KR20040075847A - small size dermis tissue culture method, covering tissue with slide glass - Google Patents

small size dermis tissue culture method, covering tissue with slide glass Download PDF

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KR20040075847A
KR20040075847A KR1020040063523A KR20040063523A KR20040075847A KR 20040075847 A KR20040075847 A KR 20040075847A KR 1020040063523 A KR1020040063523 A KR 1020040063523A KR 20040063523 A KR20040063523 A KR 20040063523A KR 20040075847 A KR20040075847 A KR 20040075847A
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slide glass
cells
dermis tissue
petri dish
culturing
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KR100567535B1 (en
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김창현
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김창현
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

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  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Dermatology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)

Abstract

PURPOSE: A method for culturing small sized dermis tissue using slide glass is provided, thereby culturing dermis tissue with high success rate without chemical treatment, and effectively culturing the small sized dermis tissue. CONSTITUTION: The method for culturing small sized dermis tissue using slide glass comprises the steps of: placing the dermis tissue collected on a collagen type-1 coated petri dish; covering the dermis tissue on the collagen type-1 coated petri dish with a slide glass to fix the dermis tissue; supplying the culturing medium to the dermis tissue; and culturing the dermis tissue, wherein 5 to 10 microliter of collagen gel per 1 dermis tissue is coated on the petri dish.

Description

작은크기로 채취된 동물세포를 슬라이드 글라스 유리판을 이용해 세포배양하는 방법 {small size dermis tissue culture method, covering tissue with slide glass}A method of culturing small sized animal cells using a slide glass glass plate {small size dermis tissue culture method, covering tissue with slide glass}

특정 세포주나 세포배양기법은 수많은 방법이 있지만 통상적으로 화학적 효소처리를 거친 후에 분리된 각각의 세포들이 배양 페트리디쉬 저면에 자연적인 방법으로 부착하여 배양하는 방법이 가장 보편적인 방법이며 인체 피부세포 배양 또는 희귀 동물세포 채취 후 이런 화학적 효소 처리 방법은 피부세포 일차배양시 실패할 확률이 있다.There are a number of methods for specific cell lines and cell cultures. However, the method of attaching and culturing each cell to the bottom of the culture petri dish after the chemical enzyme treatment is the most common method. After harvesting rare animal cells, this chemical enzyme treatment method has the potential to fail during primary culture of skin cells.

체세포 배양액은 DMEM(Dulbecco's modified Eagles medium, Gibco BRL Cat No. 11995-065)에 10% FBS(Gibco BRL Cat No. 16000-044)와 1% 항생제(Gibco BRL Cat No. 15240-062)를 첨가하여 통상적으로 사용하며 보통 피하지방층과 표피 부분을 제거한 진피를 화학적효소 처리 (dispases ,collagenase ,0.05% trypsin )에 의해 분리된 세포들을 체세포 배양액에 넣고 배양기에서 배양을 하는 방법이 통상적이다. 이러한 방법은 채취된 세포의 크기가 화학적 처리 후에도 많은 수를 얻기 위해 채취된 크기도 커야 하며 사람피부세포 및 희귀동물 세포 채취에 있어서는 채취된 세포의 크기가 상대적으로 적을 수 밖에 없는 점이 있다. 화학적 효소 처리 후 배양 할 때 단위 면적당 세포의 숫자는 7.85 * 1000000 cells/ 100 mm * 20 mm 정도가 되어야 하며 이보다 숫자가 작으면 배양이 되지 않는 경우가 많다.Somatic cell culture was added to DMEM (Dulbecco's modified Eagles medium, Gibco BRL Cat No. 11995-065) by adding 10% FBS (Gibco BRL Cat No. 16000-044) and 1% antibiotic (Gibco BRL Cat No. 15240-062). Commonly used, the dermis from the subcutaneous fat layer and the epidermis is usually removed by chemical enzyme treatment (dispases, collagenase, 0.05% trypsin) into the somatic cell culture medium and cultured in an incubator. In this method, the size of the collected cells must be large in order to obtain a large number even after chemical treatment, and in the case of collecting human skin cells and rare animal cells, the size of the collected cells is relatively small. When culturing after chemical enzyme treatment, the number of cells per unit area should be about 7.85 * 1000000 cells / 100 mm * 20 mm.

본 발명에서는 상기와 같은 문제점을 해결하기 위하여, 채취된 동물세포를 화학적 처리과정을 거치지 않고 단지 배양페트리디쉬 저면에 부착 할 수 있게 알맞은 크기로 잘려진 슬라이드 글라스를 이용해 채취된 세포가 저면에 부착하여 배양될 수 있게 일정기간동안 세포가 움직이지 못하게 눌러 주는 역할을 한다. 동시에 알맞은 크기의 슬라이드 글라스는 재질이 유리임으로 배양상태를 관찰하는데 전혀 지장을 주지 않는다.In the present invention, in order to solve the above problems, the collected cells are adhered to the bottom by using a slide glass cut to a suitable size so that the collected animal cells can be attached to the bottom of the culture petri dish without undergoing chemical treatment. It acts to press the cell to prevent movement for a certain period of time. At the same time, the slide glass of the right size does not interfere with observing the culture state because the material is glass.

또한 본 발명은 희귀 동물이나 사람에서 세포 채취는 그 크기가 상대적으로 작을 수밖에 없으며 이렇게 채취된 화학적 처리가 불가능한 작은 크기의 동물 체세포를 배양하는 방법을 제공하는데 목적이 있다.In another aspect, the present invention is to provide a method for culturing a small size animal somatic cells of the rare animals or humans can not only be relatively small in size and the chemical treatment thus obtained.

도1는 슬라이드 글라스를 잘라냄을 나타낸 도이다.1 is a diagram illustrating cutting of a slide glass.

도2는 잘려진 슬라이드 글라스 소독함을 나타낸 도이다.2 shows a slide glass disinfecting cabinet cut away.

도3은 본 발명의 세포배양을 하기위해 잘려진 슬라이드 글라스를 작업용 페트리디쉬에 배치한 도이다.3 is a view showing a slide glass cut in order to perform cell culture of the present invention in a working petri dish.

도4는 본 발명의 실시한 작업용 페트리디쉬에 배양액을 넣는 모습을 나타낸 도이다.Figure 4 is a view showing a state in which the culture medium in a working petri dish of the present invention.

도5는 본 발명의 실시예를 나타낸 도이다.5 is a diagram showing an embodiment of the present invention.

도6은 본 발명의 실시예 후 8일 후를 나타낸 도이다.6 is a view showing 8 days after the embodiment of the present invention.

도7은 본 발명의 실시 예 후 30일 후를 나타낸 도이다.7 is a view showing 30 days after the embodiment of the present invention.

<도면의 주요 부분에 대한 부호의 설명><Explanation of symbols for the main parts of the drawings>

30: 슬라이드 글라스 10: 알맞은 크기로 잘려진 슬라이드 글라스30: slide glass 10: slide glass cut to a suitable size

50: 채취된 세포 20: 콜라겐 타입-1 이 코팅된 페트리디쉬50: collected cells 20: collagen type 1 coated petri dish

90: 배양액 80: 배양된 진피세포90: culture medium 80: cultured dermal cells

본 발명은 동물 체세포 배양방법에 관한 것으로, 희귀 동물의 세포 채취 후 배양 , 사람세포 채취 후 배양 또는 체세포 크기가 작은 동물세포 채취 후 배양을 하기 위해 통상적으로 사용하는 슬라이드 글라스(30)를 배양페트리디쉬에 알맞은 크기로 연마석(70)을 이용해 잘려진 슬라이드 글라스(10) 를 (도1) 채취된 세포(50) 위에 움직이지 못하게 눌러 주어 콜라겐 타입-1 이 코팅된 페트리디쉬(20)에 일정시간 배양하여 콜라겐 타입-1 이 코팅된 페트리디쉬(20)에 화학적 효소처리를 거치지 않고 배양된 진피세포(80)를 얻는 방법이다.The present invention relates to a method for culturing animal somatic cells, and cultures Petri dishes with slide glass 30 which is commonly used for culturing after harvesting of rare animals, culturing after collecting human cells, or culturing after collecting small animal cells. Press the slide glass (10) cut using the abrasive stone (70) to a size appropriate to the collected cells (50) so as not to move to incubate for a predetermined time in the collagen type 1 coated Petri dish (20) It is a method of obtaining a cultured dermal cell 80 without undergoing chemical enzyme treatment to the collagen type-1 coated Petri dish 20.

배양을 위해 사용되는 페트리디쉬는 콜라겐 타입-1 이 코팅된 페트리디쉬(20)를 사용한다. 세포를 눌러 주기 위해 사용되는 잘려진 슬라이드 글라스(10)는알코올로 소독을 하고 알콜렘프에 소독(100) 을 해서 사용한다. (도2). 슬라이드 글라스(30)는 통상적으로 사용되는 유리제품 슬라이드 글라스(30)이며 콜라겐 타입-1 이 코팅된 페트리디쉬(20)에 포셉(60)을 이용해 채취된 세포(50)를 콜라겐 타입-1 이 코팅된 페트리디쉬(20)에 부착한다. (도3). 더 좋은 배양효과를 위해서 채취된 세포(50)를 콜라겐 타입-1 이 코팅된 페트리디쉬(20)에 부착시 채취된 세포(50) 하나당 콜라겐겔 5~10㎕ 를 도포 하여 부착한다. 채취된 동물세포(50)는 표피와 진피를 잘 구별하여 진피가 아랫부분을 향하게 하여 콜라겐 타입-1 이 코팅된 페트리디쉬(20)에 부착한다.Petri dishes used for the cultivation is a collagen type 1 coated Petri dishes 20. The cut slide glass 10 used to press the cells is sterilized with alcohol and used to sterilize the alcohol lamp 100. (Figure 2). The slide glass 30 is a glass slide glass 30 which is commonly used, and the collagen type-1 is coated with the cells 50 collected by using the forceps 60 on the collagen type-1 coated petri dish 20. Attached to the petri dish 20. (Figure 3). For better culture effect, when the cells 50 are collected and attached to the collagen type 1-coated Petri dish 20, 5-10 μl of collagen gel is applied to each of the cells 50 collected. The collected animal cells 50 are well distinguished from the epidermis and the dermis so that the dermis faces the lower part and attaches to the collagen type-1 coated petri dish 20.

체세포 배양액(90) 은 DMEM(Dulbecco's modified Eagles medium, Gibco BRL Cat No. 11995-065)에 10% FBS(Gibco BRL Cat No. 16000-044)와 1% 항생제(Gibco BRL Cat No. 15240-062)를 첨가하여 사용한다. (도4)Somatic cell culture (90) was prepared in DMEM (Dulbecco's modified Eagles medium, Gibco BRL Cat No. 11995-065) with 10% FBS (Gibco BRL Cat No. 16000-044) and 1% antibiotic (Gibco BRL Cat No. 15240-062). It is added and used. (Figure 4)

잘려진 슬라이드 글라스(10)를 채취된 세포(50)에 움직이지 못하게 눌러준 상태에서 체세포 배양액(90)을 페트리디쉬 크기에 따라 잘려진 슬라이드 글라스(10)가 잠길 정도 넣어준 후 (도5) 5% CO2, 95% 습도로 조정된 37도 온도의 배양기 내에서 배양된 진피(80)를 얻을 때 까지 배양한다. 본 발명에 의해 배양된 8일에서 10일 후 모습(도6) .After pushing the cut slide glass 10 in a state in which it is immovably pressed on the collected cells 50, the somatic cell culture solution 90 is inserted into the cut slide glass 10 according to the Petri dish size so as to be locked (FIG. 5) 5%. Cultivate the dermis 80 incubated in an incubator at 37 degrees temperature adjusted to CO 2 , 95% humidity. After 8 to 10 days cultured by the present invention (Fig. 6).

본 발명에 의해 배양된 한달 뒤 모습 (도7)After one month cultured by the present invention (Fig. 7)

배양된 진피세포(80)는 계대배양시 통상적인 배양방법을 따르며 10% FBS가 첨가된 DMEM 배지에서 4~5일 동안 배양을 실시하고 인산염완충용액으로 1~2회 세척한 후 0.05% 트립신, 1mM EDTA 용액(Gibco BRL Cat No. 15400-054)을 첨가하여 배양기 안에서 5분간 정치하면 바닥에 붙은 세포가 떨어지게 되는데, 이 세포를 인산염완충용액으로 세정후 회수하여 원심분리 세척하여 적당량을 새로운 페트리페트리디쉬에 분주하고 배지를 첨가하여 39℃, 5% CO2의 포화습도 배양기 내에서 배양한다. 페트리페트리디쉬에 세포가 가득차면 적당량을 분주하여 동결튜브에 나누어서 동결저장을 시키고 필요시 마다 하나씩 꺼내어 사용한다Cultured dermal cells (80) follow the conventional culture method in subculture, cultured in DMEM medium with 10% FBS for 4-5 days, washed 1-2 times with phosphate buffer solution, 0.05% trypsin, After adding 1 mM EDTA solution (Gibco BRL Cat No. 15400-054) and standing in the incubator for 5 minutes, the cells on the bottom will fall off.The cells are washed with phosphate buffer solution, recovered and centrifuged to wash out the appropriate amount of new Petri Petri. Dispense the dish, add the medium, and incubate in a saturated humidity incubator at 39 ° C. with 5% CO 2 . When the petri petri dish is full of cells, dispense an appropriate amount, divide it into freezing tubes, and freeze and store it.

본 발명은 희귀동물이나 사람체세포 채취 후 체세포를 다시 채취 한다는 것은 매우 힘들기 때문에 성공확률율을 높이기 위해 화학적 처리를 거치지 않고 바로 배양, 실패확률이 낮으며 세포고유의 특징을 살려서 배양할 수 있다 또한 아주 작은 크기의 세포도 효율적 세포배양을 할 수 있다.In the present invention, it is very difficult to retake somatic cells after collecting rare animals or human cells. Therefore, the cultivation and failure probability are low without chemistry treatment to increase the probability of success. Even small cells can be efficiently cultured.

Claims (2)

통상적으로 사용하는 슬라이드 글라스(30)로 부터 콜라겐 타입-1 이 코팅된 페트리디쉬(20)에 알맞은 크기로 잘려진 슬라이드 글라스(10)를 (도1) 채취된 세포(50) 위에 움직이지 못하게 눌러 주며 채취된 세포(50)와 잘려진 슬라이드 글라스(10) 사이에 배양액(90)을 넣고 일정시간 배양하여 콜라겐 타입-1 이 코팅된 페트리디쉬(20)에 화학적 효소처리를 거치지 않고 배양된 진피세포(80)를 얻을 수 있게 하는 작은 크기로 채취된 동물세포를 슬라이드 글라스 유리판을 이용해 세포 배양하는 방법.Pressing the slide glass 10 cut to a size suitable for the collagen type-1 coated Petri dish 20 from the slide glass 30 commonly used (FIG. 1) on the collected cells 50 without moving. The culture medium 90 was placed between the collected cells 50 and the cut slide glass 10 and cultured for a predetermined time, and the dermal cells cultured without undergoing a chemical enzyme treatment on the collagen type-1 coated petri dish 20 (80). A method of cell culturing animal cells collected in a small size to obtain a slide glass glass plate. 1항에 있어서 좋은 배양효과를 위해서 채취된 세포(50)를 콜라겐 타입-1 이 코팅된 페트리디쉬(20)에 부착시 채취된 세포(50) 하나당 콜라겐겔 5~10㎕ 를 도포 하여 부착하는 방법.The method of attaching 5-10 μl of collagen gel per cell 50 collected when attaching the collected cells 50 to the collagen type 1-coated Petri dish 20 for good culture effect according to claim 1 .
KR1020040063523A 2004-08-12 2004-08-12 small size dermis tissue culture method, covering tissue with slide glass KR100567535B1 (en)

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