PBS containing 4% paraformaldehyde (Nacalai Tesque) (room temperature, 20 minutes), and the slide glass was washed with PBS. PBS containing 0.5% Triton-X100 (SIGMA) was dropped onto a slide glass to permeabilize the cells (room temperature, 20 minutes), and washed with PBS.

  The TUNEL reaction was performed by dropping the TUNEL reaction solution from the in situ cell death detection kit (Roche) onto a slide glass and incubating at 37 ° C. for 60 minutes. The slide glass was washed 3 times with PBS and sealed with VECTASHIELD mounting medium (registered trademark, Vector Laboratories) containing 5 μg / ml DAPI (manufactured by SIGMA).

  The slide glass in which the cells were encapsulated was observed with a fluorescence microscope, the number of FITC signal positive cells that were apoptotic cells was counted, and the ratio of the apoptotic cells to the total cells was determined. The results are shown in Table 10.

  As shown in Table 10, F (No. 18) had high values of 5.0% and 97.1%, whereas G (No. 19) had low signal positive rates of 0.4% and 0.7%. In F (No. 18), there was a very large difference in the positive rate even though it was a sample of the same passage (p6) derived from the same cow. Therefore, some relationship was suspected between the difference in the positive rate and the loss of proliferation ability at p7 described in (1).

5). Production of nuclear transfer embryos using frozen tissue-derived cells Nuclear transfer embryos using the F (No. 18) and G (No. 19), which are cells collected from the tissue stored by the cryopreservation method of the present invention, as donor cells Were manufactured and examined for their quality. Specifically, it investigated as follows.

(1) Preparation of enucleated unfertilized ova The cattle ovaries collected at the slaughterhouse were warmed with 25 ° C physiological saline and brought back to the laboratory. Using a 10 ml syringe (Terumo) equipped with a 21G injection needle (Terumo), the cumulus ovary complex with 3 or more layers of tight cumulus cell layers attached was aspirated and collected from the cow ovary brought home. The collected cumulus oocyte complex was cultured in vitro in 199 medium (Earle salt, GIBCO) containing 5% fetal bovine serum (hereinafter abbreviated as FBS, Bio West) (5% CO ₂ , 95). % Air, 39 ° C, saturation humidity).

  The mature cumulus oocyte complex was treated with 199 medium (Hanks salt, GIBCO) containing 0.25% hyaluronidase (SIGMA) and 5% FBS for 5 minutes, and then pipetted with a Pasteur pipette whose inner diameter was processed into an egg diameter size. Petting was performed to peel and remove cumulus cells from the zona pellucida.

  After moving the cumulus-removed egg (naked egg) to 199 medium (Hanks salt) containing 5% FBS, the micromanipulator equipped with a glass needle (Narishige) was observed while viewing the naked egg with an inverted microscope. In use, the zona pellucida near the polar body of only the first polar body-released ovum contained in the naked ovum was incised. After the zona pellucida incised through the zona pellucida near the polar body was transferred to 199 medium (Hanks salt) containing 5 μg / ml cytochalasin B (SIGMA) and 5% FBS, the cytoplasm containing the nucleus was removed with a glass rod. Enucleated immature ova were prepared by extruding out of the zona pellucida and pipetting with a Pasteur pipette to separate the enucleated immature ovum and cytoplasm.

  For enucleation, the extruded cytoplasm was transferred to 199 medium (Hanks salt) containing 20 μg / ml Hoechst 33342 (SIGMA) and 5% FBS, stained for 30 minutes, and then subjected to Hoechst staining in the cytoplasm under a fluorescence microscope. This was confirmed by observing blue fluorescence indicating the formed nuclei.

(2) Preparation of nuclear transfer embryo First, the F (No. 18) and G (No. 19) were subcultured to the sixth generation (p6), and cultured until 80% confluent. Next, using a glass pipette for microinjection, the cultured cells made into a single cell by trypsin treatment as a donor cell were injected into the surrounding space of an enucleated immature ovum to produce a reconstructed embryo. The eggs into which donor cells were injected were transferred to a Zimmerman cell fusion medium, and electrical stimulation was applied twice for 2.7 kV / cm for 11 μs using a needle-type electrode to fuse the donor cells with the eggs.

Embryos with confirmed fusion were treated with Dulbecco's modified phosphate buffered saline containing 5 μM calcium ionomycin (SIGMA) for 5 minutes, and then modified synthetic oviduct fluid containing 10 μg / ml cycloheximide (hereinafter abbreviated as mSOF). ) For 6 hours (5% CO ₂ , 5% O ₂ , 90% N ₂ , 39 ° C., saturation humidity). Among the nuclear transfer embryos that were cultured, those that did not disrupt the cell membrane were selected and cultured in 0.3% BSA-mSOFaa medium for 7 days (5% CO ₂ , 5% O ₂ , 90% N ₂ , 39 ° C, saturation Humidity). In addition, instead of cryopreserved cells, the same nuclear transfer was performed using bovine auricle-derived fibroblasts that had been serum-starved for 7 to 9 days in αMEM (GIBCO) containing 0.4% FBS (experimental). Control).

  As a result, as shown in Table 11, nuclear transfer embryos were obtained from each cell line, and the proportion of embryos that developed up to the blastocyst stage in 7 days after the start of culture was F (No. 18-1, 2), G (No. 19-1, 2), and serum-starved fibroblasts as experimental controls were 3%, 7%, 20%, 8%, and 20%, respectively.

  As described above, when No. 18-1 having a high ratio of apoptotic cells in Example 4 was used as a donor cell, although the rate of occurrence up to the blastocyst stage was low, other cell lines were used as donor cells. When used as cells, they developed to the blastocyst stage at a rate similar to the experimental control. For reference, a micrograph of a nuclear transfer embryo derived from G (No. 19-1) is shown in FIG.

(3) Quality Evaluation of Nuclear Transfer Embryos Developmental Embryonicity, Total Number of Cells and Their Composition, Specifically, Inner Cell Mass (hereinafter abbreviated as ICM) constituting the embryos It is known that the number of cells of trophectoderm (hereinafter abbreviated as TE) and their constituent ratios are greatly related. Therefore, by the method described in Thouas et al. (2001 Reprod. Biomed. Online 3, 25-29), ICM and TE of the developing embryo were counterstained, and the number of cells of ICM and TE was measured, and the ratio was calculated. Calculated. The results are shown in Table 12.

  As described in Table 12, the total number of embryos derived from F (No. 18) and G (No. 19) is 110 and 103, respectively, and derived from fibroblasts, which are experimental controls. It was equivalent to the total number of cells in the developing embryo. In the embryos derived from F (No. 18) and G (No. 19), the ratio of the inner cell mass to the total number of cells (hereinafter abbreviated as% ICM / total) is 46% and 38, respectively. %, Which was equivalent to 36% of the% ICM / total of embryos derived from fibroblasts as experimental controls.

  From the above results, it was confirmed that the nuclear transfer embryo prepared from the cells contained in the tissue cryopreserved by the cryopreservation method of the present invention was a good quality embryo.

It is a photograph which shows the result of having thawed the tissue cryopreserved by the cryopreservation method of the present invention, collecting cells from the thawed tissue, and examining the proliferation rate of the collected cells. It is a photograph which shows the result of having frozen the structure | tissue from which the origin differs from the structure | tissue of FIG. 1 on different conditions, extract | collecting a cell from the structure | tissue thaw | decompressed on different conditions, and investigated the proliferation rate of the extract | collected cell. It is the photograph which shows the result of having frozen the structure | tissue from which the origin differs from the structure | tissue of FIG.1 and FIG.2 on different conditions, extract | collecting the cell from the structure | tissue thaw | decompressed on different conditions, and examining the proliferation rate of the extract | collected cell. It is a photograph which shows the result of having observed the form of the cell with the optical microscope, after collect | recovering the cell from the tissue frozen and thawed by the cryopreservation method of this invention, and subcultured 6 times. It is a photograph which shows the result of having observed the cell with a normal chromosome number with the fluorescence microscope, after collect | recovering the cell from the tissue frozen and thawed by the cryopreservation method of this invention, and subcultured 6 times. From a tissue cryopreserved and thawed by the cryopreservation method of the present invention, cells were collected and passaged six times, and then a nuclear transfer embryo was obtained by nuclear embryo transfer using the cells as donor cells. It is a photograph which shows the result of having observed the nuclear transfer embryo with the optical microscope.

Claims (3)

A collection step of collecting tissue from the mammal;
Freezing step of freezing the collected tissue at a temperature of -60 ° C. or less without cell dispersion treatment and cell culture and without using a cryoprotectant;
A method for cryopreserving mammalian tissue, comprising:   A nuclear transfer embryo of a non-human mammal, wherein a cell contained in a non-human animal tissue cryopreserved by the tissue cryopreservation method according to claim 1 is used as a donor cell.   A cloned non-human mammal produced from the nuclear transfer embryo of the non-human animal according to claim 2.