KR20090111706A - Device and method for cells transplantation - Google Patents

Abstract

PURPOSE: A method and an apparatus for transplanting cell are provided, which can obtain sperm in which a specific gene is introduced from a recipient testis. CONSTITUTION: An apparatus for transplanting cell comprises a speed measurement vessel equipped with an upper vessel in which a scale which has the receiving space of the liquid materials is indicated and a lower vessel in which observation of the liquid materials falling from the upper vessel is possible; a communication pipe(150) which is connected to the speed measurement vessel and in which a control valve is adhered so that the diameter length adjustment of the pipe can be possible; and a needle member(140) which is connected to the end part of the communication pipe and is inserted in the transplanting organ.

Description

Device and method for cells transplantation

The present invention relates to an implantation device for use in transplanting a substance containing cells such as porcine garden stem cells into an organ such as the genital organs, and a transplantation method using the device.

More specifically, the present invention is characterized in that the lower part is opened so that the injected liquid material can fall into the lower container, the upper container marked with a scale having a receiving space of the liquid material; And a lower container marked with a graduation, the lower container being connected to the upper container and including a drop space capable of observing the liquid substance falling from the upper container. A distribution pipe connected to the speed measuring container, the control valve being attached to enable adjustment of the diameter of the pipe; And a needle member connected to the distal end of the distribution pipe and including a needle member to be inserted into an implantation organ, the implantation apparatus being capable of adjusting the injection speed of the liquid material through the capacity of the liquid material falling from the upper container to the lower container of the speed measuring container. It is about.

In another aspect, the present invention is the control of the flow pipe of the implantation device through the step of filling a liquid material into the upper container of the speed measuring vessel using the implantation device, inserting the needle member of the implantation device into the organ to be implanted By regulating the valve, the invention relates to a method of implantation comprising the step of implanting a liquid material into the organ.

Sperm, a germ cell in cells, especially in mammals, including humans, is a vehicle that carries male-side genetic information to the next generation. Thus, spermatogenesis, which involves a series of complex processes that produce sperm, is an essential process for species conservation and genetic diversity. The spermatogenesis process is the most productive of adult cell production systems. In humans, normal males are known to produce 100x10 ⁶ sperm per day. In this complex and productive spermatogenesis process, spermatogonial stem cells are important cells that provide the basis and foundation for maintaining spermatogenesis throughout the male life. Garden stem cells possess the ability of self-renewal and differentiation as well as the characteristics of adult stem cells. The first stage of spermatogenesis begins with the fate decision of garden stem cells capable of producing different stages of differentiated progeny that eventually differentiate into sperm.

Thus, through transplantation of garden stem cells, a new technique for transplanting donor germ cells into other testes, garden stem cells can colonize transplanted testes and resume spermatogenesis and produce fertilized sperm. . Thus, New Moon stem cell transplantation technology can provide a framework for scientists to conduct fundamental spermogenesis research. In addition, a method for regenerating sperm from infertile patients can be provided, and genetically engineered germ cells can be provided for making transgenic animals.

Thus, although it was known that garden stem cells exist among single spermatogonia (A _s spermatogonia) group, the biochemical or molecular biological properties that can be used as morphological criteria or markers to accurately distinguish garden stem cells are clearly revealed. However, in 1994, Brinster (Proc Natl Acad Sci US A. 1994, 22; 91 (24): 11303-7) developed a technique for transplanting mouse testis cells to demonstrate the presence of garden stem cells in the testes of mice. At the same time, electricity was prepared to directly study the functional characteristics of garden stem cells. Since the testis cells and garden stem cells, this dish method is used as essential and the most essential technology in the development of biological characteristics and utilization of garden stem cells.

However, the transplantation technique has been widely used in mice and rats to date, but the anatomical structure of the reproductive organs in livestock is different from that of experimental animals. It is impossible.

 In addition, isolated germ cells can be injected into the testes through various methods. Most commonly used methods include inserting a needle through a tube protruding outside the testis and passing a needle through the testicular network. This reticular bundle is a bundle of tubes that is connected to seminiferous tubules that carry sperm to the epididymis. This injection is performed via micropipettes without the use of microscopic or micromanipulators. The micropipette is held by hand and the pressure is operated by hand using a syringe connected to the micropipette.

Micropipettes (hereinafter referred to as pipettes) are a generic term for relatively simple glass instruments for measuring and discharging the volume of liquids or gases of about 100 ml or less and about 0.001 ml, and include glass and plastic products. Its capacity is from 1 to 100 ml. The pipette is a single-piece pipette with a large bulge in the center of the glass tube, a hole pipette with a single gland, and a volume pipe, and a morph pipette that has a fine scale on the glass tube and allows the volume to be seen during spillage. Or mespipette). Usually, simply a pipette refers to a single pipette. A rubber nipple is attached to the top of a glass tube with a safety tool, and a glass scale has a simple scale, which is a kind of morph pipette.

However, the hole pipette is inconvenient to take any amount of liquid, and the morph pipette has a problem of low precision. Bull pipettes of other structures also have the advantage of being able to simply collect toxic liquids, etc., but this also has a problem of low precision.

Depending on the purpose of use, it is divided into air replaceable and direct replaceable pipettes. In other words, air displacement pipettes are used for aqueous solutions or general experiments, and direct displacement pipettes are used for specific samples (viscosity, density, volatility, radioactivity, and corrosiveness). In addition, there are micropipettes when the volume to be measured is extremely small, ultra-small pipettes when the trace amount is smaller, and the syringe is graduated.

Accurate pipettes are required to increase the reproducibility of experiments in small-scale experiments, and it is important to use the pipette correctly to begin biochemical or molecular biology experiments. However, the pipette has a lot of difficulty in accurately measuring the scale and injecting and correcting the correct amount to be injected.

Recently, Honaramooz et al. (Biol Reprod. 2002, 66 (1): 21-8) reported on how to implant testis cells in livestock pigs, thereby contributing to the expansion of transplantation into useful livestock, but the methods they have developed have been used in practical applications. It is not a perfect solution, and there is an urgent need for improved technology development to improve porting efficiency.

Therefore, the present inventors continued to develop a method for efficiently injecting cells, in particular, pig stem cells into animal organs, and as a result, the present invention was completed.

One object of the present invention is to provide a useful analytical method for verifying the biological properties and activity of the cells by implanting a material, for example, a testicular cell into the organ of the animal that can constitute a liquid material.

Another object of the present invention is to introduce or target a specific gene in the garden stem cells collected from the testes of the donor and then transplanted into recipient testes, sperm with a specific gene introduced or hit from the recipient testis can be obtained, It can be used in the method of producing a conversion animal.

Still another object of the present invention is to extract the garden stem cells from the testis of excellent breeding animals that are excellent in heredity but unable to collect semen due to disease or aging, and transplanted into the recipient recipient testes, and then the garden stems of the transplanted breeding animal from recipient testes. It is to provide a way to improve the utilization of excellent mammoths by producing sperm derived from cells.

In order to achieve the above object, the implantation apparatus of the present invention, the lower container is open so that the injected liquid material can fall into the lower container, the upper container marked with a scale having a receiving space of the liquid material; And a lower container connected to the upper container and a scaled lower container including a drop space capable of observing a liquid substance falling from the upper container. A distribution pipe connected to the speed measuring container, the control valve being attached to enable adjustment of the diameter of the pipe; And a needle member connected to the distal end of the distribution pipe and including a needle member to be inserted into an implantation organ, the implantation apparatus being capable of adjusting the injection speed of the liquid material through the capacity of the liquid material falling from the upper container to the lower container of the speed measuring container. It is characterized by.

Here, the three-way valve is mounted to the flow pipe, it is also possible to implant device further comprising an air pressure control unit consisting of a cylinder and a push rod, which is installed to be selectively used with the speed measuring container by the mounted three-way valve.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

In the accompanying drawings, an implantation apparatus according to a preferred embodiment of the present invention is shown.

Referring to Figure 2, the implantation apparatus according to the preferred embodiment of the present invention capable of controlling the injection rate of the liquid material through the capacity of the liquid material falling from the upper container to the lower container of the speed measuring vessel is the lower container An upper container marked with a scale including a receiving space 190 characterized in that the lower part is opened so as to fall down; And a lower container marked with a scale including a drop space 210 for observing the liquid substance falling from the upper container, characterized in that the upper container is connected to a portion of the upper container. A liquid material distribution pipe 150 connected to the speed measuring container and capable of adjusting the diameter of the pipe 160; And a needle member 140 connected to the distal end of the distribution pipe and inserted into the transplantation organ.

The lower opening of the receiving space 190 in the upper container of the implantation device may be achieved by including a drop pipe 200 is opened in the lower portion so that the injected liquid material falls into the lower container.

The injection device may include an injection tube 220 to connect the speed measuring container and the distribution pipe in the configuration of the lower container.

The speed measuring container may measure the volume of the liquid material injected into the upper container, and any material, any size, but may be 5 cm or more in length, as long as the droplet of the liquid material falling from the upper container to the lower container may be observed. It is preferable that the radius is 0.5 cm or more, and it is preferable that it is a transparent plastic material container.

In addition, if the drop pipe 200 of the upper container is open so that the liquid material can fall from the upper container to the lower container, the cross section may be any shape including an ellipse, a circular semicircle, a streamline, etc., but is usually circular. desirable.

In addition, the radius of the open drop pipe of the upper container may be any size as long as the liquid material can smoothly control the rate of injection by observing the drop of the liquid material falling from the upper container to the lower container in consideration of the size of the components in the container. It is preferable that the radius of the tube of the open drop tube is 3 mm or less.

In addition, the distribution pipe 150 provides a path for moving the liquid material from the speed measuring container to the needle member, and observes the drop of the liquid material falling from the upper container to the lower container of the speed measuring container, By adjusting the size 160, it is possible to adjust the dose of the liquid material to be implanted. If necessary, the size of the pipe as well as the opening and closing itself can be adjusted. Therefore, it is preferable that a raw material consists of synthetic resins, such as a soft polyurethane, or silicone.

In addition, among the organs of animals, the genitals are fragile and susceptible to injury, and once wounded, it is very difficult to be cured by the occurrence of inflammation. Rather, soft materials should be used. Therefore, the needle member 140 inserted into the genitalia and in direct contact is not limited thereto, but is preferably made of synthetic resin such as polyurethane or silicone.

In addition, when the cells are transplanted into the reproductive organs, they pass a needle member into the network of testes, which are bundled with tubes and connected with seminiferous tubules that carry semen to the epididymis. It is preferable that the needle member has a fine size of about 18 to 25 gauge so as to be inserted into the cleaning tube.

On the other hand, referring to Figure 2, the air pressure control unit 180 in the present preferred embodiment is installed to be used selectively with the speed measuring container by a three-way valve mounted on the distribution pipe, it is composed of a cylinder and a pusher. The three-way valve (3 way valve) is installed between the distribution pipe to rotate the valve as necessary to make the state of the speed measurement vessel in the configuration of the invention or not to the state of the air pressure control unit.

The air pressure control unit may be any type as long as it can control the pressure when the cell clogging is observed during cell transplantation, but the length of the cylinder is preferably 10 cm or more so that the pressure can be injected to an appropriate level.

In addition, the push rod may be anything as long as it can adjust the air pressure, and is not limited to being able to adjust the pressure by hand.

Referring to the effects of the preferred embodiment consisting of the above configuration as follows.

In the implantation apparatus, in order to efficiently implant the components in the liquid substance to the target organ, it is essential to control the speed of the liquid substance injected into the target organ. In the present invention, the lower part of the upper container and the lower container of the speed measuring container are connected to each other. Since it is superimposed, the liquid material falling from the upper container to the lower container through the lower part of the non-overlapping lower container can be monitored to smoothly control the rate at which the liquid material is injected.

Therefore, by adjusting the radius of the flow pipe of the present invention according to the drop rate of the liquid material or open and close as needed, it can be efficiently implanted in the target organ without damaging the components in the liquid material.

In addition, the air pressure control unit of the present invention according to the preferred embodiment in the manner of adjusting the air pressure through the push rod when the component in the liquid material is not implanted into the target transplant organ during the injection process of the liquid material, when the component is clogged The blockage can be removed. For example, when the liquid material is a cell suspension, the air pressure control unit may rotate the three-way valve mounted on the distribution pipe when the cell clogging occurs to prevent the inflow of the cell suspension from the speed measuring vessel and adjust the air pressure from the air pressure controller. It is a configuration to ensure that.

In another aspect, in the implantation method using the implantation device for achieving the object of the present invention, the present invention comprises the steps of filling a liquid material into the injection vessel of the implantation device; Inserting a needle member of the implantation device into an organ to be implanted; It provides a transplant method comprising the step of implanting a liquid material into the trachea by adjusting the valve diameter control valve of the flow pipe of the implantation device.

The step of inserting the needle member to the organ to be implanted is characterized in that it is carried out while observing the image of the ultrasound device.

 In addition, the implantation method of the present invention is characterized in that it further comprises the step of identifying the implantation of the liquid material while implanting the liquid material in the target organ.

The liquid substance is not limited to a specific substance as long as it is intended to be implanted into a liquid substance, but is preferably cell suspension.

The component in the liquid substance is not limited to a specific substance as long as it is intended for transplantation, but is preferably a cell.

The cells include garden stem cells transformed through the introduction or targeting of a specific gene.

Hereinafter, the present invention will be described in detail.

Definition of terms

As used herein, the term "liquid substance" refers to a liquid or dilution of a component to be implanted.

As used herein, the term "cell suspension" refers to a dilution of a cell to be transplanted into a culture solution.

As used herein, the term "reproductive organs" is also referred to as the genitals, there is a distinction between male and female and gonads and appendages. The gonad is the ovary in females and the testis in males.

As used herein, the term “testis” refers to an organ that functions to produce sperm, transport sperm produced, and secrete testosterone to the gonads of males or males of animals. The testis shape of a mammal may generally have an oval or spherical shape, and there is a pair. The testis is covered by a membrane, and when the testis is cut in cross sections, it is divided into parenchyma and interstitial tissue. The parenchyma consists of a seminiferous tubule and testicular network.

As used herein, the term "testis network" refers to the reticulated tissue of the testis located at the central site of the testis.

As used herein, the term "seminiferous tubule" is a thin tube twisted like a coil in several compartments inside the testis, which is very long and curved, 150-250 μm in diameter, and spermatogonia, primary hair cells. (primary spermatocyte), secondary spermatocyte (sencondary spermatocyte), sperm cells (spermatid) and spermatozoa (spermatozoa), such as where you can see the process of spermatogenesis. Mammalian spermatogenesis occurs in the seminiferous epithelium, located in the testis, which consists of many lavage tubes.

As used herein, the term "testicular cell" refers to garden stem cells, garden cells including all germ cells derived from garden stem cells, Sertoli cells, lydig cells and other connective cells. It means a cell group in the testis tissue including muscle cells and the like related to the tissue.

As used herein, the term "garden stem cell" is a kind of male germ-line stem cells, and refers to a cell known to have a single differentiation capacity capable of making sperm only through self-proliferation and differentiation. Germ stem cells are very small in number, are present at rest between the supporting cells in the testis in a resting state, and their proliferation and differentiation are controlled by the exchange of external signals including the supporting cells.

While most other stem cells in vivo are difficult to distinguish from other somatic cells such as hematopoietic stem cells in the bone marrow, garden stem cells are relatively easy to distinguish from other male germ cells. Since spermatogenesis occurs sequentially in the testis, it is easy to observe that garden stem cells continue to produce germ cells as they differentiate. Germ cells move toward the lumen through spermatogenesis until sperm are secreted into the central lumen.

As used herein, the term "vector" refers to a plasmid, cosmid, phage, virus, retrovirus or other carrier that allows insertion, delivery or expression of nucleic acids encoding a transgene.

As used herein, the term “promoter” refers to a regulatory DNA sequence that regulates the transcription of the transgene cDNA.

As used herein, the term “transformation” refers to altering some of the genetic traits of an organism by artificially inserting an external gene on a chromosome that the organism does not originally possess. There are several methods of transformation, but the most commonly used methods include fertilized egg microinjection and somatic cell cloning.

As used herein, the term "gene hit" refers to knocking out some genes such that the genetic trait to be changed through transformation is not expressed.

As used herein, the term “gene introduction” refers to the transplantation of some useful genes to express the genetic traits to be altered through transformation.

Specifically, the cell transplantation method using the transplant device of the present invention

Filling a liquid material into an injection container of the implantation device; Inserting a needle member of the implantation device into an organ to be implanted; And adjusting the pipe diameter adjusting valve of the distribution pipe of the implantation device, thereby implanting the liquid material into the trachea.

Referring to the implantation method using the implantation device according to the invention in detail for each step as follows.

First step; Liquid material to the injection container of the implantation device of the present invention Filled  step

If the component to be transplanted is a cell,

The cells to be transplanted can be prepared by extraction by surgical methods known in the art.

Cells that can be used in the present invention include embryonic cells, fetal cells, flow cells, adult cells, forms such as eggs, skin, oral mucosa, blood, bone marrow, liver, lung kidney, muscle and reproductive organs that can be obtained from adult cells It may be derived from the tissue of. Any cell may be any cell to be transplanted, but garden stem cells derived from mammalian testis may be used.

Mammals include Mononotmata, Marsupialia, Edentata, Plotidota, Lagomorpha, Rodentia, Primates, Demoptera, and Dip Ceoptera, Carnivorous tree (Insectivora), Carnivorous tree (Camivora), Whale tree (Oetacea), Ornamental tree (Tubuildentata), Joiner (Artiodactyla), Horse tree (Perissodactyla), Hyracoidea, Proboscidea , Sea joiner (Sirenia) may be included, the present invention is preferably a pig belonging to the joiner (Artiodactyla).

More specifically, the extraction of garden stem cells in the testis of mammals may go through the following process.

1) obtaining a testis of mammals

In the case of the present invention, it is more preferable to extract the testis from a 3 to 16 week old pig by a surgical method.

2) separating the testis cells from the testes

The above procedure removes the connective tissue and the membrane around the testis, and removes the white film surrounding the testis. Subsequently, the testes are finely cut using an anatomical knife, and then digested through various digestion methods, and single cells including garden stem cells are separated.

3) culturing the garden stem cells included in the population of testis cells

The testis cells obtained by the above procedure are cultured in a medium containing a cell growth factor on the basal cells, and porcine garden stem cells are included in the population of testis cells.

4) Identification of Porcine Garden Stem Cells

In order to confirm whether the porcine pig garden stem cells cultured by the above process is true, an identification step may be performed.

Cell suspension is made from the extracted cells.

The cell suspension is obtained by diluting the cells to be transplanted with a culture medium, and the culture may include a cell growth factor, and preferably, a fibroblast growth factor (eg, basic fibroblast growth factor) and insulin-like cells. It may include an insulin-like growth factor-1, a stem cell factor, a glial derived neurotrophic factor or a combination thereof. More preferably, it may further include a differentiation inhibitory factor, and most preferably, may include a leukemia inhibitory factor. In addition, antioxidants (e.g. β-mercaptoethanol), antibiotic-antimycobacterial agents (antibiotics antimycotics), non-essential amino acids (e.g. arginine, asparagine, aspartic acid, glutamic acid, glycine, proline and serine), buffers (e.g. Hepes Buffer) or mixtures thereof.

The temperature of the cell suspension is preferably maintained at 4 ° C.

1-1 Dyeing Steps to Identify Liquid Substances

To make sure that the liquid material is properly implanted into the target organ, the dye in the liquid material may be dyed or a dye may be added to the liquid in the liquid material.

For example, in the case of cell suspension, the cells may be preferably stained with a fluorescent substance, and more preferably with a fluorescent substance emitting red or green color.

In addition, a dye solution to be added to the liquid material liquid may preferably use trypan blue.

Transformation step by 1-2 gene introduction or hit

When the component to be transplanted is a cell, the cells in the cell suspension may be transformed through specific gene introduction or targeting before the cells are extracted and filled in the cell transplant apparatus.

Transformation can be carried out using the most appropriate method according to the gene to be introduced or targeted, known in the art. The transformation method is not limited thereto, but may be performed through the following steps.

1) separating the cells from the donor;

2) preparing an expression vector containing the gene to be transplanted and then introducing the cell into the cell of step 1;

3) selecting and culturing the cloned cells into which the expression vector is introduced;

4) removing the nucleus of the egg taken from the donor and fusing with the cell; And

5) transplanting the fused cloned eggs into surrogate mothers

Can be rough.

More preferably, the genes can be introduced or targeted to the garden stem cells, and may be performed by removing the unwanted stem cells from the testis and removing (deleting) unwanted genes or inserting the desired genes to recombine them.

In the gene targeting of the present invention, in particular, the transfer of foreign specific genes to porcine garden stem cells can be carried out by gene transfer methods commonly known in the art. For example, electroporation, liposome-mediated transfer methods (Wong, et al., 1980) and retrovirus-mediated transfer methods (Chen et al., 1990; Kopchick et al., 1991; Lee & Shuman, 1990). The foreign gene includes an antibiotic resistance gene as a selection marker, and the selection marker that can be used may be any gene that confers antibiotics on eukaryotic cells, and includes, for example, neomycin, puromycin and zemycin resistance genes.

Herein, a specific gene refers to a gene to be expressed or not expressed through gene targeting. The gene to be expressed is not limited thereto, but specifically, the human hematopoietic promoter (EPO) genome DNA, Europlakin II structural gene, expression vector PCX-EGFP / NEO, HLA-G gene, blood coagulation factor ( hvWF) gene, plasminogen activator (htPA) gene, CSF (COLONY STIMULATING FACTOR), US2 gene are preferred.

Second step; Of the implantation device of the present invention Needle member  Insertion into the organ to be transplanted

The needle member of the implant device is placed in the organ to be implanted so that the liquid material is injected into the target organ. In order to check whether the needle member is properly inserted into the target organ to be implanted, it is preferable to perform the structural observation through the ultrasound apparatus 100.

The target organ to be implanted with the liquid substance is not limited to a specific organ as long as the organ is to be implanted with the component in the liquid substance. However, if the component is a cell, the digestive, respiratory, and urinary organs that constitute the biological structure of mammals including pigs are included. , Circulatory organs, skin, and the like, preferably the reproductive organs, more preferably the testes network 110 in the testes 120.

The third step; Implanting the liquid material into the trachea by adjusting the tube size of the implantation vessel

In implanting the liquid material into the target organ using the implantation device, the flow rate of the implanted device is adjusted by observing the drop rate of the liquid material falling from the upper container to the lower container of the implantation device to adjust the size of the flow pipe of the implanted device. I can regulate it. If necessary, the speed can be adjusted by opening and closing the distribution pipe. Although not limited thereto, it is preferable to maintain an injection rate of 0.5 to 2 ml per minute.

If the blockage phenomenon occurs due to the entanglement of the components in the liquid material during implantation to remove the blockage in a manner to arbitrarily control the air pressure through the air pressure control unit 180 mounted on the implantation device of the present invention to perform the transplantation process smoothly can do. If clogging occurs, it is possible to solve the blockage by pulling the pusher of the air pressure control unit to suck the air, and then slowly push the entangled components using the air pressure inside the sucked cylinder through this.

The fourth step; Cell suspension  Steps to Identify a Transplant

In addition, it is a step to confirm the identification of the cell suspension is implanted in the target organ.

This step may preferably be performed by measuring the rate of infusion into the trachea or confirming that it is correctly implanted into the subject trachea. This can be done by staining the cells or adding the stain to the culture in the cell suspension and grafting it into the target organ or observing it under a microscope. Preferably, the cells stained with a fluorescent material may be performed by observing with a phase contrast microscope or a fluorescence microscope, and the dyeing solution added to the culture medium is preferably added through a trytry blue as a staining solution through a stereoscopic microscope, a fluorescence microscope, or the like. By observation.

In another aspect, the present invention provides a mammalian germ cell formed through the cell transplantation method of the present invention described above using the cell transplant apparatus described above.

Mammals include Monomoterata, Marsupialia, Edentata, Mandarin, Pholidota, Lagomorpha, Rodentia, Primates, Demoptera (Chiroptera), Carnivorous tree (Insectivora), Carnivorous tree (Camivora), Whale tree (Oetacea), Ornamental tree (Tubuildentata), Joiner (Artiodactyla), Horse tree (Perissodactyla), Hyracoidea, Proboscidea, Sea Joiner (Sirenia) may be included, and the present invention is preferably a pig belonging to the joiner (Artiodactyla).

The organ to be transplanted is preferably a mammalian reproductive organ, more preferably a mammalian male reproductive organ. More preferably, it may be a test tube of testes.

The cells to be transplanted are preferably mammalian stem cells or oocyte stem cells, and more preferably mammalian garden stem cells.

In addition, as described above, the garden stem cells can be introduced or targeted to the gene to be expressed or removed. The transformation may provide germ cells in which some specific genes are expressed or not. Specific genes that can be hit or introduced are as described above.

According to the implantation apparatus and the implantation method using the same of the present invention, it is possible to efficiently implant into the organ.

In particular, in the case of cells, it can be expected that the development of active applications such as the production of transgenic animals, as well as the development of the biological characteristics and activity of the cells, can be expected to breakthrough.

In particular, as the biological characteristics and activity of garden stem cells can be analyzed, the development of garden stem cells in livestock can be expected to be a breakthrough.

In addition, garden stem cells are collected from testimonials from excellent donors, which cannot be semen-collected due to disease or aging, and transplanted into young recipient testes to produce sperm derived from garden stem cells from excellent donors. This can be made possible. As a result, the economic effect of reducing the time and cost of selecting excellent donors can be expected.

Furthermore, by introducing or targeting specific genes to garden stem cells and transplanting them into recipient testes, it is possible to develop innovative new technologies for producing transgenic animals by using sperm with specific genes. Industrial ripple effects may be expected.

Those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. Therefore, it should be understood that the embodiments to be described below are exemplary in all respects and not restrictive. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the following detailed description are included in the scope of the present invention.

1. Implantation device

Two vessels from which the piston was removed were connected to a flow pipe connected with an 18-25 gauge needle member to connect a speed measuring container and an air pressure control unit (see FIG. 2).

The feature of this implantation device is that it is possible to measure the volume of the liquid material injected by double connection of two containers, and has the advantage of smoothly controlling the rate of injection by observing the falling liquid material drops. In addition, when clogging of the components occurs during the injection process is characterized in that the blockage can be removed by adjusting the pressure by using the air pressure control unit connected to the left in FIG.

2. to be transplanted donor  Preparation of Garden Stem Cells

The testis was surgically extracted from pigs aged 3 to 16 weeks of age and then transported to a laboratory in a container containing 4 ° C cell culture fluid. The white membrane of the testis was removed and single cells containing garden stem cells were separated from the testes by enzyme treatment. Then, the testis cells were transplanted into recipient testes, and then stained with a fluorescent substance emitting red light under a fluorescence microscope to accurately observe whether the testes were transplanted into the lavage tube. The cells were diluted with cell culture solution containing trypan blue stain solution at 4 ° C until transplantation. Stored in.

3. Transplantation method (garden stem cells recipient In place  transplantation)

After fixing the anesthetized recipient boar on a fixed frame and removing the testis from the scrotum, while observing the image of the ultrasound apparatus, the cell suspension (diluted testis cells stained with fluorescent substance emitting red color under fluorescent microscope) was diluted with a culture solution containing trypan blue dye. The needle member of the implantation device filled with) is placed in the testis network, which is the central part of the testes, and cell suspension was injected using a drop (Figs. 3B-F).

In the case of rapid injection, cell suspension was not injected into the testes of the testes. The cell suspension was successfully injected into the wash tube when the rate of injection of 0.5 ~ 2 ml of cell suspension per minute was maintained. When the transplantation caused clogging caused by the entanglement of the cells in the cell suspension, it was possible to perform the transplantation process smoothly by removing the blockage by arbitrarily adjusting the air pressure using the air pressure control unit mounted on the implantation apparatus designed in the present invention.

4. Transplanted Cell suspension  Sympathy confirmation

The degree of injection of the cell suspension into the testis was observed using the blue color of the trypan blue staining solution added to the cell suspension during transplantation, and it was confirmed whether the testicular cells were correctly implanted into the lavage tube by fluorescence microscopy. It was observed that cell suspension was injected into more than 50% of the testes in the testes and that the fluorescent stained testis cells were evenly transplanted in all the wash tubes in which the cell suspension was injected (see FIGS. 4A-D).

1 is a view showing the process of forming the garden stem cells of the mouse.

2 shows a cell transplantation device.

3 is a diagram illustrating a cell transplantation apparatus and a transplantation process according to an embodiment of the present invention, in which a cell member is inserted by inserting a needle member connected to the implantation device into the testis network of the testis under the observation of an ultrasound image using an ultrasound probe. It shows the process of injection into the cleaning tube.

(A) is a diagram showing an overview of the implantation device developed in the present invention, the implantation device was manufactured by connecting a speed measuring container and an air pressure control unit in which two vessels are dually connected to a tube to which an 18-25 gauge needle member is connected.

(B) is a monitor for observing the ultrasound image,

(C) is an ultrasound image of the testis network observed on the monitor and the needle member attached to the implantation device injected into the testis,

(D ~ F) shows the process of injecting the cell suspension into the testes of the actual recipient sequentially.

Figure 4 is a view showing the cut surface of the testis cells containing the garden stem cells stained with fluorescent material before transplantation and recipient testis observed after transplantation,

(A) shows the fluorescence stained cells observed under a phase contrast microscope,

(B) is the appearance of red fluorescent light by observing the cells of Figure (A) under a fluorescence microscope,

(C) is a cross-sectional view of recipient testis injected with cell suspension, in which fluorescently stained testis cells were diluted with a cell culture solution with typan blue staining solution, and the degree of injection was confirmed through typan blue staining solution added to the cell suspension. As shown in the figure, cell suspension is injected more than 50% of the recipient testis,

(D) shows the cleavage plane of the recipient testis to which cell suspension has not been injected,

(E) shows an enlarged observation of a part of the lavage tube injected with the cell suspension in the diagram of (C) under a stereoscopic microscope,

(F) shows the figure of (E) under fluorescence microscopy to show that the testis cells stained with the fluorescent material are implanted into the lavage tube and spread evenly.

Claims (13)

An upper container whose lower part is opened to allow the injected liquid material to fall into the lower container and is marked with a scale having a receiving space of the liquid material; And a lower container connected to the upper container and a scaled lower container including a drop space capable of observing a liquid substance falling from the upper container. A distribution pipe connected to the speed measuring container, the control valve being attached to enable adjustment of the diameter of the pipe; And Is connected to the distal end of the distribution pipe, including a needle member to be inserted into the transplant organ, Implantation apparatus capable of adjusting the injection rate of the liquid material through the capacity of the liquid material falling from the upper container to the lower container of the speed measuring container. According to claim 1, The three-way valve is mounted on the flow pipe, characterized in that it further comprises an air pressure control unit consisting of a cylinder and a push rod, which is installed to be selectively used with the speed measuring container by the three-way valve mounted. Implanted device. Characterized in that the implantation device according to claim 1 or 2 is used, (A) filling a liquid material into the upper container of the speed measuring container; (B) inserting a needle member of the implantation device into an organ to be implanted; And (c) implanting a liquid material into the trachea by adjusting a control valve of the implantation device distribution pipe. The implantation method of claim 3, wherein step (b) is performed while observing an image of the ultrasound apparatus. The method of claim 3, further comprising the step (d) of identifying the process of implanting the liquid material during or after the step (c). 6. The method of claim 5, wherein step (d) confirms the rate of injection into the trachea and the accuracy of implantation into the target trachea. The method of any one of claims 3 to 6, wherein the liquid substance is cell suspension. 8. The method of claim 7, wherein the cell suspension cells are cells transformed by the targeting or introduction of a specific gene. 8. The method of claim 7, wherein the cell suspension cells are cells harvested from a mammal. The method of claim 9, wherein the mammal is a pig. 8. The method of claim 7, wherein the cell suspension cells are garden stem cells or garden stem cells transformed through introduction or targeting of specific genes. 7. The method of any one of claims 3 to 6, wherein said transplant organ is an intramuscular lavage tube of a mammal. The method of claim 12, wherein the mammal is a pig.

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