KR20240052103A - Method of producing tonsillar mesenchymal stem cells - Google Patents
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Abstract
본 발명은 편도 유래 중간엽 줄기세포 제조 방법에 관한 것으로, 구체적으로피콜-하이파크(Ficoll-Hypaque) 및 퍼콜(Percoll) 구배로 편도 조직에서 세포를 분리, 정제 및 배양하여 편도 유래 중간엽 줄기세포를 제조하는 방법에 관한 것이다.
The present invention relates to a method for producing tonsil-derived mesenchymal stem cells, and specifically, to produce tonsil-derived mesenchymal stem cells by isolating, purifying, and culturing cells from tonsil tissue using Ficoll-Hypaque and Percoll gradients. It relates to a method of manufacturing.
Description
본 발명은 편도 유래 중간엽 줄기세포 제조 방법에 관한 것이다.The present invention relates to a method for producing tonsil-derived mesenchymal stem cells.
줄기 세포를 이용한 치료법은 자가증식력과 다분화능을 가진 줄기세포를 이용하여 손상된 세포의 재생을 도움으로써 환자의 질병을 치료하고 증상을 개선하는 치료법으로, 세포치료에 이용될 수 있는 줄기세포는 크게 성체줄기세포, 배아줄기세포, 역분화줄기세포로 나눌 수 있다. 각 세포들 마다 장단점이 있으나 현재 임상에서 치료목적으로 주로 이용되고 있는 것은 성체줄기세포이다.Stem cell therapy is a treatment method that treats a patient's disease and improves symptoms by helping to regenerate damaged cells using stem cells with self-proliferation and multipotency. Stem cells that can be used in cell therapy are largely adult cells. It can be divided into stem cells, embryonic stem cells, and pluripotent stem cells. Although each cell has its pros and cons, adult stem cells are currently mainly used for therapeutic purposes in clinical practice.
성체줄기세포는 우리 몸의 다양한 장기에 있으면서, 신체가 손상되었을 때 재생작용을 하는 세포로서 골수, 탯줄을 포함해서 거의 모든 장기에 존재하는 다양한 줄기세포를 통틀어 성체줄기세포라고 한다. 성체줄기세포는 분화될 수 있는 세포의 범위가 비교적 정해져 있어 조직-특이적 줄기세포라고도 하며, 의학적으로 비교적 안전한 세포로 알려져 있어 다양한 임상시험에 사용되고 있다.Adult stem cells are cells that exist in various organs of our body and perform regenerative functions when the body is damaged. The various stem cells that exist in almost all organs, including the bone marrow and umbilical cord, are collectively called adult stem cells. Adult stem cells are also called tissue-specific stem cells because the range of cells that can be differentiated is relatively limited. They are known to be medically relatively safe cells and are used in various clinical trials.
한편, 인간의 편도선은 일종의 림프상피 면역조직으로서 인체의 구인두 비인두에 존재하는데, 사춘기까지 그 면역기능을 수행하여 이후에는 서서히 퇴화하므로, 편도선이 비정상적으로 비대해지거나(아데노이드편도 과형성: adenotonsillar hyperplasia), 감염(편도염: tonsillitis)되는 등의 문제가 발생하면 편도선 절제수술(tonsillectomy)을 통해 제거하게 된다.Meanwhile, the human tonsils are a type of lymphoepithelial immune tissue that exists in the oropharynx of the human body. They perform their immune function until puberty and then gradually degenerate, causing the tonsils to become abnormally enlarged (adenotonsillar hyperplasia). If problems such as infection (tonsillitis) occur, the tonsils are removed through tonsillectomy.
최근 들어, 편도선의 조직에서 중간엽 줄기세포(Mesenchymal stem cells)를 추출한 연구결과가 보고되어, 상기 편도 조직이 성체줄기세포의 새로운 공급원으로 주목받고 있다. 상기 편도 조직으로부터 유래된 줄기세포는 다른 성체줄기세포와 동일한 수준의 줄기세포능을 나타내어, 중배엽성 세포(지방세포형성(adipogenesis), 골세포형성(osteogenesis), 연골세포형성(chondrogenesis))로 분화될 수 있다. 이러한 편도 조직의 장점은 편도선 절제수술을 통해 버려지는 조직이기 때문에 재료의 수급이 비교적 원활하다는 점이다. 그러나, 편도 조직으로부터 유래된 줄기세포는 치료적으로 유효한 양으로 확보하기 어렵기 때문에 그의 활용이 제한되고 있다.Recently, research results on extracting mesenchymal stem cells from tonsil tissue were reported, and tonsil tissue is attracting attention as a new source of adult stem cells. Stem cells derived from the tonsil tissue exhibit the same level of stem cell ability as other adult stem cells and differentiate into mesodermal cells (adipogenesis, osteogenesis, chondrogenesis). It can be. The advantage of this tonsil tissue is that since it is tissue discarded through tonsillectomy surgery, the supply and demand of the material is relatively smooth. However, since it is difficult to secure stem cells derived from tonsil tissue in a therapeutically effective amount, their use is limited.
이러한 배경하에, 본 발명자들은 편도 조직으로부터 치료적으로 유효한 양으로 줄기세포를 확보할 수 있는 방법을 개발하기 위해 노력한 결과, 피콜-하이파크(Ficoll-Hypaque) 및 퍼콜(Percoll) 구배로 편도 조직에서 세포를 분리, 정제 및 배양하여 다량의 편도 유래 중간엽 줄기세포를 제조할 수 있음을 확인하였다. 이에 상기 방법 및 이로부터 제조된 중간엽 줄기세포를 줄기세포치료제 개발에 유용하게 이용할 수 있음을 밝힘으로써, 본 출원에 이르게 되었다.Against this background, the present inventors made efforts to develop a method to secure stem cells in a therapeutically effective amount from tonsil tissue, and as a result, they were obtained from tonsillar tissue using Ficoll-Hypaque and Percoll gradients. It was confirmed that a large amount of tonsil-derived mesenchymal stem cells could be produced by separating, purifying, and culturing the cells. Accordingly, by revealing that the above method and the mesenchymal stem cells produced therefrom can be usefully used in the development of stem cell therapy, the present application has been made.
본 발명의 목적은 편도 유래 중간엽 줄기세포 제조 방법을 제공하는 것이다.The purpose of the present invention is to provide a method for producing tonsil-derived mesenchymal stem cells.
본 발명의 목적을 달성하기 위하여, 본 발명은In order to achieve the purpose of the present invention, the present invention
1) 분리된 편도 조직에 평형염액(balanced salt solution)을 넣고 세절 및 여과하여 세포 부유액을 획득하는 단계;1) Adding a balanced salt solution to the separated tonsil tissue, chopping and filtering to obtain a cell suspension;
2) 단계 1)에서 획득한 세포 부유액에서 피콜-하이파크(Ficoll-Hypaque)로 단핵세포를 분리하는 단계;2) separating mononuclear cells from the cell suspension obtained in step 1) using Ficoll-Hypaque;
3) 단계 2)에서 분리한 단핵세포를 65 내지 55%, 55 내지 45%, 35 내지 25% 및 20 내지 10% 농도의 퍼콜(Percoll) 구배한 후 35 내지 25% 및 20 내지 10% 농도의 퍼콜 사이의 세포를 회수하여 단핵세포를 정제하는 단계; 및3) The mononuclear cells isolated in step 2) were subjected to a Percoll gradient at concentrations of 65 to 55%, 55 to 45%, 35 to 25%, and 20 to 10%, and then subjected to a Percoll gradient at concentrations of 35 to 25% and 20 to 10%. Recovering cells between percolls and purifying mononuclear cells; and
4) 단계 3)의 정제한 단핵세포를 배양하는 단계를 포함하는, 편도 유래 중간엽 줄기세포 제조 방법을 제공한다.4) It provides a method for producing tonsil-derived mesenchymal stem cells, comprising culturing the purified mononuclear cells of step 3).
본 발명에서는피콜-하이파크(Ficoll-Hypaque) 및 퍼콜(Percoll) 구배로 편도 조직에서 세포를 분리, 정제 및 배양하여 다량의 편도 유래 중간엽 줄기세포를 제조할 수 있음을 확인하였으므로, 본 발명에 따른 방법 및 이로부터 제조된 중간엽 줄기세포는 줄기세포치료제 개발에 유용하게 이용될 수 있다.In the present invention, it has been confirmed that a large amount of tonsil-derived mesenchymal stem cells can be produced by separating, purifying, and culturing cells from tonsil tissue using Ficoll-Hypaque and Percoll gradients. The method according to the method and the mesenchymal stem cells produced therefrom can be usefully used in the development of stem cell therapy.
도 1은 본 발명의 일 실시예에 따라 편도 조직을 세절하는 과정을 나타낸 도이다.
도 2는 도 1의 과정에서 세절한 편도 조직을 본 발명의 일 실시예에 따라 더 잘게 세절하는 과정을 나타낸 도이다.
도 3은 본 발명의 일 실시예에 따라 세절한 조직을 인큐베이션(incubation)하는 과정을 나타낸 도이다.
도 4는 본 발명의 일 실시예에 따라 편도 조직을 세절 및 여과하여 획득한 세포 부유액을피콜-하이파크(Ficoll-Hypaque)에 적재하는 과정을 나타낸 도이다.
도 5는 본 발명의 일 실시예에 따라 세포 부유액에서 단핵세포를 분리하는 과정을 나타낸 도이다.
도 6은 본 발명의 일 실시예에 따라 분리한 단핵세포를 포함하는 세포 침전물을 퍼콜(Percoll) 구배 상층액에 적재하는 과정을 나타낸 도이다.
도 7은 본 발명의 일 실시예에 따라 정제된 단핵세포를 회수하는 과정을 나타낸 도이다.
Figure 1 is a diagram showing the process of cutting tonsil tissue according to an embodiment of the present invention.
Figure 2 is a diagram showing a process of cutting the tonsil tissue finely chopped in the process of Figure 1 into finer pieces according to an embodiment of the present invention.
Figure 3 is a diagram showing the process of incubating finely chopped tissue according to an embodiment of the present invention.
Figure 4 is a diagram showing the process of loading the cell suspension obtained by cutting and filtering tonsillar tissue into Ficoll-Hypaque according to an embodiment of the present invention.
Figure 5 is a diagram showing the process of separating mononuclear cells from a cell suspension according to an embodiment of the present invention.
Figure 6 is a diagram showing the process of loading cell sediment containing mononuclear cells isolated according to an embodiment of the present invention into a Percoll gradient supernatant.
Figure 7 is a diagram showing the process of recovering purified mononuclear cells according to an embodiment of the present invention.
이하, 본 발명을 보다 상세히 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명은This invention
1) 분리된 편도 조직에 평형염액(balanced salt solution)을 넣고 세절 및 여과하여 세포 부유액을 획득하는 단계;1) Adding a balanced salt solution to the separated tonsil tissue, chopping and filtering to obtain a cell suspension;
2) 단계 1)에서 획득한 세포 부유액에서 피콜-하이파크(Ficoll-Hypaque)로 단핵세포를 분리하는 단계;2) separating mononuclear cells from the cell suspension obtained in step 1) using Ficoll-Hypaque;
3) 단계 2)에서 분리한 단핵세포를 65 내지 55%, 55 내지 45%, 35 내지 25% 및 20 내지 10% 농도의 퍼콜(Percoll) 구배한 후 35 내지 25% 및 20 내지 10% 농도의 퍼콜 사이의 세포를 회수하여 단핵세포를 정제하는 단계; 및3) The mononuclear cells isolated in step 2) were subjected to a Percoll gradient at concentrations of 65 to 55%, 55 to 45%, 35 to 25%, and 20 to 10%, and then subjected to a Percoll gradient at concentrations of 35 to 25% and 20 to 10%. Recovering cells between percolls and purifying mononuclear cells; and
4) 단계 3)의 정제한 단핵세포를 배양하는 단계를 포함하는, 편도 유래 중간엽 줄기세포 제조 방법을 제공한다.4) It provides a method for producing tonsil-derived mesenchymal stem cells, comprising culturing the purified mononuclear cells of step 3).
본 발명에서, 용어 "편도 조직"이란, 목의 안쪽과 코의 뒷부분에 위치하여, 외부에서 침입하는 세균 등의 물질로부터 일차적으로 우리 몸을 방어함과 동시에 림프상피 면역조직으로 작용을 수행하는 조직을 의미한다. 상기 편도 조직은 인두편도, 귀인두관편도, 구개편도, 혀편도 등을 포함한다. 본 발명의 목적상 상기 편도 조직은 편도 유래 중간엽 줄기세포를 제공하는 원천조직으로 사용되지만, 특별히 이에 제한되지는 않는다.In the present invention, the term "tonsil tissue" refers to a tissue located inside the throat and the back of the nose that primarily defends the body from substances such as bacteria invading from the outside and at the same time acts as a lymphoepithelial immune tissue. means. The tonsillar tissue includes pharyngeal tonsils, otic pharyngeal tonsils, palatine tonsils, and lingual tonsils. For the purpose of the present invention, the tonsil tissue is used as a source tissue for providing tonsil-derived mesenchymal stem cells, but is not particularly limited thereto.
본 발명에서, 용어 "중간엽 줄기세포(mesenchymal stem cells; MSCs)"란, 골, 연골, 지방, 골수간질, 근육, 신경 등으로 분화될 수 있는 미분화된 줄기세포를 의미하는데, 성인에서는 일반적으로 골수에 머물러 있지만 제대혈, 말초혈액, 기타 조직 등에도 존재한다. 본 발명의 목적상 상기 중간엽 줄기세포는 특별히 이에 제한되지 않으나, 사람의 편도로부터 유래되어 서로 다른 개체에서 유래된 각각의 중간엽 줄기세포와 혼합배양이 가능한 세포가 될 수 있고, 지방세포, 골세포, 연골세포 등의 중배엽성 조직세포로 분화될 수 있을 뿐만 아니라, 부갑상선 조직세포 등의 내배엽성 조직세포로도 분화될 수 있다.In the present invention, the term "mesenchymal stem cells (MSCs)" refers to undifferentiated stem cells that can be differentiated into bone, cartilage, fat, bone marrow stroma, muscle, nerve, etc., and in adults, they are generally It remains in the bone marrow, but is also present in umbilical cord blood, peripheral blood, and other tissues. For the purpose of the present invention, the mesenchymal stem cells are not particularly limited thereto, but may be cells derived from human tonsils and capable of mixed culture with respective mesenchymal stem cells derived from different individuals, adipocytes, bone Not only can it be differentiated into mesodermal tissue cells such as cells and cartilage cells, but it can also be differentiated into endodermal tissue cells such as parathyroid tissue cells.
이하, 본 발명을 실시예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by examples.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.
[실시예 1] 시료 준비[Example 1] Sample preparation
편도 유래 줄기세포 생산을 위해 하기의 시료를 제조 및 준비하였다.The following samples were prepared and prepared for the production of tonsil-derived stem cells.
세포배양배지로10% FBS(Cat No. FP-0500-A, ATLAS) 및 1% Antibiotic-Antimycotic(Cat No. 15240-062, gibco)를 포함하는 DMEM-low glucose(Cat No. 12-707F, Lonza) 배지를 제조하였다.As a cell culture medium, DMEM-low glucose (Cat No. 12-707F, Lonza) medium was prepared.
1X HBSS(Hank's Balanced Salt Solution, gibco), 1 mM HEPES(gibco) 및 멸균수를 혼합하여 총 50 ml의 HBSS 용액을 제조하였다.A total of 50 ml of HBSS solution was prepared by mixing 1X HBSS (Hank's Balanced Salt Solution, gibco), 1 mM HEPES (gibco), and sterilized water.
콜라겐분해효소(collagenase)로 1형 콜라겐분해효소(collagenase type I, Cat No. LS004194, Worthington) 1통에 5 ml의 상기 1X HBSS 용액을 넣고 잘 섞은 후, 0.2 μm 필터(Sartorius)로 여과하여 2% 1형 콜라겐분해효소를 제조하였다. 제조한 콜라겐분해효소는 -20℃에 동결한 후 사용 시 꺼내어 아이스에서 녹였다.Add 5 ml of the 1 % Type 1 collagenase was prepared. The prepared collagenase was frozen at -20°C, then taken out and thawed on ice when used.
DNA 분해효소로 DNase I(Cat No. D4527-10KU, Sigma Aldrich)을 500 μl의 0.9% NaCl를 넣고 잘 섞은 후, 0.2 μm 필터로 여과하여 20 U/μl의 DNase I을 제조하였다. 제조한 DNA 분해효소는 20℃에 동결한 후 사용 시 꺼내어 아이스에서 녹였다.As a DNA decomposition enzyme, DNase I (Cat No. D4527-10KU, Sigma Aldrich) was added with 500 μl of 0.9% NaCl, mixed well, and filtered through a 0.2 μm filter to prepare 20 U/μl of DNase I. The prepared DNA degrading enzyme was frozen at 20°C, taken out when used, and thawed on ice.
70% 에탄올은 99.9% 에탄올을 멸균 증류수로 희석하고 0.2 μm 필터로 여과하여 제조하였다.70% ethanol was prepared by diluting 99.9% ethanol with sterile distilled water and filtering it through a 0.2 μm filter.
피콜-하이파크(Ficoll-Hypaque)로 밀도 1.077 ~ 1.0800 g/ml의 LSM(Lymphocyte Separation Medium, Cat. 50494X, mpbio)을 준비하였다.LSM (Lymphocyte Separation Medium, Cat. 50494X, mpbio) with a density of 1.077 to 1.0800 g/ml was prepared with Ficoll-Hypaque.
퍼콜(Percoll) 구배를 위해 퍼콜 원액(Cat No. P4937, Sigma Aldrich)과 10X 인산완충용액(phosphate-buffered saline, PBS, gibco)을 9 : 1 비율로 혼합하여 100% 퍼콜 용액을 제조하고, 100% 퍼콜을 1X PBS(Sigma Aldrich) 및 DMEM 배지로 희석하여 하기 [표 1]과 같이 15%, 30%, 50% 및 60% 퍼콜 용액을 제조하였다. DMEM 배지는 퍼콜의 색깔을 달리하여 층을 확인하기 위해 사용하였다.For the Percoll gradient, a 100% Percoll solution was prepared by mixing Percoll stock solution (Cat No. P4937, Sigma Aldrich) and 10X phosphate-buffered saline (PBS, gibco) at a ratio of 9:1, and 100% % Percoll was diluted with 1X PBS (Sigma Aldrich) and DMEM medium to prepare 15%, 30%, 50% and 60% Percoll solutions as shown in Table 1 below. DMEM medium was used to identify the layers by varying the color of Percoll.
[실시예 2] 편도 유래 줄기세포 생산[Example 2] Production of tonsil-derived stem cells
[2-1] 편도 조직 적출 및 세절[2-1] Tonsil tissue extraction and morcellation
환자를 대상으로 편도적출술을 시행하여 편도 조직을 획득하였다. 획득한 편도 조직은 70% 에탄올(25 ml)이 담긴 50 ml 튜브에 1분간 방치 후, HBSS 용액(25 ml)이 담긴 50 ml 튜브로 옮기고 1분간 방치하여 잔류 에탄올을 제거하였다.Tonsillectomy was performed on the patient and tonsil tissue was obtained. The obtained tonsil tissue was left in a 50 ml tube containing 70% ethanol (25 ml) for 1 minute, then transferred to a 50 ml tube containing HBSS solution (25 ml) and left for 1 minute to remove residual ethanol.
도 1과 같이 직경 100 mm의 세포배양 디쉬에 상기 편도 조직을 옮기고 5 ml의 HBSS 용액을 부어준 후 트위져(tweezer)를 이용하여 잘게 세절하였고, 이 과정에서 처음 흘러나온 세포들이 포함된 용액은 석션(suction)하여 제거하였다.As shown in Figure 1, the tonsil tissue was transferred to a cell culture dish with a diameter of 100 mm, poured with 5 ml of HBSS solution, and finely chopped using a tweezer. The solution containing the cells that first flowed out during this process was It was removed by suction.
그 다음, 도 2와 같이 새로운 HBSS 용액 5 ml 넣고, 수술용 가위로 조직을 5 mm 이내로 잘게 세절하였다.Next, as shown in Figure 2, 5 ml of a new HBSS solution was added, and the tissue was finely chopped to within 5 mm using surgical scissors.
도 3과 같이 HBSS 용액을 포함한 세절한 조직을 50 ml 코니칼 튜브(conical tube)에 옮겨 넣은 다음, 총 부피 18 ml가 되도록 HBSS 용액을 맞추고, 상기 [실시예 1]에서 제조한 콜라겐분해효소 2 ml 및 DNA 분해효소 200 μl을 첨가하였다. 이후, 7℃ CO2인큐베이터(incubator)에서 2시간 배양하였다. 배양하는 동안 15분마다 주기적으로 흔들어 주었다.As shown in Figure 3, the finely chopped tissue containing the HBSS solution was transferred to a 50 ml conical tube, then the HBSS solution was adjusted to a total volume of 18 ml, and collagenase 2 prepared in [Example 1] above was added. ml and 200 μl of DNA degrading enzyme were added. Afterwards, the cells were cultured in a 7°C CO 2 incubator for 2 hours. During incubation, the cells were shaken periodically every 15 minutes.
[2-2] 피콜-하이파크를 이용한 단핵세포의 분리[2-2] Separation of mononuclear cells using Ficoll-Hypark
상기 실시예 [2-1]에서 2시간 배양을 마친 세포 조직액을 세포 스트레이너(cell strainer)를 장착한 새로운 50 ml 코니칼 튜브에 천천히 부어 조직을 여과하여 세포 부유액을 획득하였다. 이때 막혀서 여과되지 않는 경우, 팁(tip)으로 조심히 긁어주거나 새로운 세포 스트레이너를 사용하여 여과하였다. 또한, 세포 조직액이 들어있던 튜브에 HBSS 용액(20 ml)을 넣은 뒤 상기 조직이 남아있는 세포 스트레이너에 부어 추가로 세포 부유액을 획득하였다.In Example [2-1], the cell tissue fluid that had been cultured for 2 hours was slowly poured into a new 50 ml conical tube equipped with a cell strainer, and the tissue was filtered to obtain a cell suspension. At this time, if it was clogged and could not be filtered, it was carefully scraped with a tip or filtered using a new cell strainer. Additionally, HBSS solution (20 ml) was added to the tube containing the cell tissue fluid and poured into a cell strainer containing the remaining tissue to obtain an additional cell suspension.
상기 획득한 세포 부유액을 300 ×g에서 5분 동안 원심분리한 후 상층액을 제거하고 상기 [실시예 1]의 세포배양배지 10 ml를 넣고 피펫으로 현탁하였다. 그 다음, 도 4와 같이 새로운 50 ml 튜브에 LSM(15 ml)을 넣고 상기 세포배양배지로 현탁한 세포 부유액을 층이 생기게끔 조심스럽게 적재하고, 900 ×g에서 20분 동안 원심분리하였다.The obtained cell suspension was centrifuged at 300 × g for 5 minutes, the supernatant was removed, and 10 ml of the cell culture medium of [Example 1] was added and suspended with a pipette. Next, as shown in Figure 4, LSM (15 ml) was added to a new 50 ml tube, the cell suspension suspended in the cell culture medium was carefully loaded to form a layer, and centrifuged at 900 × g for 20 minutes.
도 5와 같이 원심분리로 생성된 중간 세포층을 회수한 후 HBSS 용액(30 ml)을 넣어준 다음, 300 ×g에서 5분 동안 원심분리하였다. 원심분리 후 상층액을 제거하고 세포배양배지 5 ml를 넣고 피펫으로 현탁하여 단핵세포를 포함한 세포 침전물을 획득하였다.As shown in Figure 5, the intermediate cell layer generated by centrifugation was recovered, HBSS solution (30 ml) was added, and then centrifuged at 300 × g for 5 minutes. After centrifugation, the supernatant was removed, 5 ml of cell culture medium was added and suspended with a pipette to obtain cell precipitate including mononuclear cells.
[2-3] 퍼콜을 이용한 단핵세포 정제 및 세포 배양[2-3] Mononuclear cell purification and cell culture using Percoll
단핵세포를 정제하기 위해 도 6과 같이 상기 [실시예 1]에서 제조한 퍼콜 용액을 15 ml 코니칼 튜브에 60%, 50%, 30%, 15% 퍼콜 용액 순으로 2 ml씩 적재하고, 상기 실시예 [2-2]에서 획득한 세포 침전물을 15% 퍼콜 용액 상층에 조심스럽게 부유하였다. 그 다음, 도 7과 같이 1,200 ×g에서 10분 동안 원심분리하여 분리된 세포층 중 50% 및 30% 퍼콜 용액 사이의 세포층의 세포를 회수하였다.In order to purify mononuclear cells, 2 ml of the Percoll solution prepared in [Example 1] was loaded into a 15 ml conical tube in the order of 60%, 50%, 30%, and 15% Percoll solution, as shown in Figure 6. The cell sediment obtained in Example [2-2] was carefully suspended in the upper layer of 15% Percoll solution. Next, as shown in Figure 7, cells were centrifuged at 1,200 × g for 10 minutes to recover cells in the cell layer between 50% and 30% Percoll solution.
회수한 세포를 세척하기 위해 10 ml PBS를 넣고 300 ×g에서 5분 동안 원심분리한 후 상층액을 제거하고, 세포배양배지 30 ml를 넣고 피펫으로 현탁하였다. 세포를 계수한 후 1×106개/ml가 되도록 배지를 추가하고 T-75 플라스크(flask)에 20 ml씩 분주하였다. 2 ~ 3일 마다 배양배지를 교환하였다. 배양 시작 후 2주 정도 경과하여 약 70 ~ 80% 세포밀도가 된 세포를 편도 조직 유래 중간엽 줄기세포(tonsillar mesenchymal stem cells, T-MSC)로 간주하고 계대 배양하였다.To wash the recovered cells, 10 ml of PBS was added and centrifuged at 300 × g for 5 minutes. The supernatant was removed, and 30 ml of cell culture medium was added and suspended with a pipette. After counting the cells, medium was added to reach 1×10 6 cells/ml, and 20 ml of each was dispensed into T-75 flasks. Culture medium was changed every 2 to 3 days. About 2 weeks after the start of culture, cells that reached a cell density of about 70 to 80% were considered tonsillar tissue-derived mesenchymal stem cells (T-MSC) and subcultured.
Claims (1)
2) 단계 1)에서 획득한 세포 부유액에서 피콜-하이파크(Ficoll-Hypaque)로 단핵세포를 분리하는 단계;
3) 단계 2)에서 분리한 단핵세포를 65 내지 55%, 55 내지 45%, 35 내지 25% 및 20 내지 10% 농도의 퍼콜(Percoll) 구배한 후 35 내지 25% 및 20 내지 10% 농도의 퍼콜 사이의 세포를 회수하여 단핵세포를 정제하는 단계; 및
4) 단계 3)의 정제한 단핵세포를 배양하는 단계를 포함하는, 편도 유래 중간엽 줄기세포 제조 방법.
1) Adding a balanced salt solution to the separated tonsil tissue, chopping and filtering to obtain a cell suspension;
2) separating mononuclear cells from the cell suspension obtained in step 1) using Ficoll-Hypaque;
3) The mononuclear cells isolated in step 2) were subjected to a Percoll gradient at concentrations of 65 to 55%, 55 to 45%, 35 to 25%, and 20 to 10%, and then subjected to a Percoll gradient at concentrations of 35 to 25% and 20 to 10%. Recovering cells between percolls and purifying mononuclear cells; and
4) A method of producing tonsil-derived mesenchymal stem cells, comprising culturing the purified mononuclear cells of step 3).
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Non-Patent Citations (2)
| Title |
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| Janjanin et al. Human palatine tonsil: a new potential tissue source of multipotent mesenchymal progenitor cells. Arthritis Research & Therapy 2008, 10:R83 |
| Seong-Ho Koh. Strategy for Maximizing Therapeutic Efficacy of Adult Stem Cells. Hanyang Med Rev 2012;32:159-162 |
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