KR20110091078A

KR20110091078A - The stem cell incubating container which prevent the contamination of microbe

Info

Publication number: KR20110091078A
Application number: KR1020100010723A
Authority: KR
Inventors: 전민용
Original assignee: 전민용
Priority date: 2010-02-05
Filing date: 2010-02-05
Publication date: 2011-08-11
Also published as: WO2011096659A2; WO2011096659A3

Abstract

The present invention forms an elastic membrane made of rubber or silicone in culture and propagation of cells in a container for culturing microorganisms or cell tissues, particularly stem cells, and thus, when inoculating and culturing microorganisms or cell tissues, particularly stem cells, It provides a culture container that prevents the invasion of various germs and prevents the loss of work or the increase of cost due to contamination of biological tissue or stem cells generated during microbial manipulation.

Culture vessel, filter, elastic membrane, second elastic membrane, stopper, micropipette tip,

Description

Stem cell incubating container which prevent the contamination of microbe

The present invention prevents the invasion of bacteria or various bacteria using the elastic membrane in the culture propagation device such as microorganisms or living tissues, and can culture and multiply the desired microorganisms, tissue cells or stem cells more stably without contamination, It relates to a stem cell culture vessel that blocks the possibility of biological contamination.

Culture growth of biological materials is the most fundamental work of bio industry such as identification and amplification of DNA as well as material analysis in microbiology genetic engineering and medicine. Currently, medicine is trying to obtain various therapeutic effects using stem cells. Stem cells include embryonic stem cells from early blastocysts (Blstocyst) and adult stem cells from adult or placenta after the developmental process.

There are three main methods of obtaining these adult stem cells: first through bone marrow, second through umbilical cord blood, and then peripheral blood. easily obtained through mesenchymal blood. In addition, techniques for obtaining adult stem cells from adipocytes or other tissues of the body have been researched and developed.

However, the stem cells thus collected are so small that they make a large amount of stem cells through culture and inject them into tissues that require cell regeneration such as skin, cardiovascular, and organ tissues. Thus, the stem cells collected are inoculated and cultured in culture vessels of various shapes and types by the traditional culture method. The most basic method is to sterilize the inoculation needle with an alcohol lamp on a work bench that is blocked from outside air called cleanbench in the laboratory, open the lid or stopper of the culture vessel containing the strain or biological tissue to be inoculated, and then use a needle. After removing some stem cells, smear the culture vessel in the culture vessel with the culture vessel lid or stopper, such as a chalena or triangular flask containing culture medium or a culture bottle or cell culture well or plate, and then cover the culture vessel lid. The inoculation process is performed by sealing or covering the lid or lid. Subsequently, cultivation is carried out by incubating in an incubator capable of controlling the environment, that is, temperature or atmosphere, suitable for the proper growth conditions of the stem cells to be cultured, and exchanging the culture medium or subcultured as necessary. Will be repeated.

However, in the culture propagation process, bacteria attached to the surface of the air or work tool in the working space during the process of opening and closing the lid of the culture vessel, inoculating stem cells, or injecting the culture solution, Cells or spores of various bacteria can be infiltrated into the culture vessel. Therefore, in the culture propagation process, a lot of costs are consumed to control the contamination of the culture according to the skill of the operator or the contamination of the work space.

Therefore, in order to prevent this, microbiological laboratories are equipped with a microbiological aseptic workbench, which occupies a large space called a clean bench. Waste is happening.

This culture propagation process is described in detail in the inoculation and cultivation process of the conventional strain as shown in the domestic patent publication 2005-0079197, and also in the patent publication 2001-0029122 is attached to a special filter to prevent microbial contamination in the culture process Animal and plant cell culture vessels have been developed to prevent contamination of external microorganisms. However, these devices do not have a method or device that can prevent the possibility of contamination during inoculation culture of animal and plant cells. In addition, to prevent such contamination, clean bench, a microbiological aseptic workbench equipped with a device which blocks the inflow of external air and prevents and sterilizes the fall of microorganisms in the air or its spores, is required. In small medical institutions with severe constraints, there are difficulties that cannot be provided.Even if a medical institution, a microbiological laboratory, or a bio-industrial company equipped with such equipment is used, tissue culture occurs when culturing microorganisms or stem cells according to the skill of the operator or the degree of microbial contamination in the clean bench. Pollution of water is inevitable, resulting in a waste of time and an increase in work cost.

Literature Information of the Prior Art

[Document 1] KR 10-2005-0079197 (Mushtech Co., Ltd.) 2005. 8. 9

[Document 2] KR 10-2001-0029122 (Korea Research Institute of Bioscience and Biotechnology) 2001. 4. 6

[Reference 3] KR 10-0629588 (Daikin High School Co., Ltd.) September 27, 2006

[US 4] US 2010/0015694 A1 (CARLO ACOSTA) 2010. 1. 21

Therefore, the present invention is to provide a culture vessel such as stem cells and the like by preventing the contamination of microorganisms that can occur in the above process in the apparatus for inoculating and cultivating a plant or plant tissues, strains and stem cells.

In order to achieve the above object, in the present invention, a medium, an enzyme, a reagent, a culture medium, or a container for collecting, culturing, or culturing microorganisms or living tissues, that is, a body of a culture vessel such as a tissue culture well or chalet, a Erlenmeyer flask, a culture bottle, or a plate. The stopper is formed with an elastic membrane or elastic stopper formed of rubber, silicone, or the like, and a culture medium, a bacterium, a living tissue, or the like may be formed using an operation mechanism such as an inoculation needle, a needle, or a scraper microneedle tip that passes through the elastic membrane or the elastic stopper. This collection and collection is carried out so that a closed manipulation operation is performed in which the culture solution or animal or plant tissues, strains, stem cells, or the like is transplanted and propagated.

Stem cell culture vessel to prevent the contamination of microorganisms according to the present invention has the following effects.

First, without opening and closing the container containing the culture solution or strains and animal and plant tissues and withdraw the contents to prevent the contamination of stem cells and microorganisms due to the inflow of microorganisms present in the air.

Second, by inoculating the contents of the culture medium or strain and animal and plant tissues without opening and closing the container to prevent contamination of stem cells and microbial culture proliferation due to the inflow of microorganisms in the air.

Third, the culture and proliferation of strains, animal and plant tissues and stem cells in an enclosed space prevents contamination that can occur in open spaces, occupies a large space and eliminates sterility even in the absence of a clean bench, which increases the cost burden. Microbial and stem cell manipulation operations can be performed.

Fourth, to reduce the biological pollution caused by microbial and stem cell work, the highly skilled research personnel should pay close attention to the mental effort and reduce the loss of working time caused by microbial contamination. There is an effect of preventing the rise of the part, even non-skilled people can easily perform culture and growth operations such as microorganisms and stem cells without contamination.

Fifth, by injecting sterile distilled water, physiological saline or other solvents through the elastic membrane immediately before inoculating the microorganisms or animals and cells of the culture vessel stored in the powder state it is possible to increase the shelf life of the culture vessel.

Sixth, the tip of the tip of the micropipette can penetrate the elastic membrane of the culture vessel and quantitatively inject the strain or solution in a closed state.

Seventh, the gas generated from the cells or strains in culture is discharged, and the required gas flows into the culture vessel, and at the same time a filter is formed to block the infiltration of microorganisms or spores that cause contamination of the culture, thereby preventing contamination in microbial culture. Block it.

Eighth, by forming a plurality of layers of elastic membrane, it is fundamentally to prevent the external contaminating microorganisms or their spores are buried on the surface of the operating mechanism, such as the needle or inoculation needle micropipette tip inserted when the microorganisms are manipulated.

Ninth, the presence of microorganisms penetrating through the primary elastic membrane due to the coating or formation of the germicidal layer between the elastic layers of the plurality of layers, even if the microorganisms infiltrate the secondary elastic membrane and limited infiltration, the contact with the germicidal layer, the activity is naturally Suppressed and killed.

Figure 1 is an exemplary view showing the process of the conventional microbial work, showing the inoculation and incubation process of the microbial strain in the traditional manner, in the open space sterilized tweezers or inoculation needles in the flame of the alcohol lamp, containing the cells Open the lid (2) of the chalet (1), buried the strain in the inoculation needle, and then open the lid (2) of the chalet (1) to be cultured strain on the inoculation needle (3) on the medium (17) After coating, the lid 3 is covered and the tape is sealed. However, when this process is carried out in an open space, the contamination of the culture is considerably high, so in order to reduce the microbial contamination to the minimum, the microbes including stem cells are manipulated in a sterile work device called cleanbench. The device that occupies the space also forms an open space, and in order to increase its pollution prevention efficiency, an expensive clean bench with complicated facilities is being developed. However, even this complex clean bench cannot be a completely closed space where microorganisms, which may be polluted in a continuous process, cannot be completely closed, so no matter how skilled microbial operators operate, The disadvantage is that the cost is increased due to the loss of time and the waste of highly technical personnel.

2 and 3 is a view showing an example of the culture vessel in which the antifouling elastic membrane of the present invention was formed, the elastic membrane 4 was formed on one side of the lid 2 of the general chalet (1). The elastic membrane 4 is usually an elastic material such as rubber or silicone, and the edge portion of one side of the body 16 or the lid 2 of the culture vessel is fused to the container by heat or ultrasonic high frequency fusion or an adhesive layer. (6) can be formed and attached. The material of such an elastic membrane is not limited to rubber or silicone, and a material having such elastic force is a thin plastic film having different characteristics, for example, as a synthetic plastic that acts as a filter. As long as the perforation is closed, the material can restore the closure properties of the culture vessel. In addition, the rubber or silicon-coated composite functional elastic film 4 may be formed on a part of the material. In addition, the elastic membrane 4 may be formed in a fine fiber shape so as to perform the role of the filter 15 at the same time. The elastic membrane 4 is formed in at least one hole 5 formed in the body 16 or the lid 2 made of synthetic plastic or glass.

4 is a view showing another embodiment of the present invention, wherein the elastic membrane 4 is formed on the container body 16 or the lid 2 or part of the elastic membrane 4 in the form of a stopper 7. Of course, it can also be formed in the form fitted in the hole (5). Such a stopper 7 may be formed in an integral manner in which a part of the lid 2 or the container body 16 and a part of the stopper 7 are fused together as well as a removable type, or a stopper on a part of the elastic membrane 4. A part of (7) may be formed by fusion.

5 and 6 show another embodiment of the present invention, the elastic membrane 4 is a needle (8) or inoculation needle (3) or the tip of the micropipette tip 9 is formed as sharp as the needle Penetrating through the inside of the culture vessel such as the chalet (1), it is possible to perform operations such as inoculating the medium 17 with the strain, animal or plant cells and stem cells, or supplement the culture solution. On the other hand, in general, the end section of the conventional micropipette tip 9 is rounded or cut at an angle of 90 degrees, but the micropipette tip 9 of the present invention penetrates the elastic membrane 4 to the chalet ( 1) The tip is formed to be inclined like a needle so as to penetrate into the inside to easily perforate the elastic membrane 4, and the material of the micropipette tip 9 may be made of synthetic plastic or metal. Alternatively, the end portion penetrating and penetrating the elastic membrane 4 may be formed of a metal, and the portion inserted into the micropipette or the pipette may be formed of a synthetic plastic such as PE, PP, or PET. In addition, the grooves 10 are formed so that the rubber or silicone rings 11 are fitted to the side edges of the micropipettes or pipettes so that the rings 11 are engaged with each other so that the micropipette tips 9 may be strongly attached to the pipettes or the micropipettes. So that it does not fall easily during microbial manipulation.

The penetrating elastic membrane 4 is not limited to the needle 8, the inoculation needle 3, the micropipette tip 9, and the culture of a chalet 1, a conical flask, a culture bottle, a tissue culture well, and the like. Instruments, such as tweezers, scissors, knives, tongs, scrapers, etc. that are deemed necessary for a variety of manipulations within the vessel, and do not open the stopper 7 or lid 2 of the culture vessel outside the culture vessel. In addition, all instruments and the like that can be used for the operation work can be used.

This can be easily explained by minimizing infection and shortening the patient's recovery period by making small holes in the skin of the abdomen and inserting an endoscope or arthroscopy through the holes without surgically cutting the abdomen. It can be seen that the effect. That is, through the elastic membrane 4 of the culture vessel with a small hole, the instruments used for the operation work are put into the container, and inoculated and injected with the cells of the required strain or animal or plant or the necessary culture solution, cell differentiation inducing solution, enzyme reagent, or the like. Alternatively, the microbial and stem cell manipulation operations can be performed in a conventional culture vessel or a culture vessel by performing an operation for separating and transplanting cultured cells or strains and replenishing the necessary culture solution in a closed culture vessel. Compared to contaminated through the open and closed operation of the open and closed process, the contamination is significantly reduced, eliminating the cost and time waste, and also reducing the need for a clean space that takes up a large space. When used in hospitals, schools, laboratories, etc. Specify also is close to perfect.

Figure 7 shows an embodiment showing an aspect of the elastic membrane. That is, the elastic membrane 4 indicates that depending on the shape and use of the culture vessel, the position thereof may be formed on the body 16 of the culture vessel as well as the lid 2. The culture vessel may also be a round or square dish shape as well as a triangular flask type or bottle type or a cell culture plate having a plurality of compartment cells or wells formed therein, as shown in FIG. 8. It may be a three-dimensional design of the uneven treatment, or may be coated with a material that helps the cell adhesion to the inner surface of the culture vessel, three-dimensional cell proliferation by putting a three-dimensional artificial material therein It may be in the form of a culture vessel to induce. In addition, the elastic membrane 4 may be formed on the lid 2 of the dual culture vessel in which a plurality of chalets 1 are disposed at least one inside, and the medium 17 of the liquid or powder is pre-injected. In addition, such a culture vessel may form a ring of silicone or rubber material between the culture vessel and the lid (2) to enhance the sealing effect between the body (16) and the lid (2), formed on the body and the lid It can be attached to each other with a screw or a coupling jaw. The inside of the culture vessel may be a form in which the above-described plurality of cell culture wells are formed. Alternatively, the culture vessel may be in the form of a plurality of vessels. In addition, it can be applied to the case of the culture vessel configured to apply electrical energy to either side of the culture vessel. In addition, although the elastic membrane 4 is not shown, it is used for storing the culture medium which affects the cell growth of microorganisms and animals or animals, differentiation culture medium or enzymes and reagents that induce differentiation of various cells, and use them little by little. It may also be applied to the stopper portion of the culture vessel or reagent vessel. Although a stopper formed of a material such as plastic or glass is contained in a culture container containing a conventional culture solution, there is a possibility of causing microbial contamination during the opening and closing of the stopper to obtain its contents, and a reagent containing a liquid enzyme or reagent. The same holds true for containers. In addition to the above liquid contents, the lyophilized medium 17 component in powder form may also be applied. In this case, by injecting sterile distilled water or physiological saline or other solvents through the elastic membrane 4 into a culture container stored in a powder state. The shelf life of culture vessels can be extended. Therefore, the culture vessel or reagent vessel sealed with the stopper 7 on which the elastic membrane 4 of the present invention is formed may be formed by using the needle 8 or the tip 9 of the micropipette without opening the stopper 7. The contents can be removed and applied or injected into the culture vessel, or, on the contrary, the necessary solvent components can be injected into the culture vessel, thereby freeing the microorganisms through such a closed operation.

9 and 10, the elastic membrane 4 may be formed on the filter 15 in the form of a film. It is shown that a filter 15 may be attached for the release of the gas generated during the at least one microbial metabolism or growth of animal and plant cells and the inflow of the required gas in the lid 2 or the body of the culture vessel. At this time, the filter 15 formed is formed of a material having a permeability enough to block the inflow of microorganisms or spores of the pollutant and to distribute the gas generated during the growth and cultivation of cultured cells or strains. On the other hand, the elastic membrane 4 may be coated on a portion of the filter 15.

As can be seen in Figure 11, the lid 2 portion of the culture vessel containing the medium 17 is fused or bonded to the body 16 of the container by the elastic membrane 4 as a whole. At this time, the elastic membrane 4 may be attached to the portion of the jaw 12 formed in the body 16. It automatically injects a certain amount of medium (17) into the culture vessel in the factory for efficient mass production, and the edge of the elastic membrane (4) on the jaw (12) portion formed in the upper portion of the culture vessel in the next process by the conveyor system. The parts will be fused or glued.

Meanwhile, an elastic membrane 4 through which an operation mechanism such as a needle 8, an inoculation needle 3, and a micropipette tip 9 penetrates may be doubled. That is, the microorganisms or spores that may be buried on the surface of the operating mechanism are primarily blocked by the external second elastic membrane 13, or the remaining spores or microorganisms passing therethrough are formed on the elastic membrane (4). ) Will block the effect once more. In addition, the sterilization layer 14 may be formed between the double elastic membranes 4 and 13. As such, since the germicidal layer 14 is coated or formed between the double elastic membranes 4 and 13, the microorganisms penetrating through the primary second elastic membrane 13 are blocked by the elastic membrane 4 and infiltration is prevented. Limited contaminating microorganisms are in contact with the bactericidal layer, so that their activity is naturally inhibited and killed.

The culture vessel thus formed has the effect of blocking the contaminating microorganisms close to perfection, thereby reducing the space of the clean bench which occupies a large volume. It can be done.

Stem cell culture apparatus for preventing the microbial contamination of the present invention is not limited to the above-described embodiments, anyone of ordinary skill in the art without departing from the gist of the present invention and various modifications can be carried out It will be said that the technical spirit of this invention is to the extent possible.

1 is an exemplary view showing a process of a conventional microbial operation

2 illustrates an embodiment of the present invention.

Figure 3 shows an embodiment of the present invention

4 illustrates an embodiment of the present invention.

Figure 5 shows an embodiment of the invention

Figure 6 illustrates an embodiment of the present invention.

7 illustrates an embodiment of the present invention.

8 illustrates an embodiment of the present invention.

9 illustrates an embodiment of the present invention.

Figure 10 illustrates an embodiment of the present invention.

Figure 11 illustrates an embodiment of the present invention.

※ Explanation of code about main part of drawing ※

1 chalet, 2 lids, 3 needles, 4 elastic membranes, 5 holes, 6 adhesive layers,

7 stoppers, 8 needles, 9 micropipette tips, 10 grooves, 11 rings, 12 jaws, 13 second elastic film,

14 sterilization layers, 15 filters, 16 bodies, 17 medium

Claims

Stem cell culture vessels to prevent contamination of microorganisms, characterized in that forming a hole in one side of the culture vessel and formed at least one elastic membrane

The stem cell culture vessel of claim 1, wherein the elastic membrane of the culture vessel is formed of an elastic stopper.

The stem cell culture vessel according to claim 1 or 2, wherein the stopper is fused to a part of the elastic membrane or the container.

The stem cell culture vessel of claim 1, wherein the elastic membrane of the culture vessel has a plurality of layers formed therein.

The stem cell culture container of claim 1 and 4, wherein a sterilization layer is formed between the plurality of elastic membranes.

The stem cell culture vessel of claim 1, wherein a filter is formed on one surface of the lid or body of the culture vessel.

The stem cell culture vessel of claim 1 and 6, wherein an elastic membrane is formed on a part of the filter.

The stem cell culture vessel of claim 1, wherein an adhesive layer is coated on the edge of the elastic membrane.

The stem cell culture vessel of claim 1, wherein one or more cellar wells are formed inside the culture vessel.

[Claim 2] The stem cell culture vessel of claim 1, wherein the three-dimensional unevenness treatment is performed at the bottom of the culture vessel.

According to claim 1, Stem cell culture vessels to prevent the contamination of microorganisms characterized in that the three-dimensional cell culture artificial material in the culture vessels.

[Claim 2] The stem cell culture vessel of claim 1, wherein the microorganism is coated with a substance to help adhesion of cells to the inner surface of the culture vessel.

[Claim 2] The stem cell culture vessel of claim 1, wherein the microorganisms contain a solid or liquid medium in the culture vessel.

The stem cell culture vessel of claim 1, wherein an electric or magnetic energy is applied to the culture vessel.

The stem cell culture vessel of claim 1, wherein the filter of the culture vessel serves as an elastic membrane.

The stem cell culture vessel of claim 1, wherein a barrier rib is formed at the bottom of the culture vessel.

Stem cell culture vessel to prevent microbial contamination, characterized in that the tip of the micropipette tip is sharply formed like a needle

18. The stem cell culture container of claim 17, wherein the needle portion of the micropipette tip is formed of metal and the portion attached to the micropipette or the pipette is formed of synthetic plastic.