KR102627833B1 - Medium composition for 3D culture of lung cancer-derived cell - Google Patents
Medium composition for 3D culture of lung cancer-derived cell Download PDFInfo
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- KR102627833B1 KR102627833B1 KR1020230093258A KR20230093258A KR102627833B1 KR 102627833 B1 KR102627833 B1 KR 102627833B1 KR 1020230093258 A KR1020230093258 A KR 1020230093258A KR 20230093258 A KR20230093258 A KR 20230093258A KR 102627833 B1 KR102627833 B1 KR 102627833B1
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Abstract
본 발명은 폐암 유래 세포의 3D 배양용 배지 조성물에 관한 것으로, 본 발명에 따른 배지 조성물을 이용하는 경우, 폐암 환자의 암 조직을 모방한 3차원 오가노이드를 효과적으로 배양할 수 있어, 폐암 발병 또는 치료 기전 연구, 유전체 분석, 단백체 분석을 비롯하여, 폐암 치료제 스크리닝 및 치료제 투여량 등을 확인하는데 유용하게 활용될 수 있을 뿐만 아니라, 치료제 투여 전 치료 효과를 미리 예측할 수 있어 개인 맞춤형 치료제를 선택하는데 유용하게 활용될 수 있는 장점이 있다. The present invention relates to a medium composition for 3D culture of lung cancer-derived cells. When using the medium composition according to the present invention, it is possible to effectively culture 3D organoids that mimic cancer tissues of lung cancer patients, thereby improving the mechanism of lung cancer development or treatment. Not only can it be useful for research, genome analysis, proteome analysis, lung cancer treatment screening, and confirmation of treatment dosage, but it can also be used to predict treatment effects before administering treatment, making it useful in selecting personalized treatments. There are advantages to this.
Description
본 발명은 폐암 유래 세포의 3D 배양용 배지 조성물에 관한 것으로, 더 상세하게는 폐암 환자의 암 조직을 모방한 3차원 오가노이드를 단시간에 효과적으로 배양 할 수 있는 폐암 유래 세포 배양용 배지 조성물에 관한 것이다.The present invention relates to a medium composition for 3D culture of lung cancer-derived cells, and more specifically, to a medium composition for culture of lung cancer-derived cells that can effectively culture 3D organoids mimicking cancer tissues of lung cancer patients in a short time. .
암 연구에서는 환자의 특성을 잘 나타내는 암 모델이 중요하다. 지금까지 in vitro에서 주로 이용되었던 암 모델은 상업적으로 이용 가능한 암 세포주이다. 암 세포주는 환자의 암 조직에서 유래한 암세포를 2차원 배양으로 적응시켜 만든 모델로, 실험적으로 다루기 쉬울 뿐만 아니라, 한꺼번에 많은 유전적, 약물학적 스크리닝을 하기에 적합하다는 장점이 있다. 그러나, 환자 유래 암세포를 긴 시간 동안 2차원 계대배양한 결과, 환자의 암에서 나타나는 이질성이나 돌연변이와 같은 조직학적 구조들에서 발생할 수 있는 특성이 사라지고, 덩어리로 뭉쳐서 존재하는 환자의 암 조직을 재현할 수 없는 것과 같이 이질성을 잃은 세포주를 이용하여 항암제의 효과를 테스트하는 경우 원래 환자의 특성을 제대로 반영할 수 없다는 문제점이 있다.In cancer research, a cancer model that well represents patient characteristics is important. The cancer models that have been mainly used in vitro so far are commercially available cancer cell lines. Cancer cell lines are models created by adapting cancer cells derived from a patient's cancer tissue to two-dimensional culture. Not only are they easy to handle experimentally, but they also have the advantage of being suitable for many genetic and pharmacological screenings at once. However, as a result of two-dimensional subculture of patient-derived cancer cells for a long period of time, characteristics that may occur in histological structures such as heterogeneity or mutation that appear in the patient's cancer disappear, and it is impossible to reproduce the patient's cancer tissue that exists in lumps. When testing the effectiveness of anticancer drugs using cell lines that have lost their heterogeneity, there is a problem that the characteristics of the original patient cannot be properly reflected.
암 세포주의 이러한 단점을 보완하는 모델로서, 환자 유래 세포를 3D 배양하는 기술이 개발되었지만, 기존에 알려진 폐암 유래 세포는 3D 배양 성공률이 낮고, 작은 양의 시료로 빠른 시간 내에 항암제 감수성 실험을 하기에 충분한 양으로 세포 배양하는데 문제점을 보이고 있다. As a model to compensate for these shortcomings of cancer cell lines, a technology for 3D culturing patient-derived cells has been developed. However, the success rate of 3D culturing of previously known lung cancer-derived cells is low, and it is difficult to conduct anticancer drug susceptibility tests in a short time with a small amount of sample. There are problems with cultivating cells in sufficient quantities.
한편, 폐암 환자로부터 직접 암 조직을 분리하여 3D 배양한 오가노이드를 이용하면, 면역조직화학, 면역형광, 면역블롯팅, 유세포분석, 정량적 중합효소 연쇄 반응 및 RNA 시퀀싱 등과 같은 방법을 이용하여, 개별 환자 특유의 형태학적 및 기능적 분석이 가능하다. 또한, 배양에 사용된 배양 배지(conditioned media)는 ELISA 분석에 이용될 수 있으며, 배양된 오가노이드는 중간에서 높은 처리량 방식(moderate-to-high throughput manner)으로 개별 환자에게 적합한 기존의 또는 신규한 치료방법을 스크리닝 할 수 있도록 한다. 뿐만 아니라, 사용하고자 하는 치료제를 다양한 농도로 처리하여 치료제의 IC50를 결정함으로써 치료제 처리량을 예측할 수 있다. Meanwhile, by using organoids that are 3D cultured by isolating cancer tissue directly from lung cancer patients, methods such as immunohistochemistry, immunofluorescence, immunoblotting, flow cytometry, quantitative polymerase chain reaction, and RNA sequencing can be used to Patient-specific morphological and functional analysis is possible. Additionally, the conditioned media used for culturing can be used for ELISA analysis, and the cultured organoids can be used in existing or novel methods suitable for individual patients in a moderate-to-high throughput manner. Allows screening of treatment methods. In addition, the therapeutic agent throughput can be predicted by treating the therapeutic agent to be used at various concentrations and determining the IC 50 of the therapeutic agent.
본 발명자들은 폐암 환자 유래 오가노이드(PDO)를 생채 외에서 신속하고 안정적으로 배양할 수 있는 배지 조성물을 개발하고자 예의 노력하였으며, 그 결과 종래 알려진 폐암 환자 유래 오가노이드 배양용 배지 조성물과 비교하여 짧은 시간 내에 폐암 유래 세포의 생장이 진행되어 3차원 종양 형상의 오가노이드가 형성되는 것을 확인함으로써 본 발명을 완성하였다. The present inventors have made diligent efforts to develop a medium composition that can quickly and stably culture lung cancer patient-derived organoids (PDO) in vitro. As a result, compared to conventionally known medium compositions for culturing lung cancer patient-derived organoids (PDO) in a short period of time. The present invention was completed by confirming that the growth of lung cancer-derived cells progressed and a three-dimensional tumor-shaped organoid was formed.
본 발명은 폐암 유래 세포 배양에 최적화된 배지 조성물, 이를 이용한 폐암 오가노이드 배양방법, 상기 폐암 오가노이드를 이용한 폐암 치료제 스크리닝 방법을 제공하는 것을 목적으로 한다. The purpose of the present invention is to provide a medium composition optimized for culturing lung cancer-derived cells, a method for cultivating lung cancer organoids using the same, and a method for screening therapeutic agents for lung cancer using the lung cancer organoids.
상기 목적을 달성하기 위하여, 본 발명은 성장인자, ROCK 억제제, Wnt 작용물질, BMP 억제제 및 항산화제를 포함하는 폐암 유래 세포 배양용 배지 조성물을 제공한다.In order to achieve the above object, the present invention provides a medium composition for culturing lung cancer-derived cells containing growth factors, ROCK inhibitors, Wnt agonists, BMP inhibitors, and antioxidants.
본 발명에 있어서, 상기 배지 조성물은 Advanced DMEM/F12 배지를 기본 배지로, 성장인자, ROCK 억제제, Wnt 작용물질, BMP 억제제 및 항산화제를 포함하는 것을 특징으로 할 수 있다. In the present invention, the medium composition may be characterized as containing Advanced DMEM/F12 medium as a base medium, growth factors, ROCK inhibitors, Wnt agonists, BMP inhibitors, and antioxidants.
본 발명에 있어서, 상기 성장인자는 FGF7, FGF10 및 EGF인 것을 특징으로 할 수 있다. In the present invention, the growth factors may be FGF7, FGF10, and EGF.
본 발명에 있어서, 상기 조성물은 인간 재조합 FGF-7을 1ng/㎖ 내지 20ng/㎖, 인간 재조합 FGF-10을 1ng/ml 내지 50 ng/ml, 인간 재조합 EGF를 1ng/㎖ 내지 20ng/㎖로 포함하는 것을 특징으로 할 수 있다. In the present invention, the composition contains 1 ng/ml to 20 ng/ml of human recombinant FGF-7, 1 ng/ml to 50 ng/ml of human recombinant FGF-10, and 1 ng/ml to 20 ng/ml of human recombinant EGF. It can be characterized as:
본 발명에 있어서, 상기 조성물은 항생제, 글루타민, B-27, 니코틴아마이드, 뉴레귤린1 및 A-83-01로 구성된 군에 선택되는 하나 이상의 성분을 더 포함하는 것을 특징으로 할 수 있다. In the present invention, the composition may further include one or more ingredients selected from the group consisting of antibiotics, glutamine, B-27, nicotinamide, neuregulin 1, and A-83-01.
본 발명에 있어서, 상기 조성물은 유기 완충액을 더 포함하는 것을 특징으로 할 수 있다. In the present invention, the composition may further include an organic buffer.
본 발명에 있어서, 상기 ROCK 억제제는 Y-27632이고, 상기 Y-27632은 1uM 내지 20uM로 포함되는 것을 특징으로 할 수 있다. In the present invention, the ROCK inhibitor is Y-27632, and Y-27632 may be contained in an amount of 1uM to 20uM.
본 발명에 있어서, 상기 Wnt 작용물질은 R-스폰딘 3이고, 상기 R-스폰딘 3는 1ng/㎖ 내지 1000ng/㎖로 포함되는 것을 특징으로 할 수 있다. In the present invention, the Wnt agonist is R-spondin 3, and the R-spondin 3 may be contained in an amount of 1 ng/ml to 1000 ng/ml.
본 발명에 있어서, 상기 BMP 억제제는 Noggin이고, 상기 Noggin은 10 내지 1000 ng/㎖로 포함되는 것을 특징으로 할 수 있다. In the present invention, the BMP inhibitor is Noggin, and the Noggin may be contained in an amount of 10 to 1000 ng/ml.
본 발명에 있어서, 상기 항산화제는 N-아세틸시스테인이고, 상기 N-아세틸시스테인은 0.1mM 내지 10 mM로 포함되는 것을 특징으로 할 수 있다. In the present invention, the antioxidant is N-acetylcysteine, and the N-acetylcysteine may be contained in an amount of 0.1mM to 10mM.
본 발명에 있어서, 상기 조성물은 종양-구(tumor-sphere) 형성을 촉진시키는 것을 특징으로 할 수 있다. In the present invention, the composition may be characterized as promoting tumor-sphere formation.
본 발명은 또한, 상기 조성물을 이용하여 폐암 유래 세포를 배양하는 단계를 포함하는 폐암 오가노이드 배양 방법을 제공하는 것을 목적으로 한다.Another object of the present invention is to provide a lung cancer organoid culture method comprising culturing lung cancer-derived cells using the composition.
본 발명에 있어서, 상기 폐암 유래 세포는 In the present invention, the lung cancer-derived cells are
세포 배양액이 수용될 수 있는 수용 공간이 형성되어 있고, 배양 대상체인 세포가 놓여지는 부분으로, 평평한 바닥으로부터 일정 높이를 갖도록 기둥 형태로 돌출된 복수의 필라부들; 및A receiving space in which a cell culture medium can be accommodated is formed, and a plurality of pillars protruding from a flat floor in the form of a pillar to have a certain height are formed as a part where cells, which are culture objects, are placed; and
수용 공간에 놓여진 배양액이 평평한 바닥에 흩어져 잔류하지 않고 한쪽에 모여 있을 수 있도록, 평평한 바닥에 대해 볼록하게 형성된 필라부들과는 반대로 오목하게 형성되는 트렌치홈부;를 포함하는 필라 플레이트에서 배양하는 것을 특징으로 할 수 있다. Characterized by culturing on a pillar plate including a trench groove formed concavely as opposed to the pillars formed convexly with respect to the flat floor so that the culture medium placed in the receiving space can be gathered on one side rather than scattered and remaining on the flat floor. can do.
본 발명은 또한, 상기 배양 방법에 의해 배양된 폐암 오가노이드를 제공하는 것을 목적으로 한다.The present invention also aims to provide lung cancer organoids cultured by the above culture method.
본 발명은 또한, 다음 단계를 포함하는 폐암 치료제 스크리닝 방법을 제공하는 것을 목적으로 한다:The present invention also aims to provide a lung cancer therapeutic screening method comprising the following steps:
(a) 상기 폐암 오가노이드에 폐암 치료제 후보 물질을 처리하는 단계; 및(a) treating the lung cancer organoid with a candidate lung cancer treatment material; and
(b) 상기 후보 물질 중 폐암 세포사멸 효과를 유도하는 물질을 폐암 치료제로 선별하는 단계. (b) Selecting a substance that induces a lung cancer cell death effect among the candidate substances as a treatment for lung cancer.
본 발명에 있어서, 상기 폐암 치료제는 환자 맞춤형 폐암 치료제인 것을 특징으로 할 수 있다. In the present invention, the lung cancer treatment may be characterized as a patient-tailored lung cancer treatment.
본 발명은 폐암 환자 유래 세포를 배양할 수 있는 특화된 배지 조성물을 제공할 수 있으며, 본 발명에 따른 배지 조성물을 이용하는 경우, 폐암 환자의 암 조직을 모방한 3차원 오가노이드를 효과적으로 배양할 수 있어, 폐암 발병 또는 치료 기전 연구, 유전체 분석, 단백체 분석을 비롯하여, 폐암 치료제 스크리닝 및 치료제 투여량 등을 확인하는데 유용하게 활용될 수 있을 뿐만 아니라, 치료제 투여 전 치료 효과를 미리 예측할 수 있어 개인 맞춤형 치료제를 선택하는데 유용하게 활용될 수 있는 장점이 있다.The present invention can provide a specialized medium composition capable of culturing cells derived from lung cancer patients, and when using the medium composition according to the present invention, three-dimensional organoids that mimic cancer tissues of lung cancer patients can be effectively cultured. Not only can it be useful for research on lung cancer development or treatment mechanisms, genome analysis, and proteome analysis, as well as lung cancer treatment screening and treatment dosage confirmation, but it can also predict treatment effects before administering treatment, so you can select a personalized treatment. There are advantages that can be usefully utilized.
도 1은 본 발명에 따른 배지 조성물과 비교예 1, 비교예 2에 따른 배지 조성물을 이용하여 3개 환자 샘플에서 유래한 폐암 세포의 오가노이드 배양 효과를 광학현미경으로 비교한 결과이다.
도 2는 본 발명에 따른 배지 조성물과 비교예 1, 비교예 2에 따른 배지 조성물을 이용하여 3개 환자 샘플에서 유래한 폐암 세포의 오가노이드 배양 효과를 형광현미경으로 비교한 결과이다.
도 3은 도 2의 결과를 정량화한 결과이다.
도 4는 본 발명에 따른 배지 조성물과 비교예 3, 비교예 4, 비교예 5에 따른 배지 조성물을 이용하여 환자 샘플에서 유래한 폐암 세포의 오가노이드 배양 효과를 광학현미경으로 비교한 결과이다.
도 5는 도 4의 결과를 정량화한 결과이다. Figure 1 shows the results of comparing the effect of organoid culture of lung cancer cells derived from three patient samples using the medium composition according to the present invention and the medium composition according to Comparative Example 1 and Comparative Example 2 using an optical microscope.
Figure 2 shows the results of comparing the effect of organoid culture of lung cancer cells derived from three patient samples using the medium composition according to the present invention and the medium composition according to Comparative Example 1 and Comparative Example 2 using a fluorescence microscope.
Figure 3 is a result of quantifying the results of Figure 2.
Figure 4 shows the results of comparing the effect of organoid culture of lung cancer cells derived from patient samples using the medium composition according to the present invention and the medium compositions according to Comparative Examples 3, 4, and 5 using an optical microscope.
Figure 5 is a result of quantifying the results of Figure 4.
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로, 본 명세서에서 사용된 명명법 및 이하에 기술하는 실험 방법은 본 기술분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by a person skilled in the art to which the present invention pertains. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.
명세서 및 특허청구범위에서 사용된 특징부의 크기, 양 및 물리적 특성을 표현하는 모든 수는 모든 경우 용어 "약"에 의해 수식되는 것으로 이해되어야 한다. 따라서, 반대로 나타내지 않는 한, 명세서 및 특허청구범위에 개시된 수치 파라미터는 본 명세서에 개시된 교시 내용을 이용하여 당업자가 얻고자 하는 원하는 특성에 따라 달라질 수 있는 특정 수치의 ±10%를 나타내는 근사치이다. All numbers expressing size, quantity and physical properties of features used in the specification and claims are to be understood in all instances as being modified by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters disclosed in the specification and claims are approximations representing ±10% of the specific numerical value that may vary depending on the desired properties sought to be achieved by one of ordinary skill in the art using the teachings disclosed herein.
본 발명에서는 폐암 유래 환자로부터 폐암 세포를 분리하고 이를 특정 조합의 배지 조성물로 배양하는 경우 폐암 오가노이드를 효과적으로 형성시킬 수 있음을 확인하였다. In the present invention, it was confirmed that lung cancer organoids can be effectively formed when lung cancer cells are isolated from lung cancer-derived patients and cultured with a specific combination of medium composition.
따라서, 본 발명은 일 관점에서 성장인자, ROCK 억제제, Wnt 작용물질, BMP 억제제 및 항산화제를 포함하는 폐암 유래 세포 배양용 배지 조성물에 관한 것이다. Accordingly, in one aspect, the present invention relates to a medium composition for culturing lung cancer-derived cells containing growth factors, ROCK inhibitors, Wnt agonists, BMP inhibitors, and antioxidants.
본 발명의 바람직한 양태로서 상기 배지 조성물은 임의의 기본 배지를 포함한다. 상기 배지 조성물을 사용하여 본 발명의 폐암 유래 세포는 5% 내지 10% CO2, 바람직하게는 5% CO2를 포함하는 대기에서 배양될 수 있다. 바람직한 상기 배지 조성물은 pH 약 7.4 (바람직하게는 pH 7.2 내지 7.6)에서 버퍼링되는 유기 완충액, 예컨대 HEPES를 포함할 수 있다. 바람직한 양태로서, 본 발명은 약 1mM 내지 약 50mM의 HEPES를 사용할 수 있고, 더 바람직하게는 약 5mM 내지 약 20mM, 가장 바람직하게는 약 10mM의 HEPES를 사용할 수 있다. In a preferred embodiment of the present invention, the medium composition includes any basic medium. Using the above medium composition, lung cancer-derived cells of the present invention can be cultured in an atmosphere containing 5% to 10% CO 2 , preferably 5% CO 2 . Preferred medium compositions may include an organic buffer, such as HEPES, buffered at pH about 7.4 (preferably pH 7.2 to 7.6). In a preferred embodiment, the present invention may use about 1mM to about 50mM of HEPES, more preferably about 5mM to about 20mM of HEPES, and most preferably about 10mM of HEPES.
본 발명의 바람직한 일 양태로서, 상기 배지 조성물은 Advanced DMEM/F12 배지를 기본 배지로, 상기 성장인자, ROCK 억제제, Wnt 작용물질, BMP 억제제 및 항산화제를 포함하는 것을 특징으로 할 수 있으나, 이에 한정되지는 않는다. In a preferred embodiment of the present invention, the medium composition may be characterized as containing Advanced DMEM/F12 medium as a base medium and the growth factors, ROCK inhibitors, Wnt agonists, BMP inhibitors, and antioxidants, but is limited thereto. It doesn't work.
즉, 본 발명의 폐암 유래 세포 배양용 배지 조성물은 당업계에서 통상적으로 이용되는 동물세포용 배지를 기초로 하여 제조될 수 있다. 본 발명에서 이용될 수 있는 배지는 동물세포의 배양에 통상적으로 이용되는 어떠한 배지도 가능하며, 예를 들어, NBA(NeuroBasal medium A, Xie C. et al., Free RadicBiolMed 28:665-737(2000), Eagles's MEM (Eagle's minim um essential medium, Eagle, H. Science 130:432(1959)), α-MEM (Stanner, C.P. et al., Nat. NewBiol. 230:52(1971)), Iscove's MEM (Iscove, N. et al., J. Exp. Med. 147:923(1978)), 199 배지 (Morgan et al., Proc. Soc. Exp. Bio. Med., 73:1(1950)), CMRL 1066, RPMI 1640 (Moore et al., J. Amer. Med. Assoc. 199:519(1967)), F12 (Ham, Proc. Natl. Acad. Sci. USA 53:288(1965)), F10 (Ham, R.G. Exp. Cell Res. 29:515(1963)), DMEM (Dulbecco's modification of Eagle's medium, Dulbecco, R. et al., Virology 8:396(1959)), DMEM과 F12의 혼합물 (Barnes, D. et al., Anal. Biochem. 102:255(1980)), Way-mouth's MB752/1 (Waymouth, C. J. Natl. Cancer Inst. 22:1003(1959)), McCoy's 5A (McCoy, T.A., et al., Proc. Soc. Exp. Biol. Med. 100:115(1959)) 및 MCDB 시리즈 (Ham, R.G. et al., In Vitro 14:11(1978)) 등이 이용될 수 있다. 바람직하게는 NBA, α-MEM, Eag les's MEM, Iscove's MEM, 199 배지, CMRL 1066, RPMI 1640, F12, F10, DMEM, Way-mouth's MB752/1 및 McCoy's 5A로 구성된 군으로부터 선택되는 어느 하나 이상이며, 가장 바람직하게는 Advanced DMEM/F12 배지일 수 있다. That is, the medium composition for culturing lung cancer-derived cells of the present invention can be prepared based on the medium for animal cells commonly used in the art. The medium that can be used in the present invention can be any medium commonly used for culturing animal cells, for example, NBA (NeuroBasal medium A, Xie C. et al., Free RadicBiolMed 28:665-737 (2000) ), Eagles's MEM (Eagle's minimum essential medium, Eagle, H. Science 130:432 (1959)), α-MEM (Stanner, C.P. et al., Nat. NewBiol. 230:52 (1971)), Iscove's MEM ( Iscove, N. et al., J. Exp. Med. 147:923 (1978)), 199 medium (Morgan et al., Proc. Soc. Exp. Bio. Med., 73:1 (1950)), CMRL 1066, RPMI 1640 (Moore et al., J. Amer. Med. Assoc. 199:519(1967)), F12 (Ham, Proc. Natl. Acad. Sci. USA 53:288(1965)), F10 (Ham , R.G. Exp. Cell Res. 29:515 (1963)), DMEM (Dulbecco's modification of Eagle's medium, Dulbecco, R. et al., Virology 8:396 (1959)), mixture of DMEM and F12 (Barnes, D. et al., Anal. Biochem. 102:255 (1980)), Way-mouth's MB752/1 (Waymouth, C. J. Natl. Cancer Inst. 22:1003 (1959)), McCoy's 5A (McCoy, T.A., et al., Proc. Soc. Exp. Biol. Med. 100:115 (1959)) and MCDB series (Ham, R.G. et al., In Vitro 14:11 (1978)), etc. can be used. Preferably NBA, α - Any one or more selected from the group consisting of MEM, Eagles's MEM, Iscove's MEM, 199 medium, CMRL 1066, RPMI 1640, F12, F10, DMEM, Way-mouth's MB752/1 and McCoy's 5A, most preferably Advanced It may be DMEM/F12 medium.
상기 세포 배양 배지에 정제된 천연, 반-합성 및/또는 합성 성장 인자를 보충하고 확실하지 않은 성분, 예를 들어 소 태아 혈청 또는 송아지 태아 혈청은 포함하지 않는 것이 특징일 수 있다. 다양한 상이한 혈청 보충 제형들을 상업적으로 입수할 수 있으며 이는 당업자에게 공지어 있다. The cell culture medium may be supplemented with purified natural, semi-synthetic and/or synthetic growth factors and may be characterized as being free of uncertain components, such as fetal bovine serum or fetal calf serum. A variety of different serum supplementation formulations are commercially available and are known to those skilled in the art.
본 발명에 있어서, 상기 배지 조성물은 성장인자로 FGF-7, FGF-1 및 EGF를 포함할 수 있다. 상기 성장 인자는 유사분열 촉진 성장 인자로서, 폐암 유래 세포의 성장을 촉진한다. 본 발명에 있어서, FGF-7은 약 1ng/ml 내지 약 20ng/ml, 바람직하게는 2ng/ml 내지 약 15ng/ml, 더 바람직하게는 약 3ng/ml 내지 약 7ng/ml, 가장 바람직하게는 약 5ng/ml의 농도로 포함될 수 있다. 본 발명에 있어서, FGF-10은 약 1ng/ml 내지 약 50ng/ml, 바람직하게는 5ng/ml 내지 약 40ng/ml, 더 바람직하게는 약 10ng/ml 내지 약 30ng/ml, 가장 바람직하게는 약 20ng/ml의 농도로 포함될 수 있다. 본 발명에 있어서, EGF는 약 1ng/ml 내지 약 20ng/ml, 바람직하게는 2ng/ml 내지 약 15ng/ml, 더 바람직하게는 약 3ng/ml 내지 약 7ng/ml, 가장 바람직하게는 약 5ng/ml의 농도로 포함될 수 있다.In the present invention, the medium composition may include FGF-7, FGF-1, and EGF as growth factors. The growth factor is a mitogenic growth factor and promotes the growth of lung cancer-derived cells. In the present invention, FGF-7 is present in an amount of about 1 ng/ml to about 20 ng/ml, preferably about 2 ng/ml to about 15 ng/ml, more preferably about 3 ng/ml to about 7 ng/ml, most preferably about It may be included at a concentration of 5ng/ml. In the present invention, FGF-10 is present in an amount of about 1 ng/ml to about 50 ng/ml, preferably about 5 ng/ml to about 40 ng/ml, more preferably about 10 ng/ml to about 30 ng/ml, most preferably about It may be included at a concentration of 20ng/ml. In the present invention, EGF is about 1 ng/ml to about 20 ng/ml, preferably 2 ng/ml to about 15 ng/ml, more preferably about 3 ng/ml to about 7 ng/ml, most preferably about 5 ng/ml. It can be included in a concentration of ml.
본 발명의 바람직한 양태로서, 상기 배지 조성물은 Rock (Rho-kinase) 억제제를 포함한다. 상기 Rock 억제제는 바람직하게는, (R)-(+)-trans-4-(1-aminoethyl)-N-(4-Pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), 5-(1,4-diazepan-1-ylsulfonyl)isoquinoline (fasudil 또는 HA1077), 및 (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (H-1152)로 구성된 군에서 선택될 수 있다. 상기 Rock 억제제는 바람직하게는 Y-27632일 수 있으며, 상기 Y-27632는 약 1uM 내지 20uM, 바람직하게는 약 2uM 내지 약 10uM, 가장 바람직하게는 약 5uM 농도로 첨가될 수 있으나, 이에 한정되지는 않는다. In a preferred embodiment of the present invention, the medium composition includes a Rock (Rho-kinase) inhibitor. The Rock inhibitor is preferably (R)-(+)-trans-4-(1-aminoethyl)-N-(4-Pyridyl)cyclohexanecarboxamide dihydrochloride monohydrate (Y-27632), 5-(1,4-diazepan) -1-ylsulfonyl)isoquinoline (fasudil or HA1077), and (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]-hexahydro-1H-1,4-diazepine dihydrochloride (H-1152). The Rock inhibitor may be preferably Y-27632, and Y-27632 may be added at a concentration of about 1uM to 20uM, preferably about 2uM to about 10uM, and most preferably about 5uM, but is not limited thereto. No.
Wnt 작용물질은 Wnt-1/Int-1; Wnt-2/Irp(Int-1 관련 단백질); Wnt-2b/13; Wnt-3/Int-4; Wnt-3a(R&D sytems); Wnt-4; Wnt-5a; Wnt-5b; Wnt-6(Kirikoshi H et al. 2001. Biochem Biophys Res Com 283: 798-805); Wnt-7a(R&D sytems); Wnt-7b; Wnt8a/8d; Wnt-8b; Wnt-9a/14; Wnt-9b/14b/15; Wnt-10a; Wnt-10b/12; Wnt-11; 및 Wnt-16을 포함하는 분비 당단백질을 포함할 수 있다. 인간 Wnt 단백질들에 대해서는 문헌["THE WNT FAMILY OF SECRETED PROTEINS", R&D Systems Catalog, 2004]에 상세히 설명되어 있으며, 해당 문헌은 본 명세서에 참조로서 포함된다. 추가의 Wnt 작용물질로는 Wnt 신호전달 경로의 활성화 및 조절에 관련되고 4개의 구성원(R-스폰딘 1(NU206, Nuvelo, San Carlos, CA), R-스폰딘 2(R&D sytems), R-스폰딘 3 및 R-스폰딘-4)을 포함하는 분비 단백질의 R-스폰딘 패밀리; 및 노린(또한 노리병 단백질 또는 NDP라고도 칭함)(R&D systems)(상기 프리즐드-4 수용체에 높은 친화성으로 결합하고 상기 Wnt 신호전달 경로의 활성화를 유도한다는 점에서 Wnt 단백질과 같은 기능을 하는 분비 조절 단백질이다)(Kestutis Planutis et al.(2007) BMC Cell Biol. 8:12)을 사용할 수 있다. 상기 Wnt 신호전달 경로의 소-분자 작용물질인 아미노피리미딘 유도체가 최근에 동정되었으며, 이 또한 Wnt 작용물질로 본 발명에 사용될 수 있다(Liu et al.(2005) Angew Chem Int Ed Engl. 44, 1987-90). 본 발명에 있어서, 상기 Wnt 작용물질은 바람직하게는 R-스폰딘3일 수 있다. 본 발명에 있어서, R-스폰딘3은 약 1 내지 약 1000ng/㎖, 바람직하게는 약 10ng/㎖ 내지 약 500ng/㎖, 더 바람직하게는 약 100ng/㎖ 내지 약 400ng/㎖, 가장 바람직하게는 약 250ng/㎖로 포함될 수 있으나, 이에 한정되지는 않는다. Wnt agonists include Wnt-1/Int-1; Wnt-2/Irp (Int-1 related protein); Wnt-2b/13; Wnt-3/Int-4; Wnt-3a (R&D systems); Wnt-4; Wnt-5a; Wnt-5b; Wnt-6 (Kirikoshi H et al. 2001. Biochem Biophys Res Com 283: 798-805); Wnt-7a (R&D systems); Wnt-7b; Wnt8a/8d; Wnt-8b; Wnt-9a/14; Wnt-9b/14b/15; Wnt-10a; Wnt-10b/12; Wnt-11; and secreted glycoproteins including Wnt-16. Human Wnt proteins are described in detail in “THE WNT FAMILY OF SECRETED PROTEINS”, R&D Systems Catalog, 2004, which is incorporated herein by reference. Additional Wnt agonists are involved in the activation and regulation of the Wnt signaling pathway and include four members: R-spondin 1 (NU206, Nuvelo, San Carlos, CA), R-spondin 2 (R&D sytems), and R-spondin 2 (R&D sytems). R-spondin family of secreted proteins, including spondin 3 and R-spondin-4); and Norin (also called Nori disease protein or NDP) (R&D systems) (a secreted protein that functions like a Wnt protein in that it binds with high affinity to the Frizzled-4 receptor and induces activation of the Wnt signaling pathway. It is a regulatory protein) (Kestutis Planutis et al. (2007) BMC Cell Biol. 8:12) can be used. Aminopyrimidine derivatives, which are small-molecule agonists of the Wnt signaling pathway, were recently identified, and can also be used as Wnt agonists in the present invention (Liu et al. (2005) Angew Chem Int Ed Engl. 44, 1987-90). In the present invention, the Wnt agonist may preferably be R-spondin3. In the present invention, R-spondin3 is about 1 to about 1000 ng/ml, preferably about 10 ng/ml to about 500 ng/ml, more preferably about 100 ng/ml to about 400 ng/ml, most preferably It may be included at about 250 ng/ml, but is not limited thereto.
본 발명의 바람직한 양태로서, 상기 배지 조성물은 BMP 억제제를 더 포함할 수 있다. BMP는 이량체성 리간드로서 2 개의 상이한 수용체 세린/쓰레오닌 키나제, 유형 I 및 유형 II 수용체로 이루어지는 수용체 복합체에 결합한다. 상기 유형 II 수용체는 상기 유형 I 수용체를 인산화하여, 상기 수용체 키나제를 활성화시킨다. 상기 유형 I 수용체는 특이적인 수용체 기질(SMAD)을 후속으로 인산화하여, 신호 전달 경로를 생성시켜 전사 활성을 유도한다. BMP 억제제를 BMP 분자에 결합하여 복합체를 형성하는 작용제로서 정의하며, 여기에서 상기 BMP 활성은 예를 들어 상기 BMP 분자의 BMP 수용체에의 결합을 방지하거나 억제함으로써 중화된다. 한편으로, 상기 억제제는 길항물질 또는 역 작용물질로서 작용하는 작용제이다. 이러한 유형의 억제제는 BMP 수용체와 결합하고 상기 수용체에 대한 BMP의 결합을 방지한다. 상기 후자 작용제의 예는, BMP 수용체와 결합하고 항체-결합된 수용체에의 BMP의 결합을 방지하는 항체이다. In a preferred embodiment of the present invention, the medium composition may further include a BMP inhibitor. BMP is a dimeric ligand that binds to a receptor complex consisting of two different receptor serine/threonine kinases, type I and type II receptors. The type II receptor phosphorylates the type I receptor, activating the receptor kinase. The type I receptor subsequently phosphorylates its specific receptor substrate (SMAD), creating a signaling pathway that induces transcriptional activity. A BMP inhibitor is defined as an agent that binds to a BMP molecule to form a complex, wherein the BMP activity is neutralized, for example by preventing or inhibiting the binding of the BMP molecule to the BMP receptor. On the one hand, the inhibitor is an agonist that acts as an antagonist or inverse agonist. This type of inhibitor binds to the BMP receptor and prevents binding of BMP to the receptor. An example of the latter agent is an antibody that binds to the BMP receptor and prevents binding of the BMP to the antibody-bound receptor.
BMP 억제제를 상기 배지에, 세포 중의 BMP-의존성 활성을, 동일한 세포 유형에서 평가 시, 상기 억제제 부재 하의 BMP 활성 수준에 비해 90% 이하, 보다 바람직하게는 80% 이하, 보다 바람직하게는 70% 이하, 보다 바람직하게는 50% 이하, 보다 바람직하게는 30% 이하, 보다 바람직하게는 10% 이하, 보다 바람직하게는 0%까지 억제하기에 유효한 양으로 첨가할 수 있다. 숙련가에게 공지된 바와 같이, BMP 활성을, 예를 들어 문헌[Zilberberg et al., 2007. BMC Cell Biol. 8:41]에 예시된 바와 같이, BMP의 전사 활성을 측정함으로써 측정할 수 있다.A BMP inhibitor is added to the medium, and the BMP-dependent activity in the cells, when assessed in the same cell type, is 90% or less, more preferably 80% or less, more preferably 70% or less compared to the level of BMP activity in the absence of the inhibitor. , more preferably 50% or less, more preferably 30% or less, more preferably 10% or less, and more preferably 0% or less. As known to those skilled in the art, BMP activity can be measured, for example, in Zilberberg et al., 2007. BMC Cell Biol. 8:41], by measuring the transcriptional activity of BMP.
천연 BMP-결합 단백질의 여러 부류, 예를 들어 Noggin(Peprotech), 코르딘 및 코르딘 도메인을 포함하는 코르딘-유사 단백질(R&D systems), 폴리스타틴 및 폴리스타틴 도메인을 포함하는 폴리스타틴-관련된 단백질(R&D systems), DAN 및 DAN 시스테인-노트 도메인을 포함하는 DAN-유사 단백질(R&D systems), 스클레로스틴/SOST(R&D systems), 데코린(R&D systems), 및 알파-2 마크로글로불린(R&D systems)이 공지되어 있다. 본 발명에 있어서, 바람직한 상기 BMP 억제제는 Noggin일 수 있다. 본 발명에 있어서, Noggin은 약 10ng/㎖ 내지 약 1000ng/㎖, 바람직하게는 약 50ng/㎖ 내지 약 500ng/㎖, 가장 바람직하게는 약 100ng/㎖로 포함될 수 있으나, 이에 한정되지는 않는다.Several classes of natural BMP-binding proteins include Noggin (Peprotech), Chordin-like proteins containing Chordin and Chordin domains (R&D systems), Follistatin and Follistatin-related proteins containing Follistatin domains. (R&D systems), DAN-like protein containing DAN and DAN cysteine-knot domains (R&D systems), sclerostin/SOST (R&D systems), decorin (R&D systems), and alpha-2 macroglobulin (R&D systems) are known. In the present invention, the preferred BMP inhibitor may be Noggin. In the present invention, Noggin may be included in an amount of about 10ng/ml to about 1000ng/ml, preferably about 50ng/ml to about 500ng/ml, and most preferably about 100ng/ml, but is not limited thereto.
본 발명의 바람직한 양태로서, 상기 배지 조성물은 항산화제를 추가로 포함할 수 있다. 상기 항산화제는 바람직하게는 N-아세틸시스테인(NAC), L-시스테인, 카탈라아제, 슈퍼옥시드 디스뮤타아제 및 2-메르캅토에탄올로 구성된 군으로부터 선택되는 적어도 1종, 더욱 바람직하게는 N-아세틸시스테인 및 L-시스테인으로 구성된 군으로부터 선택되는 적어도 1종을 들 수 있다. 상기 항산화제는 세포사멸 저해 작용을 갖는다. 본 발명에 있어서, 가장 바람직한 항산화제는 N-아세틸시스테인으로, 폐암 유래 세포 배양을 위해서 상기 N-아세틸시스테인은 약 0.1mM, 내지 약 10mM, 바람직하게는 약 0.5mM, 내지 약 5mM, 더 바람직하게는 약 1mM, 내지 약 3mM, 가장 바람직하게는 약 1.25 mM로 포함될 수 있으나, 이에 한정되지는 않는다. In a preferred embodiment of the present invention, the medium composition may further include an antioxidant. The antioxidant is preferably at least one selected from the group consisting of N-acetylcysteine (NAC), L-cysteine, catalase, superoxide dismutase and 2-mercaptoethanol, more preferably N-acetylcysteine. and at least one selected from the group consisting of cysteine and L-cysteine. The antioxidant has an apoptosis inhibitory effect. In the present invention, the most preferred antioxidant is N-acetylcysteine, and for lung cancer-derived cell culture, the N-acetylcysteine is about 0.1mM, to about 10mM, preferably about 0.5mM, to about 5mM, more preferably. It may be included at about 1mM, to about 3mM, and most preferably at about 1.25mM, but is not limited thereto.
본 발명에 있어서, 상기 조성물은 항생제, 글루타민, B-27, 니코틴아마이드(Nicotinamide), 뉴레귤린1(NRG1 또는 Neuregulin 1) 및 A-83-01로 구성된 군에서 하나 이상의 성분을 더 포함하는 것을 특징으로 할 수 있다. In the present invention, the composition further comprises one or more ingredients from the group consisting of antibiotics, glutamine, B-27, nicotinamide, Neuregulin 1 (NRG1 or Neuregulin 1), and A-83-01. You can do this.
본 발명에 있어서, 상기 항생제는 폐암 유래 세포의 성장을 저해하지 않으며, 폐암 유래 세포 이외의 균 성장을 억제할 수 있는 것이라면 어떠한 항생제라도 사용할 수 있다. 예를 들어, 상기 항생제는 페니실린/스트렙토마이신(penicillin/streptomyciin), 사이클로헥사미드(cycloheximide), 세포퍼라존(cefoperazon), 암포테리신(amphotericin) 및 이들의 염(salt)으로 구성된 군으로부터 선택되는 화합물일 수 있으나, 이에 한정되지 않는다. 바람직하게는 상기 항생제는 페니실린/스트렙토마이신(penicillin/streptomyciin) 일 수 있으며, 상기 페니실린과 스트렙토마이신은 각각 약 10㎍/㎖ 내지 약 1000㎍/㎖, 바람직하게는 약 50㎍/㎖ 내지 약 500㎍/㎖, 가장 바람직하게는 약 100㎍/㎖로 포함될 수 있으나, 이에 한정되지는 않는다. In the present invention, the antibiotic does not inhibit the growth of lung cancer-derived cells, and any antibiotic can be used as long as it can inhibit the growth of bacteria other than lung cancer-derived cells. For example, the antibiotic is selected from the group consisting of penicillin/streptomycin, cycloheximide, cefoperazon, amphotericin, and salts thereof. It may be a compound, but is not limited thereto. Preferably, the antibiotic may be penicillin/streptomycin, and the amount of penicillin and streptomycin is each about 10 μg/ml to about 1000 μg/ml, preferably about 50 μg/ml to about 500 μg. /ml, most preferably about 100㎍/㎖, but is not limited thereto.
또한, 본 발명에 바람직한 항생제로서 PrimocinTM을 추가로 포함할 수 있다. PrimocinTM은 세포 배양용 항균제(예를 들면 Invivogene#ant-pm-1, 또는 그것과 동일한 조성을 가지는 다른 제품)이며 미생물 오염에서 세포를 보호하기 위해 사용되는 항생 물질로 그람 양성균, 그람 음성균, 마이코플라스마 및 진균을 죽이는 효과가 있다. 상기 PrimocinTM은 약 1㎍/㎖ 내지 약 1000㎍/㎖, 바람직하게는 약 5㎍/㎖ 내지 약 500㎍/㎖, 더 바람직하게는 약 10㎍/㎖ 내지 약 100㎍/㎖, 가장 바람직하게는 약 50㎍/㎖로 포함될 수 있으나, 이에 한정되지는 않는다. Additionally, Primocin TM may be additionally included as a preferred antibiotic for the present invention. Primocin TM is an antibacterial agent for cell culture (e.g. Invivogene#ant-pm-1, or another product with the same composition) and is an antibiotic used to protect cells from microbial contamination, including gram-positive bacteria, gram-negative bacteria, and mycoplasma. and has the effect of killing fungi. The Primocin TM is about 1㎍/㎖ to about 1000㎍/㎖, preferably about 5㎍/㎖ to about 500㎍/㎖, more preferably about 10㎍/㎖ to about 100㎍/㎖, most preferably It may be included at about 50㎍/㎖, but is not limited thereto.
본 발명에서 상기 글루타민은 GlutaMaxTM(Gibco)를 사용할 수 있고, 이는 0.85% NaCl 상의 200mM L-alanyl-L-glutamine dipeptide (100X)를 1X로 희석하여 사용하거나, 이와 동일한 작용을 하는 상용화된 대체 물질을 사용할 수 있다. In the present invention, the glutamine can be used as GlutaMax TM (Gibco), which is 200mM L-alanyl-L-glutamine dipeptide (100X) in 0.85% NaCl diluted to 1X, or a commercially available alternative with the same action. can be used.
본 발명에서 사용되는 'B27 보충물'(인비트로젠 (미국 캘리포니아주 칼스바드 소재); www.invitrogen.com; 현재 카탈로그 번호 17504-044; 및 PAA 레보라토리즈 GmbH(오스트리아 Pasching 소재); www.paa.com; 카탈로그 번호 F01-002; 문헌[Brewer et al., J Neurosci Res., 35(5):567-76, 1993]로부터 입수할 수 있다)을 사용하여 비오틴, 콜레스테롤, 리놀레산, 리놀렌산, 프로제스테론, 푸트레신, 레티놀, 레티닐 아세테이트, 나트륨 셀레나이트, 트라이-요오도티로닌(T3), DL-알파 토코페롤(비타민 E), 알부민, 인슐린 및 트랜스페린을 포함하는 배양 배지를 제형화할 수 있다. B27 보충물은 PAA 레보라토리즈 GmbH에 의해, 다른 성분들 중에서도 비오틴, 콜레스테롤, 리놀레산, 리놀렌산, 프로제스테론, 푸트레신, 레티놀, 레티닐 아세테이트, 나트륨 셀레나이트, 트라이-요오도티로닌(T3), DL-알파 토코페롤(비타민 E), 알부민, 인슐린 및 트랜스페린을 함유하는 액체 50x 농축물로서 공급된다. 이들 성분 중에서 적어도 리놀렌산, 레티놀, 레티닐 아세테이트 및 트라이-요오도티로닌(T3)은 핵 호르몬 수용체 작용물질이다. B27 보충물을 농축물로서 배양 배지에 첨가하거나 또는 배양 배지에 첨가하기 전에 희석할 수도 있다. 상기 보충물을 1x 최종 농도로 또는 다른 최종 농도로 사용할 수도 있다. B27 보충물의 사용은 비오틴, 콜레스테롤, 리놀레산, 리놀렌산, 프로제스테론, 푸트레신, 레티놀, 레티닐 아세테이트, 나트륨 셀레나이트, 트라이-요오도티로닌(T3), DL-알파 토코페롤(비타민 E), 알부민, 인슐린 및 트랜스페린을 본 발명의 배양 배지에 혼입시키는 편리한 방법이다.The 'B27 supplement' used in the present invention (Invitrogen, Carlsbad, CA, USA; www.invitrogen.com; current catalog number 17504-044; and PAA Laboratories GmbH, Pasching, Austria; www. biotin, cholesterol, linoleic acid, linolenic acid, Culture media can be formulated containing progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri-iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, insulin and transferrin. B27 supplement is manufactured by PAA Laboratories GmbH and contains, among other ingredients, biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri-iodothyronine (T3), DL - Supplied as a liquid 50x concentrate containing alpha tocopherol (vitamin E), albumin, insulin and transferrin. Among these components, at least linolenic acid, retinol, retinyl acetate, and tri-iodothyronine (T3) are nuclear hormone receptor agonists. B27 supplement may be added to the culture medium as a concentrate or may be diluted prior to addition to the culture medium. The supplement may be used at 1x final concentration or other final concentrations. The use of B27 supplements includes biotin, cholesterol, linoleic acid, linolenic acid, progesterone, putrescine, retinol, retinyl acetate, sodium selenite, tri-iodothyronine (T3), DL-alpha tocopherol (vitamin E), albumin, and insulin. and a convenient method for incorporating transferrin into the culture medium of the present invention.
본 발명에 있어서, B27은 B-27 Serum-Free Supplement (50X) liquid (Gibco)를 최종 1X가 되도록 배지에 첨가하는 것이 바람직하나, 이에 한정되지는 않는다. In the present invention, it is preferable to add B-27 Serum-Free Supplement (50X) liquid (Gibco) to the medium so that the final concentration of B27 is 1X, but it is not limited to this.
본 발명은 니코틴아미드(nicotinamide)는 수용성 비타민이며 비타민 B군 중 하나로서 알려진 물질이다. 본 발명에 사용된 니코틴아미드는, 또한 니아신아미드, 니코틴산 아미드, 피리딘-3-카르복실산 아미드, 비타민 B3, 비타민 PP, 3-피리딘카르복사미드, CAS 넘버 98-92-0, 또는 C6H6N2O으로도 지칭될 수 있다. 본 발명에 있어서, 폐암 유래 세포 배양을 위해서 니코틴아미드는 약 0.1mM 내지 약 100mM, 바람직하게는 약 0.5mM 내지 약 50mM, 더 바람직하게는 약 1mM 내지 약 10mM, 가장 바람직하게는 약 5mM로 포함될 수 있으나, 이에 한정되지는 않는다.The present invention relates to nicotinamide, which is a water-soluble vitamin and is known as one of the B vitamins. Nicotinamide used in the present invention may also be niacinamide, nicotinic acid amide, pyridine-3-carboxylic acid amide, vitamin B3, vitamin PP, 3-pyridinecarboxamide, CAS No. 98-92-0, or C 6 H It may also be referred to as 6 N 2O . In the present invention, for lung cancer-derived cell culture, nicotinamide may be included in an amount of about 0.1mM to about 100mM, preferably about 0.5mM to about 50mM, more preferably about 1mM to about 10mM, and most preferably about 5mM. However, it is not limited to this.
본 발명의 바람직한 배지 조성물은 뉴레귤린 1을 추가로 포함할 수 있다. 본 발명의 뉴레귤린 1은 NRG1 (neuregulin-1)와 상호 교환적으로 사용될 수 있으며, NRG1 유전자에 의해 암호화되는 단백질로, EGFR 수용체에 작용한다. 뉴레귤린 1의 단백질 서열 등에 대한 정보는 공지된 데이터베이스(NCBI Reference Sequence: NP_001153467, NP_001153468, NP_001153471, NP_001153473, NP_001153474) 등을 통해 확인할 수 있다. 상기 뉴레귤린 1은 약 0.1nM 내지 약 100nM, 바람직하게는 약 0.5 내지 약 50nM, 더 바람직하게는 약 1nM 내지 약 10nM, 가장 바람직하게는 약 5nM로 포함될 수 있으나, 이에 한정되지는 않는다. A preferred medium composition of the present invention may further include neuregulin 1. Neuregulin 1 of the present invention can be used interchangeably with NRG1 (neuregulin-1), a protein encoded by the NRG1 gene, and acts on the EGFR receptor. Information on the protein sequence of Neuregulin 1 can be confirmed through known databases (NCBI Reference Sequence: NP_001153467, NP_001153468, NP_001153471, NP_001153473, NP_001153474). The neuregulin 1 may be included in an amount of about 0.1 nM to about 100 nM, preferably about 0.5 to about 50 nM, more preferably about 1 nM to about 10 nM, and most preferably about 5 nM, but is not limited thereto.
본 발명의 바람직한 양태로서, 상기 배지 조성물은 Tgf-베타 신호전달(signaling)을 차단 및 억제하는 소분자화합물로 A-83-01을 포함할 수 있다. 이는 인간 세포 또는 오가노이드를 배양하는 경우 특히 유용하다. 본 발명에 있어서, A-83-01은 약 100nM 내지 약 1000nM, 바람직하게는 약 300nM 내지 약 700nM, 가장 바람직하게는 약 500nM로 포함될 수 있으나, 이에 한정되지는 않는다.In a preferred embodiment of the present invention, the medium composition may include A-83-01, a small molecule compound that blocks and inhibits Tgf-beta signaling. This is particularly useful when culturing human cells or organoids. In the present invention, A-83-01 may be included in an amount of about 100nM to about 1000nM, preferably about 300nM to about 700nM, and most preferably about 500nM, but is not limited thereto.
본 발명의 가장 바람직한 양태로서, 상기 배지 조성물은 In the most preferred embodiment of the present invention, the medium composition
어드밴스드 DMEM/F12를 기본 배지로, Advanced DMEM/F12 as the basic medium,
유기 완충액으로, HEPES를 1mM 내지 50 mM;As organic buffer, HEPES at 1mM to 50mM;
성장인자로, 인간 재조합 FGF-7을 1ng/㎖ 내지 20ng/㎖, 인간 재조합 FGF-10을 1ng/ml 내지 50 ng/ml, 인간 재조합 EGF를 1ng/㎖ 내지 20ng/㎖;As growth factors, 1 ng/ml to 20 ng/ml of human recombinant FGF-7, 1 ng/ml to 50 ng/ml of human recombinant FGF-10, and 1 ng/ml to 20 ng/ml of human recombinant EGF;
ROCK 억제제로, Y-27632를 1uM 내지 20uM;As a ROCK inhibitor, Y-27632 at 1uM to 20uM;
Wnt 작용물질로 인간 R-스폰딘 3을 1ng/㎖ 내지 1000ng/㎖;1 ng/ml to 1000 ng/ml of human R-spondin 3 as a Wnt agonist;
BMP 억제제로, Noggin을 10ng/㎖ 내지 1000ng/㎖; As a BMP inhibitor, Noggin at 10ng/ml to 1000ng/ml;
항산화제로, N-아세틸-L-시스테인을 0.1mM 내지 10 mM;As an antioxidant, N-acetyl-L-cysteine at 0.1mM to 10mM;
항생제로, 페니실린/스트렙토마이신을 각각 10 내지 1000 ㎍/㎖ 및/또는 프리모신을 1ug/ml 내지 1000ug/ml;As an antibiotic, 10 to 1000 μg/ml of penicillin/streptomycin and/or 1 μg/ml to 1000 μg/ml of primosin, respectively;
글루타민으로, GlutaMAXTM (1X, Invitrogen);With glutamine, GlutaMAX TM (1X, Invitrogen);
B27 (1X, Gibco); B27 (1X, Gibco);
뉴레귤린1을 0.1nM 내지 100nM;Neuregulin 1 at 0.1nM to 100nM;
니코틴아마이드를 0.1mM 내지 100mM;Nicotinamide at 0.1mM to 100mM;
A-83-01을 100 내지 1000 nM로 포함할 수 있다. It may contain 100 to 1000 nM of A-83-01.
본 발명에 있어서, Noggin, FGF7, FGF10을 배지조성물에 포함하는 경우 상피세포에서 유래된 오가노이드의 성장을 촉진하고, 크기가 큰 오가노이드 생성하며, 오가노이드의 뻗어자람(branching)을 유도한다. In the present invention, when Noggin, FGF7, and FGF10 are included in the medium composition, the growth of organoids derived from epithelial cells is promoted, large-sized organoids are generated, and branching of the organoids is induced.
ROCK inhibitor (Y-27632)과 A83-01을 포함하는 경우 3차원 환경에서 암세포의 증식을 향상시키고, 오가노이드 성장을 연장시킨다. [Endo H. et al., 2013. J Thorac Oncol. Feb;8(2):131-9, Hu Q. et al., 2022. Biofabrication. Apr 13;14(3)., Lee K. et al., 2021. Cells. Nov; 10(11): 3012 ]Containing ROCK inhibitor (Y-27632) and A83-01 improves the proliferation of cancer cells in a 3D environment and prolongs organoid growth. [Endo H. et al., 2013. J Thorac Oncol. Feb;8(2):131-9, Hu Q. et al., 2022. Biofabrication. Apr 13;14(3)., Lee K. et al., 2021. Cells. Nov; 10(11): 3012 ]
특히 Neuregulin-1과 EGF를 배지조성물에 포함시키는 경우 폐암 환자 조직에서 유래된 구 (Cancer tissue-originated spheroids)의 성장 촉진, 오가노이드 크기 조절, WNT signaling 활성 증가를 통해 폐암 오가노이드 배양 및 계대배양 효율을 증진시킨다. 이는 이전에 개재된 문헌을 통해 입증되었다 [ Rabata A. et al., 2020. Front Cell Dev Biol. 8: 574., Chartier C. et al., 2016. Cancer Res. Feb 1;76(3):713-23.]. In particular, when Neuregulin-1 and EGF are included in the medium composition, lung cancer organoid culture and subculture efficiency are promoted by promoting the growth of cancer tissue-originated spheroids, controlling organoid size, and increasing WNT signaling activity. promotes This has been proven through previously published literature [Rabata A. et al., 2020. Front Cell Dev Biol. 8: 574., Chartier C. et al., 2016. Cancer Res. Feb 1;76(3):713-23.].
그리고 R-spondin 3은 LGR5와 결합하여 Wnt signaling을 활성시키는 단백질로, 폐암 환자에서 유래된 PDX 모델에서 R-Spondin 1보다 R-spondin 3가 폐암에서 발현이 높다는 것이 문헌[Chartier C. et al., 2016. Cancer Res. Feb 1;76(3):713-23.]을 통해 입증되었다. And R-spondin 3 is a protein that binds to LGR5 and activates Wnt signaling. In a PDX model derived from a lung cancer patient, R-spondin 3 is expressed more highly in lung cancer than R-Spondin 1, according to the literature [Chartier C. et al. , 2016. Cancer Res. Feb 1;76(3):713-23.].
본 발명에 있어서, R-spondin 3를 배지조성물에 포함하는 경우 WNT signaling의 활성을 통해 폐암 오가노이드 형성의 효율을 증진시킨다.In the present invention, when R-spondin 3 is included in the medium composition, the efficiency of lung cancer organoid formation is improved through the activation of WNT signaling.
본 발명에 있어서, % 농도는 v/v, w/v 또는 w/w 일 수 있다. In the present invention, the % concentration may be v/v, w/v or w/w.
상기와 같은 배지 조성물을 이용하는 경우, 본 발명은 폐암 유래 세포의 종양-구(tumor-sphere) 형성을 촉진시키는 효과가 우수하다.When using the above-described medium composition, the present invention is excellent in promoting tumor-sphere formation of lung cancer-derived cells.
그 밖에도, 본 발명의 배지 조성물에는 다양한 화합물(compound) 및 원소(element)가 포함될 수 있고, 이러한 화합물 및 원소들은 당업계에서 통상의 지식을 가진 당업자에게 공지되어 있다. 예를 들어, 본 발명의 배지 조성물에 포함되는 성분은 수소 공여자(hydrogen donor) 및 수용자(acceptor)의 소스(source), 탄소, 질소, 황(sulfur), 인(phosphorus), 무기염, 비타민 및 아미노산을 포함하나 이에 한정되지 않는다. 또한, 배지 조성물에는 탄수화물, 다양한 염(칼슘, 마그네슘, 망간, 나트륨, 칼륨, 인 및 황의 염을 포함하나 이에 한정되지 않는다)이 첨가될 수 있다.In addition, the medium composition of the present invention may contain various compounds and elements, and these compounds and elements are known to those skilled in the art. For example, the components included in the medium composition of the present invention include sources of hydrogen donors and acceptors, carbon, nitrogen, sulfur, phosphorus, mineral salts, vitamins, and Including, but not limited to, amino acids. Additionally, carbohydrates and various salts (including but not limited to salts of calcium, magnesium, manganese, sodium, potassium, phosphorus, and sulfur) may be added to the medium composition.
한편, 본 발명은 다른 관점에서, 상기 조성물을 이용하여 폐암 유래 세포를 배양하는 단계를 포함하는 폐암 오가노이드 배양 방법에 관한 것이다.Meanwhile, from another perspective, the present invention relates to a method of culturing lung cancer organoids, including culturing lung cancer-derived cells using the composition.
본 발명에 있어서, 상기 폐암 유래 세포는 In the present invention, the lung cancer-derived cells are
세포 배양액이 수용될 수 있는 수용 공간이 형성되어 있고, 배양 대상체인 세포가 놓여지는 부분으로, 평평한 바닥으로부터 일정 높이를 갖도록 기둥 형태로 돌출된 복수의 필라부들; 및A receiving space in which a cell culture medium can be accommodated is formed, and a plurality of pillars protruding from a flat floor in the form of a pillar to have a certain height are formed as a part where cells, which are culture objects, are placed; and
수용 공간에 놓여진 배양액이 평평한 바닥에 흩어져 잔류하지 않고 한쪽에 모여 있을 수 있도록, 평평한 바닥에 대해 볼록하게 형성된 필라부들과는 반대로 오목하게 형성되는 트렌치홈부;를 포함하는 필라 플레이트에서 배양하는 것을 특징으로 할 수 있다. Characterized by culturing on a pillar plate including a trench groove formed concavely as opposed to the pillars formed convexly with respect to the flat floor so that the culture medium placed in the receiving space can be gathered on one side rather than scattered and remaining on the flat floor. can do.
본 발명에 있어서, 상기 필라 플레이트는 필라부 상부에 세포외 기질이 코팅된 것일 수 있으며, 상기 세포외 기질에는 본 발명에 따른 배양 대상체인 폐암 유래 세포가 군집된 형태로 배양되는 것을 특징으로 할 수 있다. In the present invention, the pillar plate may be coated with an extracellular matrix on the upper part of the pillar, and the extracellular matrix may be characterized in that lung cancer-derived cells, which are culture subjects according to the invention, are cultured in a clustered form. there is.
즉, 본 발명에 따른 상기 조성물은 특히 상기 필라 플레이트 상에서 폐암 오가노이드를 효과적으로 배양할 수 있는 특징이 있다. That is, the composition according to the present invention has the characteristic of effectively cultivating lung cancer organoids, especially on the pillar plate.
상기 폐암 오가노이드 배양 방법에 있어서, 상기 필라 플레이트에서 배양하는 단계 전 단계에서, 상기 폐암 유래 세포는 상기 세포외 기질의 하부에 군집된 형태로 배양되기 위하여, 중력 또는 외부 힘으로 세포외 기질에 퍼져 있는 세포를 한곳으로 모으는 세포 응집 단계를 추가로 포함할 수 있다.In the lung cancer organoid culture method, in the step before culturing on the pillar plate, the lung cancer-derived cells are spread in the extracellular matrix by gravity or external force to be cultured in a clustered form at the bottom of the extracellular matrix. A cell aggregation step that brings together cells in one place may be additionally included.
구체적으로, 상기 세포 응집 단계는 0-5℃, 예컨대, 약 4℃에서 액상의 세포외 기질에 폐암 유래 세포를 첨가하고 5 내지 30분, 바람직하게는 7 내지 20분, 예컨대 약 10분간 방치하여 세포가 중력에 의해 하부로 응집되도록 하는 단계로, 세포가 하부로 응집되면, 30-40℃, 예컨대, 약 36℃에서 첨가하고 5 내지 30분, 바람직하게는 7 내지 20분, 예컨대 약 10분간 방치하여 세포외 기질을 굳히는 과정으로 진행될 수 있다.Specifically, the cell aggregation step involves adding lung cancer-derived cells to a liquid extracellular matrix at 0-5°C, for example, about 4°C, and leaving it for 5 to 30 minutes, preferably 7 to 20 minutes, for example, about 10 minutes. A step of allowing the cells to aggregate downward by gravity. When the cells aggregate downward, add at 30-40°C, such as about 36°C, and stir for 5 to 30 minutes, preferably 7 to 20 minutes, such as about 10 minutes. If left untreated, it can progress to a process of hardening the extracellular matrix.
본 발명에 있어서, 상기 필라 플레이트에서 배양하는 경우, 하나의 필라당 1000 내지 10000 개의 세포를 분주하여 배양하는 것을 특징으로 할 수 있으나, 이에 한정되지는 않는다. In the present invention, when culturing on the pillar plate, 1,000 to 10,000 cells may be distributed and cultured per pillar, but the culture is not limited to this.
본 발명에서 용어 "필라 플레이트"란 판상 구조를 가지는 기판부; 상기 기판부와 일체로 형성되고 상기 기판부의 하부면에서 하방으로 돌출되어 웰에 삽입되는 삽입 필라부; 및 상기 기판부와 일체로 형성되고 상기 삽입 필라부에 대응하여 상기 기판부의 상부면에서 상방으로 돌출되는 보상 필라부를 포함하는 세포 배양용 플레이트를 의미한다.In the present invention, the term “pillar plate” refers to a substrate portion having a plate-shaped structure; an insertion pillar portion formed integrally with the substrate portion and protruding downward from a lower surface of the substrate portion and inserted into the well; and a compensating pillar part formed integrally with the substrate part and protruding upward from the upper surface of the substrate part in response to the insertion pillar part.
본 발명에 따른 필라 플레이트로는 일 양태로서 대한민국 등록특허 제10-1632426호 또는 대한민국 등록특허 제10-2463531호에 기재된 바이오 칩용 필라 구조체를 적절히 변형하여 사용할 수 있고, 따라서 대한민국 등록특허 제10-1632426호 또는 대한민국 등록특허 제 10-2463531호에 기재된 전문이 본 발명을 실시하기 위한 하나의 양태로서 본 발명에 통합된다.In one embodiment, the pillar plate according to the present invention can be used by appropriately modifying the pillar structure for a biochip described in Republic of Korea Patent No. 10-1632426 or Republic of Korea Patent No. 10-2463531, and thus, Republic of Korea Patent No. 10-1632426. or Republic of Korea Patent No. 10-2463531 is incorporated into the present invention as an aspect for carrying out the present invention.
본 발명에 있어서, 상기 배양은 마트리겔TM을 이용한 3차원 배양일 수 있다. In the present invention, the culture may be a three-dimensional culture using Matrigel TM .
본 발명은 또 다른 관점에서, 상기 배양 방법에 의해 배양된 폐암 오가노이드 및 이의 용도에 관한 것이다.In another aspect, the present invention relates to lung cancer organoids cultured by the above culture method and their use.
본 발명에 따라 배양된 폐암 오가노이드는 치료제 선별, 독성 분석에서 이용될 수 있다. 세포 기본 독성 시험을 기관 특이성 세포 독성의 측정에 사용한다. 상기 시험에서 시험되는 화합물은 암 화학치료제일 수 있다. 본 발명에 있어서, 상기 방법은 상기 세포를 일정한 기간 동안 배수 농도의 시험제에 노출시키고, 시험제의 다양한 농도 범위를 1 내지 15일 간 노출 및 최고 용해 농도로부터의 로그 희석을 사용하여 예비 분석에서 측정하고, 상기 노출 기간의 끝에서, 상기 배양물을 생육 억제에 대해 평가할 수 있다. Lung cancer organoids cultured according to the present invention can be used in therapeutic screening and toxicity analysis. Cell-based toxicity tests are used to measure organ-specific cytotoxicity. The compound tested in the test may be a cancer chemotherapy agent. In the present invention, the method involves exposing the cells to multiple concentrations of the test agent for a period of time, exposure to a range of different concentrations of the test agent for 1 to 15 days, and log dilution from the highest lytic concentration in a preliminary assay. At the end of the exposure period, the cultures can be evaluated for growth inhibition.
따라서, 본 발명은 또 다른 관점에서, 다음 단계를 포함하는 폐암 치료제 스크리닝 방법에 관한 것이다.Accordingly, from another aspect, the present invention relates to a method for screening a therapeutic agent for lung cancer comprising the following steps.
(a) 상기 폐암 오가노이드에 폐암 치료제 후보 물질을 처리하는 단계; 및(a) treating the lung cancer organoid with a candidate lung cancer treatment material; and
(b) 상기 후보 물질 중 폐암 세포사멸 효과를 유도하는 물질을 폐암 치료제로 선별하는 단계. (b) Selecting a substance that induces a lung cancer cell death effect among the candidate substances as a treatment for lung cancer.
본 발명에 있어서, 상기 폐암 치료제는 환자 맞춤형 폐암 치료제인 것을 특징으로 할 수 있다.In the present invention, the lung cancer treatment may be characterized as a patient-tailored lung cancer treatment.
[실시예][Example]
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.
본 발명의 일 실시예에 따른 배지 조성 및 종래 폐암 유래 세포 배양을 위해 사용되는 배지 조성(비교예 1 내지 비교예 5)은 다음과 같다.The composition of the medium according to an embodiment of the present invention and the composition of the medium used for culturing conventional lung cancer-derived cells (Comparative Examples 1 to 5) are as follows.
실시예Example
100 μg/ml100 μg/ml,
100 μg/ml
비교예 1Comparative Example 1
100 μg/ml100 μg/ml,
100 μg/ml
Nat Commun 2019 Sep 5;10(1):3991Nat Commun 2019 Sep 5;10(1):3991
비교예 2Comparative Example 2
100 μg/ml100 μg/ml,
100 μg/ml
EMBO J 2019 Feb 15;38(4):e100300EMBO J 2019 Feb 15;38(4):e100300
비교예 3Comparative Example 3
100 μg/ml100 μg/ml,
100 μg/ml
비교예 4Comparative Example 4
100 μg/ml100 μg/ml,
100 μg/ml
비교예 5Comparative Example 5
100 μg/ml100 μg/ml,
100 μg/ml
실험예 1. 세포 성장능 확인 Experimental Example 1. Confirmation of cell growth ability
폐암 환자 유래 종양 조직은 환자의 동의 하에 수득하였다(승인번호 KC18TNSI0033). 각기 다른 환자 3명의 폐암 시료를 이용하여 3000cells/ul 단위로 각각 실시예 및 비교예의 배지를 이용하여 오가노이드 배양을 시도하였다.Tumor tissue derived from a lung cancer patient was obtained with the patient's consent (approval number KC18TNSI0033). Organoid culture was attempted using lung cancer samples from three different patients at 3000 cells/ul using the media of the examples and comparative examples, respectively.
종양 조직을 < ~1mm로 잘게 분해하고, HBSS-DF 1㎖를 함유한 에펜도르프 튜브에 옮겨 두었다. 37℃에서 10분간 인큐베이션 하며 800rpm으로 혼합하고, 상온에서 10초간 스핀 다운한 후, 상층액을 제거하였다. 펠렛을 1㎖의 0.25% trypsin-EDTA로 재현탁시키고, 37℃에서 10분간 인큐베이션 하며 800rpm으로 혼합하였다. 트립신 처리된 조직을 100-μm stainer로 필터링하고, 8㎖ STI를 함유하는 50㎖ 튜브에 넣어 주었다. 필터링된 세포 현탁액 8㎖을 세척하고, 188 × g로 5분간 4℃에서 원심분리하였다. Tumor tissue was broken down into pieces <~1 mm and transferred to an Eppendorf tube containing 1 ml of HBSS-DF. Incubate at 37°C for 10 minutes, mix at 800 rpm, spin down for 10 seconds at room temperature, and then remove the supernatant. The pellet was resuspended in 1 ml of 0.25% trypsin-EDTA, incubated at 37°C for 10 minutes, and mixed at 800 rpm. The trypsinized tissue was filtered with a 100-μm stainer and placed in a 50 ml tube containing 8 ml STI. 8 ml of filtered cell suspension was washed and centrifuged at 188 × g for 5 minutes at 4°C.
실험에 필요한 세포수 (필라당 5 x 103 cell)를 계산하여 세포를 마이크로튜브로 옮긴 후 Matrigel®(Corning)과 2:8 비율로 혼합하였다. 필라 플레이트(Cellvitro™(주)엠비디)의 각 필라에 세포를 1.5ul씩 토출한 후, 빈 웰 플레이트에 결합시켰다. 세포는 플레이트 채로 4℃로 옮겨져 15분간 아이싱 작업을 통해 세포를 중앙으로 군집시키고, 그 후 37℃ Incubator로 옮겨 1시간 동안 젤화 과정을 진행하였다. 젤화 과정 동안 37℃의 실시예 또는 비교예 배지를 웰 플레이트에 40ul씩 분주 하고, 젤화가 끝난 세포는 배지가 들어있는 웰 플레이트와 결합하여 3일간 배양하였다. The number of cells required for the experiment ( 5 After dispensing 1.5ul of cells into each pillar of the pillar plate (Cellvitro™ MBD Co., Ltd.), Binding was performed on an empty well plate. The cells were transferred to the plate at 4°C and clustered in the center through icing for 15 minutes. Then, they were transferred to a 37°C incubator and gelation process was performed for 1 hour. During the gelation process, 40 ul of Example or Comparative Example medium at 37°C was dispensed into each well plate, and the gelated cells were combined with the well plate containing the medium and cultured for 3 days.
필라 플레이트에서 배양한 세포의 이미지는 ASFA scanner로 촬영하여 나타내었다(도 1). Images of cells cultured on pillar plates were captured and displayed with an ASFA scanner (Figure 1).
또한, Calcein-AM staining Kit (MBD Co. 한국)를 이용하여 제조사의 지침에 따라 살아있는 세포만을 염색하고 ASFA scanner을 이용하여 형광 이미지를 관찰하였다(도 2, 도 4). 또한, 이미지의 형광강도를 ASFA analyzer를 통해 측정하고, 실시예 및 비교예의 형광강도를 염색된 세포 이미지 전체 형광강도 값(Area) 혹은 염색된 전체 세포의 평균 형광강도(Mean area) 값으로 비교하여 세포 생존율을 그래프로 나타내었다(도 3, 도 5). In addition, only living cells were stained using Calcein-AM staining Kit (MBD Co. Korea) according to the manufacturer's instructions, and fluorescence images were observed using an ASFA scanner (Figures 2 and 4). In addition, the fluorescence intensity of the image was measured using an ASFA analyzer, and the fluorescence intensity of the examples and comparative examples was compared with the total fluorescence intensity value (Area) of the stained cell image or the average fluorescence intensity (Mean area) of all stained cells. Cell viability was graphed (Figures 3 and 5).
그 결과, 도 1 내지 도 5에서 확인할 수 있듯이, 본 발명에 따른 배지 조성물은 종래 알려진 폐암 세포 배양 배지에 비해 오가노이드 생성 효과가 현저히 우수한 것이 확인되었다. As a result, as can be seen in Figures 1 to 5, it was confirmed that the medium composition according to the present invention had a significantly better organoid production effect compared to conventionally known lung cancer cell culture media.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
Claims (16)
상기 성장인자는 FGF7, FGF10 및 EGF이고, 상기 조성물은 인간 재조합 FGF-7을 1ng/㎖ 내지 20ng/㎖, 인간 재조합 FGF-10을 1ng/ml 내지 50 ng/ml, 인간 재조합 EGF를 1ng/㎖ 내지 20ng/㎖로 포함하는 것을 특징으로 하고,
상기 Wnt 작용물질은 R-스폰딘 3이고, 상기 R-스폰딘 3는 1ng/㎖ 내지 1000ng/㎖로 포함되는 것을 특징으로 하며,
상기 BMP 억제제는 Noggin이고, 상기 Noggin은 10 ng/㎖ 내지 1000 ng/㎖로 포함되는 것을 특징으로 하는, 폐암 유래 세포 배양용 배지 조성물.
A medium composition for culturing lung cancer-derived cells containing growth factors, ROCK inhibitors, Wnt agonists, BMP inhibitors, and antioxidants,
The growth factors are FGF7, FGF10, and EGF, and the composition contains 1 ng/ml to 20 ng/ml of human recombinant FGF-7, 1 ng/ml to 50 ng/ml of human recombinant FGF-10, and 1 ng/ml of human recombinant EGF. Characterized in that it contains from 20ng/ml,
The Wnt agonist is R-spondin 3, and the R-spondin 3 is contained in an amount of 1 ng/ml to 1000 ng/ml,
The BMP inhibitor is Noggin, and the Noggin is contained in an amount of 10 ng/ml to 1000 ng/ml. A medium composition for culturing lung cancer-derived cells.
The composition according to claim 1, wherein the medium composition includes Advanced DMEM/F12 medium as a base medium and growth factors, ROCK inhibitors, Wnt agonists, BMP inhibitors, and antioxidants.
The composition of claim 1, wherein the composition further comprises one or more ingredients selected from the group consisting of antibiotics, glutamine, B-27, nicotinamide, neuregulin 1, and A-83-01.
The composition of claim 1, wherein the composition further comprises an organic buffer.
The composition of claim 1, wherein the ROCK inhibitor is Y-27632, and Y-27632 is contained in an amount of 1uM to 20uM.
The composition according to claim 1, wherein the antioxidant is N-acetylcysteine, and the N-acetylcysteine is contained in an amount of 0.1mM to 10mM.
The composition according to claim 1, wherein the composition promotes tumor-sphere formation.
A method of culturing lung cancer organoids comprising culturing lung cancer-derived cells using the composition of any one of claims 1, 2, 5 to 7, 10, and 11.
세포 배양액이 수용될 수 있는 수용 공간이 형성되어 있고, 배양 대상체인 세포가 놓여지는 부분으로, 평평한 바닥으로부터 일정 높이를 갖도록 기둥 형태로 돌출된 복수의 필라부들; 및
수용 공간에 놓여진 배양액이 평평한 바닥에 흩어져 잔류하지 않고 한쪽에 모여 있을 수 있도록, 평평한 바닥에 대해 볼록하게 형성된 필라부들과는 반대로 오목하게 형성되는 트렌치홈부;를 포함하는 필라 플레이트에서 배양하는 것을 특징으로 하는, 폐암 오가노이드 배양 방법.
The method of claim 12, wherein the lung cancer-derived cells are
A receiving space in which a cell culture medium can be accommodated is formed, and a plurality of pillars protruding from a flat floor in the form of a pillar to have a certain height are formed as a part where cells, which are culture objects, are placed; and
Characterized by culturing on a pillar plate including a trench groove formed concavely as opposed to the pillars formed convexly with respect to the flat floor so that the culture medium placed in the receiving space can be gathered on one side rather than scattered and remaining on the flat floor. Method for culturing lung cancer organoids.
Lung cancer organoids cultured by the culture method of claim 12.
(a) 제14항의 폐암 오가노이드에 폐암 치료제 후보 물질을 처리하는 단계; 및
(b) 상기 후보 물질 중 폐암 세포사멸 효과를 유도하는 물질을 폐암 치료제로 선별하는 단계.
A method of screening for a treatment for lung cancer comprising the following steps:
(a) treating the lung cancer treatment candidate material in the lung cancer organoid of item 14; and
(b) Selecting a substance that induces a lung cancer cell death effect among the candidate substances as a treatment for lung cancer.
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