KR102315584B1 - Method of LGR5 positive auditory progenitor cells isolation from inner ear tissue - Google Patents

Method of LGR5 positive auditory progenitor cells isolation from inner ear tissue Download PDF

Info

Publication number
KR102315584B1
KR102315584B1 KR1020200064949A KR20200064949A KR102315584B1 KR 102315584 B1 KR102315584 B1 KR 102315584B1 KR 1020200064949 A KR1020200064949 A KR 1020200064949A KR 20200064949 A KR20200064949 A KR 20200064949A KR 102315584 B1 KR102315584 B1 KR 102315584B1
Authority
KR
South Korea
Prior art keywords
cells
auditory
lgr5
tissue
present
Prior art date
Application number
KR1020200064949A
Other languages
Korean (ko)
Inventor
이민영
장소영
나타니엘 카르페나
이재훈
정희원
정재윤
정필상
Original Assignee
단국대학교 천안캠퍼스 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 단국대학교 천안캠퍼스 산학협력단 filed Critical 단국대학교 천안캠퍼스 산학협력단
Priority to KR1020200064949A priority Critical patent/KR102315584B1/en
Application granted granted Critical
Publication of KR102315584B1 publication Critical patent/KR102315584B1/en

Links

Images

Classifications

    • 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
    • C12N5/0618—Cells of the nervous system
    • C12N5/062—Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain
    • 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
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
    • C12N2509/10—Mechanical dissociation

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Neurology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Acoustics & Sound (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Neurosurgery (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention relates to a method for isolating auditory progenitor cells using a method for isolating leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) positive cells. LGR5+ cells do not exist or are very few in the adult auditory organ, so auditory cells are not regenerated, and therefore, it is difficult to treat hearing loss. Accordingly, the auditory progenitor cell isolation through the method of the present invention has an advantage in that the isolation precision is high because the LGR5+ specific antibody is used, and the cells are not damaged during the isolation process because the MACS is used. Since the auditory progenitor cells can be differentiated into hair cells, the isolation of LGR5+ from auditory organs using MACS is expected to play a very important role in future auditory regeneration research.

Description

청각 내이 내 LGR5+ 청각 전구세포 분리방법{Method of LGR5 positive auditory progenitor cells isolation from inner ear tissue}Method of LGR5 positive auditory progenitor cells isolation from inner ear tissue

본 발명은 LGR5(Leucine-rich repeat-containing G-protein coupled receptor 5) 양성 세포를 분리하는 방법을 이용한 청각 전구세포 분리방법 등에 관한 것이다.The present invention relates to a method for isolating auditory progenitor cells using a method for isolating leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) positive cells.

청각 영역에서 감각 신경성 청력상실을 초래하는 대표적인 원인 중 하나는 음향 자극 유도 수용체로 작용하는 유모세포의 영구적인 손상이다. 인간은 약 15,000개의 내이 내 유모세포를 갖고 태어나지만 재생되지 않는다. 즉, 유모세포 주변의 청각지지세포로 불리는 상피세포로부터 새로운 유모세포가 재생되는 하등 척추동물과는 달리 포유동물의 청각기관 내 유모세포는 자발적인 재생이 불가능하기 때문에, 일단 청력이 손상되면 완전한 청력 회복이 매우 어려운 난치성 질환을 초래한다.One of the representative causes of sensorineural hearing loss in the auditory area is permanent damage to hair cells that act as acoustic stimulus-induced receptors. Humans are born with about 15,000 hair cells in the inner ear, but they do not regenerate. In other words, unlike lower vertebrates, where new hair cells are regenerated from epithelial cells called auditory support cells around hair cells, hair cells in the auditory organ of mammals cannot spontaneously regenerate, so once hearing is damaged, complete hearing recovery. This leads to a very difficult intractable disease.

청력 개선을 위한 치료법으로 음향 증폭 보청기와 음파 전기자극 인공와우수술, 줄기세포 이식과 유전자 치료가 시도되고 있으나, 보청기의 경우 자연음 재현이 어렵고, 청신경세포가 확보된 환자에게만 적용할 수 있는 인공와우도 수술과 고비용이 부담되고 있으며, 생물학적 치료는 아직까지 안정성과 효용성이 미확보 된 상태이다.Acoustic amplification hearing aids, cochlear implantation surgery, stem cell transplantation, and gene therapy are being tried as treatments for improving hearing. Also, surgery and high cost are burdened, and the safety and efficacy of biological treatment have not been secured yet.

Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5)는 줄기세포의 분화, 증식 및 유도에 중요한 역할을 하며 성체 줄기세포의 바이오 마커로 알려져 있으며, 또한 포유류에서 LGR5+ 세포로부터 내이 유모세포가 유래된다는 것이 보고되었다.Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) plays an important role in the differentiation, proliferation and induction of stem cells and is known as a biomarker of adult stem cells. Also, inner ear hair cells are derived from LGR5+ cells in mammals. It has been reported that

이전 보고는 이독성 손상을 입은 신생쥐의 청각지지세포가 새로운 유모세포로 분화될 수 있는 가능성이 있음을 제시하였고, 또한 새로운 유모세포가 LGR5+ inner pillar cell이나 LGR5+ deiters cell로부터 유래되었음을 확인하였다.A previous report suggested that auditory support cells of new mice with ototoxic damage could be differentiated into new hair cells, and it was also confirmed that the new hair cells were derived from LGR5+ inner pillar cells or LGR5+ deiters cells.

따라서, 본 발명은 새로운 난청 치료법 개발 소재로 활용될 수 있는 청각세포로 분화 가능한 Lgr5+ 청각 전구세포의 생체 내 분리방법을 확립하고자 하였다.Therefore, the present invention was intended to establish a method for in vivo isolation of Lgr5+ auditory progenitor cells capable of differentiating into auditory cells that can be used as a material for the development of a novel treatment for hearing loss.

대한민국 등록특허공보 10-0489248Republic of Korea Patent Publication No. 10-0489248

본 발명의 목적은 LGR5(Leucine-rich repeat-containing G-protein coupled receptor 5) 양성 세포를 분리하는 단계를 포함하는, 청각전구세포 분리 방법을 제공하는 것이다.It is an object of the present invention to provide a method for isolating auditory progenitor cells, comprising the step of isolating LGR5 (Leucine-rich repeat-containing G-protein coupled receptor 5) positive cells.

본 발명의 다른 목적은 상기 방법으로 분리된 청각전구세포를 제공하는 것이다.Another object of the present invention is to provide auditory progenitor cells isolated by the above method.

상기 본 발명의 목적을 달성하기 위해 본 발명은 a) 달팽이관 조직을 세포 단위로 분리시키는 단계; 및In order to achieve the object of the present invention, the present invention comprises the steps of: a) separating the cochlea tissue into cell units; and

b) 상기 분리된 세포에서 LGR5(Leucine-rich repeat-containing G-protein coupled receptor 5) 양성 세포를 분리하는 단계를 포함하는, 청각 전구세포 분리방법을 제공한다.b) isolating a Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5)-positive cell from the separated cells, there is provided a method for isolating auditory progenitor cells.

본 발명의 일 구현예로 상기 a)단계의 상기 달팽이관 조직은 코르티 기관 조직일 수 있다.In one embodiment of the present invention, the cochlear tissue in step a) may be a Corti organ tissue.

본 발명의 일 구현예로 상기 a)단계의 상기 분리는 조직에 단백질 분해효소 처리하는 화학적 분리 및 단일세포 분리 장치를 이용하는 물리적 분리 단계를 포함할 수 있다.In one embodiment of the present invention, the separation in step a) may include a chemical separation step of treating the tissue with a protease and a physical separation step using a single cell separation device.

본 발명의 일 구현예로 상기 b)단계의 LGR5 양성 세포 분리는 마그네틱 라벨링된 항-LGR5 항체를 이용할 수 있다.In one embodiment of the present invention, the separation of LGR5-positive cells in step b) may use a magnetically labeled anti-LGR5 antibody.

본 발명의 일 구현예로 상기 LGR5 양성 세포 분리는 MACS(Magnetic-assisted cell sorting)을 이용할 수 있다.In one embodiment of the present invention, the separation of LGR5-positive cells may use MACS (Magnetic-Assisted Cell Sorting).

또한 본 발명은 상기 방법으로 분리된 청각전구세포를 제공한다.The present invention also provides auditory progenitor cells isolated by the above method.

청각 전구세포 (LGR5+)세포는 성인 청각기관에는 존재하지 않거나 극소수이고 청각세포들이 재생되지 않으므로 난청은 치료되기 어렵다. 한편, 본 발명의 방법을 통한 청각전구세포 분리는 LGR5+ 특이적인 항체를 이용하므로 분리 정밀도가 높으며 MACS를 이용하므로 분리과정에서 세포가 손상되지 않는 장점이 있다. 청각전구세포는 유모세포로 분화될 수 있으므로 청각 기관에서 LGR5+ 세포를 MACS를 이용해서 분리하는 것은 향후 청각 재생 연구에 매우 중요한 역할을 할 것으로 기대된다.Hearing loss is difficult to treat because auditory progenitor cells (LGR5+) cells do not exist or are very few in the adult auditory organ, and auditory cells do not regenerate. On the other hand, the auditory progenitor cell isolation through the method of the present invention has an advantage in that the separation precision is high because the LGR5+ specific antibody is used, and the cells are not damaged during the separation process because the MACS is used. Since auditory progenitor cells can be differentiated into hair cells, the isolation of LGR5+ cells from auditory organs using MACS is expected to play a very important role in future auditory regeneration studies.

도 1은 1 일령 마우스의 모습 및 달팽이관 조직 해부 과정을 나타낸 것이다.
도 2는 청각전구세포 분리를 위한 실험 과정을 나타낸 사진이다.
도 3은 조직 분해와 여과(A), MACS의 작용원리(B)를 도식화한 것이다.
도 4는 분리한 청각 전구세포의 배양 7일째 세포 특성을 나타낸 것이다.1 shows the appearance and cochlear tissue dissection process of a 1-day-old mouse.
2 is a photograph showing an experimental procedure for isolating auditory progenitor cells.
Figure 3 is a schematic diagram of tissue degradation and filtration (A), the working principle of MACS (B).
4 shows the cell characteristics of the isolated auditory progenitor cells on the 7th day of culture.

본 발명은 새로운 난청 치료법 개발 소재로 활용될 수 있는 청각세포로 분화 가능한 Lgr5+ 청각 전구세포의 분리방법을 확립하고자 한 것이다.An object of the present invention is to establish a method for isolating Lgr5+ auditory progenitor cells capable of differentiating into auditory cells that can be used as a material for the development of a novel treatment for hearing loss.

이에, 본 발명은 a) 달팽이관 조직을 세포 단위로 분리시키는 단계; 및 b) 상기 분리된 세포에서 LGR5(Leucine-rich repeat-containing G-protein coupled receptor 5) 양성 세포를 분리하는 단계를 포함하는, 청각 전구세포 분리방법을 제공한다.Accordingly, the present invention comprises the steps of: a) separating the cochlea tissue into cell units; and b) isolating Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5)-positive cells from the separated cells.

본 발명에서 상기 달팽이관(cochlear canal)이란 내이의 달팽이(cochlear)의 전정관과 고실관 사이에 있는 유연성 막으로 된 도관이며, 달팽이관 내부에 있는 감각수용기로 청각을 감지하며, 달팽이관 조직이란 이러한 기능을 수행하기 위해 특수화된 세포들의 집합체를 일컫는다. In the present invention, the cochlear canal is a conduit made of a flexible membrane between the vestibular canal and the tympanic tube of the cochlear of the inner ear, and senses hearing with sensory receptors inside the cochlear tube, and the cochlear tissue has this function A collection of cells specialized to perform.

본 발명에서 상기 LGR5(Leucine-rich repeat-containing G-protein coupled receptor 5)란 줄기세포의 분화, 증식 및 유도에 중요한 역할을 하며 성체 줄기세포의 바이오 마커로 알려져 있으며, 또한 포유류에서 LGR5+ 세포로부터 내이 유모세포가 유래된다는 것이 보고되었다.In the present invention, the Leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5) plays an important role in the differentiation, proliferation and induction of stem cells and is known as a biomarker of adult stem cells. It has been reported that hair cells are derived.

본 발명에서 상기 a)단계에서 세포 단위로 분리시킨다는 것은 특정 조직에서 결합조직 등 세포 이외의 성분을 세포와 분리시키는 것을 의미할 수 있다. 또한, 상기 분리 결과 세포는 단독 세포로 존재하거나 세포의 집합체로 존재할 수 있다.In the present invention, the separation into cells in step a) may mean separating components other than cells, such as connective tissue, from cells in a specific tissue. In addition, as a result of the separation, the cells may exist as single cells or as an aggregate of cells.

본 발명에서 상기 전구세포(precursor cell)란 자손에 해당되는 세포가 분화형질을 발현하지 않은 미분화 친(親)세포를 의미하며, 본 발명의 청각전구세포란 분화시 청각세포로 기능하는 세포라면 제한없이 본 발명의 범위에 해당할 수 있으며 바람직하게 상기 청각전구세포는 유모세포로 분화되는 것일 수 있다.In the present invention, the precursor cell means an undifferentiated parent cell in which the cell corresponding to the progeny does not express the differentiation trait. Without it, it may fall within the scope of the present invention, and preferably, the auditory progenitor cells may be differentiated into hair cells.

본 발명에서 a)단계의 상기 달팽이관 조직은 코르티 기관 조직일 수 있다.In the present invention, the cochlear tissue in step a) may be a Corti organ tissue.

본 발명에서 상기 코르티 기관이란 달팽이관의 기저막 위에 있는 특수한 청각 수용체를 총칭하며 신경상피 유모 세포와 이들을 지지하는 내외지주 세포 및 기타 여러 세포을 포함하는 개념이다.In the present invention, the organ of Corti refers to a special auditory receptor on the basement membrane of the cochlea, and is a concept including neuroepithelial hair cells, inner and outer stalk cells supporting them, and various other cells.

본 발명에서 상기 a)단계의 상기 분리는 조직에 단백질 분해효소 처리하는 화학적 분리 및 단일세포 분리 장치를 이용하는 물리적 분리 단계를 포함할 수 있다. 상기 단백질 분해효소는 트립신일 수 있으나 이에 제한되는 것은 아니다. 상기 단일세포 분리 장치는 셀 스트레이너(cell strainer)일 수 있으나 이에 제한되는 것은 아니다.In the present invention, the separation of step a) may include a chemical separation step of treating the tissue with a protease and a physical separation step using a single cell separation device. The protease may be trypsin, but is not limited thereto. The single cell separation device may be a cell strainer, but is not limited thereto.

본 발명에서 b)단계의 LGR5 양성 세포 분리는 마그네틱 라벨링된 항-LGR5 항체를 이용할 수 있다. 상기 항체는 LGR5 특이적 결합이 가능하므로 LGR5 양성 세포의 표면에 특이적으로 부착 가능하다. 또한 상기 항체는 마그네틱 라벨링 되어있어 MACS를 이용하는 경우 높은 선택성으로 LGR5 양성 세포 분리가 가능하게 한다.In the present invention, the separation of LGR5-positive cells in step b) may use a magnetically labeled anti-LGR5 antibody. Since the antibody is capable of LGR5-specific binding, it can be specifically attached to the surface of LGR5-positive cells. In addition, since the antibody is magnetically labeled, it is possible to isolate LGR5-positive cells with high selectivity when MACS is used.

다른 양태로서 본 발명은 상기 방법으로 분리된 청각전구세포를 제공한다. 상기 청각전구세포는 유모세포로 분화될 수 있으므로 청각 재생에 매우 중요한 역할을 할 수 있다.In another aspect, the present invention provides auditory progenitor cells isolated by the above method. Since the auditory progenitor cells can be differentiated into hair cells, they can play a very important role in hearing regeneration.

이하, 본 발명을 실시예 및 시험예를 통하여 더욱 상세히 설명한다. 그러나, 하기 실시예는 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이에 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail through Examples and Test Examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

<실시예 1: 시약 및 재료><Example 1: Reagents and Materials>

내이 샘플링inner ear sampling

1-4일령 C57BL/c6 마우스를 100Φ 일회용 페트리접시에 놓고, 70% 알코올 스프레이로 소독한 다음 아이리스 가위로 단두한 후 두개골을 반으로 잘라 1X DPBS로 옮긴다. 해부현미경으로 관찰하면서 마이크로 포셉으로 양쪽 귀(N=14)를 떼어내어 otic capsule을 제거하고 달팽이관만을 해부하여 획득하였다(도 1 참조).Place 1-4-day-old C57BL/c6 mice in a 100Φ disposable Petri dish, sterilize with 70% alcohol spray, cut with iris scissors, cut the skull in half, and transfer to 1X DPBS. While observing under a dissecting microscope, both ears (N=14) were removed with micro forceps, the otic capsule was removed, and only the cochlea was dissected (see FIG. 1).

감각상피(sensory epithelia)의 세포외 매트릭스(extracellular matrix)를 분해하고, 유모세포(hair cell)과 지지세포(supporting cell)를 분리할 수 있는 non-enzymatinc 용액인 Matrisperse 세포회수용액 5mL로 1시간동안 배양하였다. 조직을 회수한 후 0.5 x g에서 5 분 동안 원심 분리하고 37 ℃에서 20 분 동안 TrypLE (Gibco)에서 반응시켰다. 0.5 x g에서 5 분 동안 추가로 원심 분리한 후, 해리된 세포를 DPBS에 현탁시키고, 1000 μL 피펫 팁을 사용하여 50-80회 분쇄한 후 40-micron cell strainer를 이용하여 단일 세포 현탁액을 생성하였다.5 mL of Matrisperse cell recovery solution, a non-enzymatinc solution that can decompose the extracellular matrix of sensory epithelia and separate hair cells and supporting cells, for 1 hour cultured. After the tissue was recovered, it was centrifuged at 0.5 x g for 5 min and reacted in TrypLE (Gibco) at 37 °C for 20 min. After further centrifugation at 0.5 x g for 5 min, the dissociated cells were suspended in DPBS, triturated 50-80 times using a 1000 μL pipette tip, and then a single cell suspension was generated using a 40-micron cell strainer. .

<실시예 2: Magnetic-assisted cell sorting(MACS)를 이용한 LGR5+ 청각 전구세포 분리><Example 2: Isolation of LGR5+ auditory progenitor cells using Magnetic-assisted cell sorting (MACS)>

단일 세포 현탁액을 상기 언급된 실시예 1 방법에 의해 수득하고, PBS에 1:100으로 희석된 토끼 항-마우스 Lgr5 폴리클로날 항체 (rabbit anti-mouse Lgr5 polyclonal antibody; Miltenyi Biotech, Bergisch Gladbach, Germany)에 45분 동안 실온에서 반응시켰다. 차가운 MACS 완충액 (Miltenyi Biotech)으로 1회 세척하고 300 x g로 10분 동안 4 ℃에서 원심분리 후, 획득한 세포 펠렛을 MACS 완충액에 재현탁시키고, 총 107 개 세포 당 20uL의 항-토끼 IgG 마이크로 비드 (anti-rabbit IgG microbeads; Miltenyi Biotech)를 넣어 4 ℃에서 15 분 동안 반응시켰다. Lgr5 양성 세포는 제조사의 지시에 따라 자기장에서 자기 활성화 세포 분류 (MACS) 자기 칼럼 (Miltenyi Biotech)에 의해 분류되었다.A single cell suspension was obtained by the above-mentioned Example 1 method and diluted 1:100 in PBS with rabbit anti-mouse Lgr5 polyclonal antibody (Miltenyi Biotech, Bergisch Gladbach, Germany) was reacted at room temperature for 45 minutes. After washing once with cold MACS buffer (Miltenyi Biotech) and centrifugation at 300 x g for 10 minutes at 4°C, the obtained cell pellet was resuspended in MACS buffer, and anti-rabbit IgG microfluidic of 20uL per 10 7 cells in total. Beads (anti-rabbit IgG microbeads; Miltenyi Biotech) were put and reacted at 4 °C for 15 minutes. Lgr5-positive cells were sorted by magnetic activated cell sorting (MACS) magnetic column (Miltenyi Biotech) in a magnetic field according to the manufacturer's instructions.

<실시예 3: 분리된 LGR5+ 청각 전구세포 세포 배양 후 특성 확인><Example 3: Confirmation of characteristics after culture of isolated LGR5+ auditory progenitor cells>

분리한 LGR5 양성 세포를 in vitro 환경 37°C, 5% CO2에서 배양하였다. The isolated LGR5-positive cells were cultured in vitro at 37°C and 5% CO 2 .

배양 7일째 세포 성격 확인 및 검증을 위하여 청각 전구세포 유전자 마커인 Lgr5 항체와 청각 지지세포 유전자 마커인 Sox2 항체를 이용하여 면역형광염색을 수행하였다. 면역 염색을 위해, 5% normal goat serum과 0.3% Triton X-100을 첨가한 1X PBS에 샘플을 넣고 실온에서 1 시간 동안 blocking 한 후, 1% normal goat serum과 0.3% Triton X-100에 희석된 1 차 항체를 사용하여 4 ℃에서 16-18시간 (overnight) 반응시켰는데, Rabbit(IgG) anti-Lgr5 (1:100희석, ab75732, Abcam)를 사용하여 전구체 세포를 확인하고, mouse (IgG1, κ) anti-Sox2를 사용하여 지지 세포를 식별하였다 (1:100희석, #561469, BD). 이후, 샘플을 1X PBS로 10분씩 3번 세척 한 다음, Alexa Fluor 488-conjugated goat anti-rabbit (IgG) (1:1000 희석, # A11034, Life Technologies) 및 Alexa Fluor 568-conjugated goat anti-mouse (1:1000 희석, # A21124, Life Technologies )를 사용한 2차 항체로 37 ℃에서 60분 동안 반응시켰다. 마지막으로, 샘플을 1X PBS로 10분씩 3번 PBS 세척 후 Vectashield (Vector Labs)를 사용하여 샘플 슬라이드를 제작하였다. 염색된 세포를 공초점 현미경 (LSM 510 META, Zeiss, Germany)으로 관찰하였다.On the 7th day of culture, immunofluorescence staining was performed using the Lgr5 antibody, which is an auditory progenitor cell gene marker, and the Sox2 antibody, which is the auditory support cell gene marker, to confirm and verify the cell characteristics. For immunostaining, samples were placed in 1X PBS containing 5% normal goat serum and 0.3% Triton X-100, blocked at room temperature for 1 hour, and diluted in 1% normal goat serum and 0.3% Triton X-100. The primary antibody was reacted for 16-18 hours (overnight) at 4 °C, and the precursor cells were identified using Rabbit (IgG) anti-Lgr5 (1:100 dilution, ab75732, Abcam), and mouse (IgG1, κ) Supporting cells were identified using anti-Sox2 (1:100 dilution, #561469, BD). Afterwards, samples were washed 3 times with 1X PBS for 10 min each, followed by Alexa Fluor 488-conjugated goat anti-rabbit (IgG) (1:1000 dilution, # A11034, Life Technologies) and Alexa Fluor 568-conjugated goat anti-mouse ( 1:1000 dilution, # A21124, Life Technologies ) was reacted for 60 minutes at 37 °C with secondary antibody. Finally, samples were washed 3 times with 1X PBS for 10 minutes each in PBS, and then sample slides were prepared using Vectashield (Vector Labs). Stained cells were observed under a confocal microscope (LSM 510 META, Zeiss, Germany).

그 결과 청각전구세포 마커인 LGR5 항체(녹색)와 청각지지세포 마커인 Sox2 항체(빨간색)를 이용한 형광면역염색 결과 co-staining 되는 것을 확인함으로써 분리된 세포가 청각전구 지지세포임을 확인하였다(도 4 참조).As a result, it was confirmed that the isolated cells were auditory progenitor support cells by confirming that they were co-stained as a result of fluorescence immunostaining using the auditory progenitor cell marker LGR5 antibody (green) and the auditory support cell marker Sox2 antibody (red) (Fig. 4). Reference).

배양 7일째의 MACS를 통해 분리된 LGR5 양성 세포는 세포수가 많이 증가한 것은 아니나, 다리를 뻗으면서 자라며 세포의 크기가 성장하였다.LGR5-positive cells isolated through MACS on day 7 of culture did not increase in cell number much, but grew as they stretched their legs.

Claims (6)

a) 달팽이관 조직을 세포 단위로 분리시키는 단계; 및
b) 상기 분리된 세포에서 LGR5(Leucine-rich repeat-containing G-protein coupled receptor 5) 양성 세포를 분리하는 단계를 포함하되,
상기 b)단계의 LGR5 양성 세포 분리는 마그네틱 라벨링된 항-LGR5 항체를 이용하고, 상기 LGR5 양성 세포 분리는 MACS(Magnetic-assisted cell sorting)을 이용하는, 청각 전구세포 분리방법.
a) isolating the cochlea tissue into cells; and
b) separating the LGR5 (Leucine-rich repeat-containing G-protein coupled receptor 5) positive cells from the separated cells,
The method for separating LGR5-positive cells in step b) uses a magnetically labeled anti-LGR5 antibody, and the separation of LGR5-positive cells uses MACS (Magnetic-Assisted Cell Sorting).
 제1항에 있어서, a)단계의 상기 달팽이관 조직은 코르티 기관 조직인 것인, 청각 전구세포 분리방법.
The method of claim 1, wherein the cochlear tissue of step a) is an organ tissue of Corti.
 제1항에 있어서, a)단계의 상기 분리는 조직에 단백질 분해효소를 처리하는 화학적 분리 및 단일세포 분리 장치를 이용하는 물리적 분리 단계를 포함하는 것인, 청각 전구세포 분리방법.
The method of claim 1, wherein the separation of step a) comprises a chemical separation step of treating a tissue with a protease and a physical separation step using a single cell separation device.
 삭제delete 삭제delete 제1항 내지 제3항 중 어느 한 항의 방법으로 분리된 청각전구세포.The auditory progenitor cells isolated by the method of any one of claims 1 to 3.
KR1020200064949A 2020-05-29 2020-05-29 Method of LGR5 positive auditory progenitor cells isolation from inner ear tissue KR102315584B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
KR1020200064949A KR102315584B1 (en) 2020-05-29 2020-05-29 Method of LGR5 positive auditory progenitor cells isolation from inner ear tissue

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
KR1020200064949A KR102315584B1 (en) 2020-05-29 2020-05-29 Method of LGR5 positive auditory progenitor cells isolation from inner ear tissue

Publications (1)

Publication Number Publication Date
KR102315584B1 true KR102315584B1 (en) 2021-10-21

Family

ID=78268729

Family Applications (1)

Application Number Title Priority Date Filing Date
KR1020200064949A KR102315584B1 (en) 2020-05-29 2020-05-29 Method of LGR5 positive auditory progenitor cells isolation from inner ear tissue

Country Status (1)

Country Link
KR (1) KR102315584B1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114231389A (en) * 2021-12-21 2022-03-25 四川大学华西医院 Rhesus monkey cochlear membrance labyrinth tissue cell nondestructive extraction device and use method

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100489248B1 (en) 2002-02-19 2005-05-11 메디포스트(주) Isolation and culture-expansion methods of mesenchymal stem/progenitor cells from umbilical cord blood, and differentiation method of umbilical cord blood-derived mesenchymal stem/progenitor cells into various mesenchymal tissues
US20170071937A1 (en) * 2014-09-03 2017-03-16 Massachusetts Institute Of Technology Compositions, Systems, and Methods for Generating Inner Ear Hair Cells for Treatment of Hearing Loss

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR100489248B1 (en) 2002-02-19 2005-05-11 메디포스트(주) Isolation and culture-expansion methods of mesenchymal stem/progenitor cells from umbilical cord blood, and differentiation method of umbilical cord blood-derived mesenchymal stem/progenitor cells into various mesenchymal tissues
US20170071937A1 (en) * 2014-09-03 2017-03-16 Massachusetts Institute Of Technology Compositions, Systems, and Methods for Generating Inner Ear Hair Cells for Treatment of Hearing Loss

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114231389A (en) * 2021-12-21 2022-03-25 四川大学华西医院 Rhesus monkey cochlear membrance labyrinth tissue cell nondestructive extraction device and use method

Similar Documents

Publication Publication Date Title
EP3865506A1 (en) Fusion protein for use in the treatment of hvg disease
Odeberg et al. Low immunogenicity of in vitro-expanded human neural cells despite high MHC expression
KR102315584B1 (en) Method of LGR5 positive auditory progenitor cells isolation from inner ear tissue
JP2011529341A (en) Method of growing natural killer cells
ES2946934T3 (en) Biomarkers for photoreceptor cells
JP2013526839A (en) Method and apparatus for using stem cell immune modulation
Mulay et al. An in vitro model of murine middle ear epithelium
CN107142242B (en) Isolated culture method of naked mole rat skeletal muscle myoblasts
Bojic et al. CD200 expression marks a population of quiescent limbal epithelial stem cells with holoclone forming ability
Lopez et al. Isolation and characterization of human optic nerve head astrocytes and lamina cribrosa cells
KR100519384B1 (en) Manufacturing method of exosomes using gene transfection and use of the same
EP2420566A1 (en) Method of preparing human lung tissue stem cells and method of inducing differentiation into human alveolar epithelial cells
RU2006126332A (en) METHOD FOR ISOLATING CELLS FROM BREAST SECRET
CN112175909B (en) VSX2 green fluorescent reporter gene human-induced pluripotent stem cell line and construction method thereof
CN111154807B (en) Construction method of secretory Laoshan mountain milk goat mammary epithelial cell line
KR102196069B1 (en) Method for Isolating of Retinal Ganglion Cell and Uses of the Cells Thereby Isolated
US10287547B2 (en) Enrichment and characterization of human corneal endothelial cells (hCENC) with novel monoclonal antibody
JP2012039946A (en) Establishment of immortalized endometriotic epithelial cell keeping characteristic of endometriotic epithelial cell
KR101994640B1 (en) Method of isolating and culturing for neural stem cell
WO2003093457A1 (en) Method for the identification and isolation of stem cells from a human tissue
CN109402041A (en) A kind of people&#39;s renal cells isolation and culture method
JP5035928B2 (en) Screening method for human neural stem cells
KR20210044626A (en) Method for isolating basal cells from epithelial cells in mucous membrane of human respiratory organ
JP4997613B2 (en) Monoclonal antibody selective for human neural stem cells and screening method using the same
RU2785136C1 (en) Method for production of matrix-bound vesicles from monolayer cell cultures and cell spheroids

Legal Events

Date Code Title Description
E701 Decision to grant or registration of patent right
GRNT Written decision to grant