Conselho Nacional de Desenvolvimento Científico e Tecnologico (480807/2011-6) 및 Fundação de Amparo à Pesquisa de Minas Gerais (APQ-01237-11)의 보조금으로 지원되는 연구. 이 연구는 PROPP UESC(073.6764.2019.0021079-85)의 일부 재정 지원을 받았습니다. MGAG와 URS는 각각 FAPESB(Fundação de Amparo à Pesquisa do Estado da Bahia)와 CAPES(Coordenação de Aperfeiçoamento de Pessoal de Nível Superior)에서 수여하는 장학금 덕분입니다.

마우스 부고환에서 유래한 지방 조직 유래 줄기 세포의 1차 배양

<ol>
	<li>Caplan, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells.">Mesenchymal stem cells.</a> J Orthop Res. 9 (5), 641-650 (1991).</li><li>Pittenger, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell+perspective:+cell+biology+to+clinical+progress.">Mesenchymal stem cell perspective: cell biology to clinical progress.</a> npj Regen Med. 4, 22 (2019).</li><li>Lin, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+and+cancer:+Clinical+challenges+and+opportunities.">Mesenchymal stem cells and cancer: Clinical challenges and opportunities.</a> BioMed Res Int. 2820853, 1-12 (2019).</li><li>Mazini, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hopes+and+limits+of+adipose-derived+stem+cells+(ADSCs)+and+mesenchymal+stem+cells+(MSCs)+in+wound+healing.">Hopes and limits of adipose-derived stem cells (ADSCs) and mesenchymal stem cells (MSCs) in wound healing.</a> Int J Mol Sci. 21 (4), 1306 (2020).</li><li>Shi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoregulatory+mechanisms+of+mesenchymal+stem+and+stromal+cells+in+inflammatory+diseases.">Immunoregulatory mechanisms of mesenchymal stem and stromal cells in inflammatory diseases.</a> Nat Rev Nephrol. 14 (8), 493-507 (2018).</li><li>Uccelli, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+in+health+and+disease.">Mesenchymal stem cells in health and disease.</a> Nat Rev Immunol. 8 (9), 726-736 (2008).</li><li>Dong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adipose+tissue-derived+small+extracellular+vesicles+promote+soft+tissue+repair+through+modulating+M1-to-M2+polarization+of+macrophages.">Human adipose tissue-derived small extracellular vesicles promote soft tissue repair through modulating M1-to-M2 polarization of macrophages.</a> Stem Cell Res Ther. 14 (1), 67 (2023).</li><li>Maffioli, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+analysis+of+the+secretome+of+human+bone+marrow-derived+mesenchymal+stem+cells+primed+by+pro-inflammatory+cytokines.">Proteomic analysis of the secretome of human bone marrow-derived mesenchymal stem cells primed by pro-inflammatory cytokines.</a> J. Proteomics. 166, 115-126 (2017).</li><li>Akiyama, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal-stem-cell-induced+immunoregulation+involves+FAS-ligand-/FAS-mediated+T+cell+apoptosis.">Mesenchymal-stem-cell-induced immunoregulation involves FAS-ligand-/FAS-mediated T cell apoptosis.</a> Cell Stem Cell. 10, 544-555 (2012).</li><li>Wang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+mesenchymal+stem+cells+in+immunomodulation:+pathological+and+therapeutic+implications.">Plasticity of mesenchymal stem cells in immunomodulation: pathological and therapeutic implications.</a> Nat Immunol. 15, 1009-1016 (2014).</li><li>Li, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Allogeneic+vs.+autologous+mesenchymal+stem/stromal+cells+in+their+medication+practice.">Allogeneic vs. autologous mesenchymal stem/stromal cells in their medication practice.</a> Cell Biosci. 11, 187 (2021).</li><li>Zhang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+challenges+and+promises+of+allogeneic+mesenchymal+stem+cells+for+use+as+a+cell-based+therapy.">The challenges and promises of allogeneic mesenchymal stem cells for use as a cell-based therapy.</a> Stem Cell Res Ther. 6, 234 (2015).</li><li>Jurado, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue-derived+mesenchymal+stromal+cells+as+part+of+therapy+for+chronic+graft-versus-host+disease:+A+phase+I/II+study.">Adipose tissue-derived mesenchymal stromal cells as part of therapy for chronic graft-versus-host disease: A phase I/II study.</a> Cytotherapy. 19 (8), 927-936 (2017).</li><li>Zhang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell-derived+exosome-educated+macrophages+alleviate+systemic+lupus+erythematosus+by+promoting+efferocytosis+and+recruitment+of+IL-17++regulatory+T+cell.">Mesenchymal stem cell-derived exosome-educated macrophages alleviate systemic lupus erythematosus by promoting efferocytosis and recruitment of IL-17+ regulatory T cell.</a> Stem Cell Res Ther. 13 (1), 484 (2022).</li><li>Fern&#225;ndez, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+mesenchymal+stem+cells+(AdMSC)+for+the+treatment+of+secondary-progressive+multiple+sclerosis:+A+triple+blinded,+placebo+controlled,+randomized+phase+I/II+safety+and+feasibility+study.">Adipose-derived mesenchymal stem cells (AdMSC) for the treatment of secondary-progressive multiple sclerosis: A triple blinded, placebo controlled, randomized phase I/II safety and feasibility study.</a> PLoS One. 13 (5), 0195891 (2018).</li><li>Abdolmohammadi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell-based+therapy+as+a+new+therapeutic+approach+for+acute+inflammation.">Mesenchymal stem cell-based therapy as a new therapeutic approach for acute inflammation.</a> Life Sci. 312, 121206 (2023).</li><li>Hoang, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell-based+therapy+for+human+diseases.">Stem cell-based therapy for human diseases.</a> Signal Transduct Target Ther. 7 (1), 272 (2022).</li><li>Lee, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+expression+analysis+of+mesenchymal+stem+cells+from+human+bone+marrow+and+adipose+tissue.">Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue.</a> Cell Physiol Biochem. 14 (4-6), 311-324 (2004).</li><li>Strem, B. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotential+differentiation+of+adipose+tissue+derived+stem+cells.">Multipotential differentiation of adipose tissue derived stem cells.</a> Keio J Med. 54 (3), 132-141 (2005).</li><li>Kern, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+mesenchymal+stem+cells+from+bone+marrow,+umbilical+cord+blood,+or+adipose+tissue.">Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue.</a> Stem Cells. 24 (5), 1294-1301 (2006).</li><li>Dominici, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.</a> Cytotherapy. 8 (4), 315-317 (2006).</li><li>Berryman, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+hormone's+effect+on+adipose+tissue:+Quality+versus+quantity.">Growth hormone's effect on adipose tissue: Quality versus quantity.</a> International Journal of Molecular Sciences. 18 (8), 1621 (2017).</li><li>Shomer, N. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+rodent+euthanasia+methods.">Review of rodent euthanasia methods.</a> J Am Assoc Lab Anim Sci. 59 (3), 242-253 (2020).</li><li>Miranda, V. H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+damage+in+schistosomiasis+is+reduced+by+adipose+tissue-derived+stem+cell+therapy+after+praziquantel+treatment.">Liver damage in schistosomiasis is reduced by adipose tissue-derived stem cell therapy after praziquantel treatment.</a> PLoS Negl Trop Dis. 14 (8), e0008635 (2020).</li><li>Li, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+primary+bone+marrow+mesenchymal+stromal+cells.">Isolation and characterization of primary bone marrow mesenchymal stromal cells.</a> Ann N Y Acad Sci. 1370 (1), 109-118 (2016).</li><li>Boregowda, S. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+mouse+bone+marrow+mesenchymal+stem+cells.">Isolation of mouse bone marrow mesenchymal stem cells.</a> Methods Mol Biol. 1416, 205-223 (2016).</li><li>Sreejit, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+mesenchymal+stem+cell+lines+from+murine+bone+marrow.">Generation of mesenchymal stem cell lines from murine bone marrow.</a> Cell Tissue Res. 350 (1), 55-68 (2012).</li><li>Bunnell, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose+tissue-derived+mesenchymal+stem+cells.">Adipose tissue-derived mesenchymal stem cells.</a> Cells. 10 (12), 3433 (2021).</li></ol>

저자는 밝힐 것이 없습니다.

MSC는 다양한 조직에서 추출할 수 있습니다. 골수가 쥐와 인간 모두에서 MSCs의 공통 공급원을 나타내고 있음에도 불구하고25,26, 우리는 MSC가 풍부하고 신체에 분포하며 접근이 용이하기 때문에 이 연구에서 지방 조직과 함께 작업하기로 결정했습니다. 대안으로, 지방 유래 줄기 세포를 사용하여 MSC와 같은 불멸화 세포를 생성할 수있다 27</su...

중간엽 줄기 세포(MSC)는 조골세포, 연골아세포, 지방세포 1,2와 같은 세포로 분화하는 성체 다전위 세포입니다. 이 세포는 여러 장기에 존재하기 때문에 골수, 근육, 지방, 모낭, 치아 뿌리, 태반, 진피, 주위, 탯줄, 폐, 간 및 비장과 같은 성인 조직에서 추출할 수 있습니다 3,4.
MSC가 생리학 및 면역 체계에 미치는 영향은 5,6으로 보고되었습니다. 이 세포는 인간 및 수의학 모두에서 여러 질병을 치료하는 데 유망했습니다. MSC는 세포-세포 접촉, 용해성 인자 및 작은 세포외 소포체와 같은 다양한 메커니즘을 통해 염증을 제어하고 혈관 신생 및 조직 항상성을 촉진할 수 있습니다 7,8,9,10. 또한, MSC는 면역학적으로 양립할 수 없는 동종 이식 수혜자에서 생존할 수 있는 면역 특권 세포인데, 이는 이들 세포가 클래스 I 주요 조직 적합성 복합체(MHC) 분자의 발현이 낮고 동종 이식을 위한 세포 기반 치료에 사용되기 때문입니다11,12. 낮은 면역원성과 재생 잠재력이 결합된 MSC는 이식편대숙주병(GvHD)13, 전신성 홍반성 루푸스(SLE)14 및 다발성 경화증15 등과 같은 세포 치료에 이상적인 후보입니다16,17.
MSC가 여러 성인 조직에 존재한다는 사실에도 불구하고, 지방 조직은 최소한의 외과적 개입으로 수확에 대한 접근성과 같은 다른 공급원에 비해 이점을 제공합니다. 높은 팽창률을 가진 많은 수의 사용 가능한 셀; 특정 장비 및 저비용 재료 없이 수행하기 쉬운 프로토콜을 사용하여 체외 확장이 용이합니다 18,19,20. 추출한 지방 조직 유래 줄기 세포(ADSC)는 국제 세포 치료 학회(International Society for Cellular Therapy, ISCT)에서 정한 대로 특성화해야 합니다21. 따라서 MSC는 엔도글린(CD105), 엑토-5'-뉴클레오티다아제(CD73) 및 Thy-1(CD90)과 같은 중간엽 마커의 높은 비율(≥95%)을 발현하고, 백혈구 공통 항원(CD45), 막관통 인산지방단백(CD34), 당지질 고정 막 당단백질(CD14), 인테그린 알파 M(CD11b), B세포 항원 수용체 복합체 관련 단백질 알파 사슬(CD79α)과 같은 조혈 마커의 낮은 비율(≤2%)을 발현하는 형태학을 보여야 합니다. B-림프구 표면 항원 B4(CD19) 및 클래스 II 인간 백혈구 항원(HLA-II). 또한, 기능적 특성화가 필요하며, 세포는 조골세포, 연골아세포 또는 지방아세포로 분화할 수 있어야 한다21.
여기에서는 in vitro 연구를 위한 기계적 해리 및 효소 소화를 사용하여 부고환 지방 조직에서 MSC를 얻는 방법과 ISTT에 의해 preconized 형태학적 특성 분석을 보여줍니다.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>140 &#176;C High Heat Sterilization CO2 Incubator</td>
			<td>RADOBIO SCIENTIFIC CO. LTD, China</td>
			<td>C180</td>
			<td></td>
		</tr>
		<tr>
			<td>3-Isobutyl-1-methylxanthine</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>I7018</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid glacial</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>PHR1748</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcian Blue 8GX</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>A9186</td>
			<td>BioReagent, suitable for detection of glycoproteins. 1% in acetic acid, pH 2.5</td>
		</tr>
		<tr>
			<td>Alcohol 70%</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>65350-M</td>
			<td>70% in water</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>PHR1662</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibodies anti-mouse anti-CD29 FITC (Clone Ha2/5)</td>
			<td>BD Biosciences, San Diego, CA, USA</td>
			<td>555005</td>
			<td>Functions in the cell: Adhesion and activation, embryogenesis, Leukocytes, DC, platelets, mast cells, fibroblasts and endothelial cells</td>
		</tr>
		<tr>
			<td>Antibodies anti-mouse anti-CD34 PE (Clone RAM34)</td>
			<td>BD Biosciences, San Diego, CA, USA</td>
			<td>551387</td>
			<td>Functions in the cell: Cell adhesion factor. Hematopoietic stem cells</td>
		</tr>
		<tr>
			<td>Antibodies anti-mouse anti-CD45 APC (Clone 30-F11)</td>
			<td>BD Biosciences, San Diego, CA, USA</td>
			<td>559864</td>
			<td>Functions in the cell: Assists in the activation of leukocytes</td>
		</tr>
		<tr>
			<td>Antibodies anti-mouse anti-CD71 FITC (Clone C2)</td>
			<td>BD Biosciences, San Diego, CA, USA</td>
			<td>553266</td>
			<td>Functions in the cell: Controls iron uptake during cell proliferation. Proliferating cells, reticulocytes and precursors</td>
		</tr>
		<tr>
			<td>Antibodies anti-mouse anti-CD90 PerCP (Clone OX-7)</td>
			<td>BD Biosciences, San Diego, CA, USA</td>
			<td>557266</td>
			<td>Functions in the cell: Signaling, adhesion. T lymphocyte, NK, monocyte, HSC, neuron, fibroblast</td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>PHR1008</td>
			<td></td>
		</tr>
		<tr>
			<td>Automatic pipettes</td>
			<td>Thermo Fisher Scientific, Waltham, Massachusetts, USA</td>
			<td>4700850N</td>
			<td>Finnpipette F1 Good Laboratory Pipetting (GLP) Kits</td>
		</tr>
		<tr>
			<td>Beaker</td>
			<td>Not applicable</td>
			<td></td>
			<td>1 unit</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates (6-well)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>Z707759</td>
			<td>07 units sterile. TPP tissue culture plates</td>
		</tr>
		<tr>
			<td>Cell culture plates (96-well. Round or V bottom)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>CLS353077</td>
			<td>01 unit sterile. Wells, 96, Tissue Culture (TC)-treated surface, round bottom clear wells, sterile</td>
		</tr>
		<tr>
			<td>Chondrogenic medium</td>
			<td>Stem Pro Chondrogenesis Differentiation&#8211;Life Technologies</td>
			<td>A1007101</td>
			<td>TGF-&#946;2, TGF-&#946;3, dexamethasone, insulin, transferrin, ITS, sodium-l - ascorbate, sodium pyruvate, ascorbate-2-phosphate</td>
		</tr>
		<tr>
			<td>Collagenase type II</td>
			<td>Life Technologies, California, USA</td>
			<td>17101015</td>
			<td></td>
		</tr>
		<tr>
			<td>cork or styrofoam board covered with aluminum</td>
			<td>Not applicable</td>
			<td></td>
			<td>1 unit</td>
		</tr>
		<tr>
			<td>cotton</td>
			<td>Not applicable</td>
			<td></td>
			<td>50 g</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>D4902</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting scissor</td>
			<td>Not applicable</td>
			<td></td>
			<td>03 units sterile</td>
		</tr>
		<tr>
			<td>DPX Mountant for histology</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>6522</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>D5523</td>
			<td>With 1000 mg/L glucose and L-glutamine, without sodium bicarbonate, powder, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Eosin B</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>861006</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>47608</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>G1397</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>H3136</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic Needle (0.3mm x 13mm)</td>
			<td>Not applicable</td>
			<td></td>
			<td>5 units</td>
		</tr>
		<tr>
			<td>Indomethacin</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>I0200000</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>I3536</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>563935</td>
			<td>70% in H2O</td>
		</tr>
		<tr>
			<td>Ketamine-D4 hydrochloride solution</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>K-006</td>
			<td>1.0 mg/mL in methanol (as free base), certified reference material, Cerilliant&#174;</td>
		</tr>
		<tr>
			<td>Neubauer chamber</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>BR718620</td>
			<td>BRAND counting chamber BLAUBRAND Neubauer pattern. With clips, double ruled</td>
		</tr>
		<tr>
			<td>Nichiryo pipette tips (0.1&#8211;10 &#956;L)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>Z645540</td>
			<td>Volume range 0.1&#8211;10 &#956;L, elongated, bulk pack. Sterile</td>
		</tr>
		<tr>
			<td>Nichiryo pipette tips (1&#8211;10 mL)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>Z717401</td>
			<td>Volume range 1&#8211;10 mL, universal, bulk pack. Sterile</td>
		</tr>
		<tr>
			<td>Nichiryo pipette tips (200 &#956;L)</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>Z645516</td>
			<td>Maximum volume 200 &#956;L, graduated, ministack. Sterile</td>
		</tr>
		<tr>
			<td>Oil-Red O solution</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>O1391</td>
			<td>0.5% in isopropanol</td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>107.151</td>
			<td>46&#8211;48, in block form</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>P4333</td>
			<td>Solution stabilized, with 10,000 units penicillin and 10 mg streptomycin/mL, 0.1 &#956;m filtered, BioReagent, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline solution 1x (PBS).</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>P3813</td>
			<td>Powder, pH 7.4, for preparing 1 L solutions. Balanced and sterile</td>
		</tr>
		<tr>
			<td>Polypropylene conical tubes (15 mL)</td>
			<td>Falcon, Fisher Scientific</td>
			<td>14-959-53A</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Polypropylene conical tubes (50 mL)</td>
			<td>Falcon, Fisher Scientific</td>
			<td>14-432-22</td>
			<td>2 units sterile</td>
		</tr>
		<tr>
			<td>scalpel (optional)</td>
			<td>Not applicable</td>
			<td></td>
			<td>1 unit</td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>85228</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium thiosulfate</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>72049</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical tweezer (15 cm)</td>
			<td>Not applicable</td>
			<td></td>
			<td>3 units sterile</td>
		</tr>
		<tr>
			<td>Swiss male mice (6&#8211;8 weeks)</td>
			<td>Bioterium, Santa Cruz State University</td>
			<td>021/22</td>
			<td></td>
		</tr>
		<tr>
			<td>syringe (1 mL)</td>
			<td>Not applicable</td>
			<td></td>
			<td>1 unit</td>
		</tr>
		<tr>
			<td>Trypan Blue Dye</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>T8154</td>
			<td>0.4%, liquid, sterile-filtered, suitable for cell culture</td>
		</tr>
		<tr>
			<td>Trypsin/EDTA (ethylenediaminetetraacetic acid)</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>T3924</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>PHR3263</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate disodium salt hydrate</td>
			<td>Sigma-Aldrich, San Luis, Missouri, USA</td>
			<td>G9422</td>
			<td>BioUltra, suitable for cell culture, suitable for plant cell culture, &#8805;99% (titration)</td>
		</tr>
	</tbody>
</table>

isolation, in vitro expansion, and characterization of mesenchymal stem cells from mouse epididymal adipose tissue

중간엽 줄기 세포(MSC)는 주로 면역 반응을 조절할 수 있는 잠재력으로 인해 악화된 염증을 막기 위한 새로운 치료 접근법으로 광범위하게 연구되었습니다. MSC는 class I major histocompatibility complex(MHC) 분자의 낮은 발현과 동종 이식을 위한 세포 기반 요법의 사용을 기반으로 면역학적으로 호환되지 않는 동종 이식 수혜자에서 생존할 수 있는 면역 특권 세포입니다. 이 세포는 여러 조직에서 분리할 수 있으며, 가장 일반적으로 사용되는 것은 골수와 지방 조직입니다. 당사는 마우스의 부고환 지방 조직에서 MSC를 분리, 배양 및 특성화할 수 있는 간편한 프로토콜을 제공합니다. 부고환 지방 조직은 외과적으로 절제하고 물리적으로 단편화한 후 0.15% 콜라겐분해효소 II형 용액으로 분해합니다. 그런 다음 1차 지방 조직 유래 줄기세포(ADSC)를 시험관에서 배양 및 증식하고 유세포 분석을 통해 표현형 특성 분석을 수행합니다. 또한 ADSC를 골형성(osteogenic), 지방형성(adipogenic) 및 연골형성(chondrogenic) 세포로 분화하는 단계를 제공하고 각 세포 계통의 기능적 특성을 규명합니다. 여기에 제공된 프로토콜은 in vivo 및 ex vivo 실험에 사용할 수 있으며, 대안으로 지방 유래 줄기 세포를 사용하여 MSC와 같은 불멸 세포를 생성할 수 있습니다.

Mesenchymal stem cells (MSCs) are adult multipotential cells differentiating into cells such as osteoblast, chondroblast, and adipocyte1,2. These cells reside in several organs, and because of that, they can be extracted from adult tissues such as bone marrow, muscle, fat, hair follicle, tooth root, placenta, dermis, perichondrium, umbilical cord, lung, liver, and spleen3,4.
The effects of MSCs on physiology and the immune system have been reported5,6. These cells have been promising for treating several diseases, both in human and veterinary medicine. The MSCs can control inflammation and promote angiogenesis and tissue homeostasis through different mechanisms, such as cell-cell contact, soluble factors, and small extracellular vesicles7,8,9,10. Furthermore, the MSCs are immune-privileged cells capable of surviving in immunologically incompatible allogeneic transplant recipients because these cells show low expression of class I major histocompatibility complex (MHC) molecules and are used in cell-based therapy for allogeneic transplant11,12. The low immunogenicity combined with the regenerative potential makes MSCs ideal candidates for cell therapy, such as graft-versus-host disease (GvHD)13, systemic lupus erythematosus (SLE)14, and multiple sclerosis15, among others16,17.
Despite the fact that MSCs reside in several adult tissues, the adipose tissue offers advantages over other sources, such as accessibility for harvesting, with minimal surgical intervention; large number of available cells with high expansion rate; and easy in vitro expansion using an easy-to-perform protocol without the need for specific equipment and low-cost materials18,19,20. Once extracted, the adipose tissue-derived stem cells (ADSCs) must be characterized as established by the International Society for Cellular Therapy (ISCT)21. Thus, MSCs must show morphology fibroblast-like, adherence to plastic culture, expressing a high percentage (&#8805;95%) of mesenchymal markers such as endoglin (CD105), ecto-5&#39;-nucleotidase (CD73) and Thy-1 (CD90), and low percentage (&#8804;2%) of hematopoietic markers such as leukocyte common antigen (CD45), transmembrane phosphoglycoprotein (CD34), glycolipid-anchored membrane glycoprotein (CD14), integrin alpha M (CD11b), B-cell antigen receptor complex-associated protein alpha chain (CD79&#945;) or B-lymphocyte surface antigen B4 (CD19) and class II human leukocyte antigen (HLA-II). Furthermore, a functional characterization is required, and the cells should be able to differentiate into osteoblast, chondroblast, or adipoblast cells21.
Here, we show how to obtain the MSCs from epididymal adipose tissue using mechanical dissociation and enzymatic digestion for in vitro studies and the morphological characterization preconized by ISCT.

저자 스포트라이트: Advancing Treatment Approaches with Adipose-Derived Stem Cells(지방 유래 줄기세포를 이용한 치료 접근법 발전)

Isolation, In Vitro Expansion, and Characterization of Mesenchymal Stem Cells from Mouse Epididymal Adipose Tissue(마우스 부고환 지방 조직에서 중간엽 줄기 세포의 분리, 체외 확장 및 특성 분석)

My research focus on adipose-derived mesenchymal stem cells as a new approach treatment associated or not associated with other therapies to improve the inflammation from several disease. We demonstrated that using adipose mesenchymal stem cells in conjunction with Praziquantel has significantly controlled the granulomatous reaction in Schistosoma mansoni infection, highlighting the benefits of this combined treatment approach. Our protocol address the research gap in the meticulous collection of epididymal adipose tissue under aseptic condictions, that the physical processing involving vigorous shaking, and identifies the optimal time for utilizing adipose-derived stem cells.Our protocol offers significant advantages, notably the abundant availability and the potential strength for isolation of mesenchymal stem cells. Additionally, mesenchymal stem cells poses potent therapeutic properties including anti-inflammatory and tissue regeneration capabilities.

여기에 제시된 프로토콜에 따라 지방 조직에서 추출한 세포는 ISCT에서 제안한 MSC에 대한 최소 기준과 일치하는 형태를 보여주었습니다. 프로토콜의 개요는 그림 1에 나와 있습니다. 표현형적으로, ADSC는 세포 배양 초기에 플라스틱 및 섬유아세포와 유사한 형태에 대한 부착을 보여주었습니다(그림 2A). 게다가, 그들은 균질하게 성장하여 식민지를 형성?...

Read this Scientific Article Protocol about Author Spotlight: Advancing Treatment Approaches with Adipose-Derived Stem Cells at JoVE.com

지방 조직은 중간엽 줄기 세포의 훌륭한 공급원입니다. 여기에서는 스위스 생쥐 부고환 지방 조직에서 지방 조직 유래 줄기 세포(ADSC)의 단계별 추출, 배양 및 특성 분석을 제공합니다.

Mesenchymal stem cells (MSCs) have been extensively studied as a new therapeutic approach, mainly to stop exacerbated inflammation due to their potential ...

제 연구는 여러 질병의 염증을 개선하기 위해 다른 치료법과 연관되거나 연관되지 않은 새로운 접근법 치료법으로 지방 유래 중간엽 줄기 세포에 중점을 둡니다. 우리는 지방 중간엽 줄기 세포를 프라지콴텔과 함께 사용하여 주혈흡충 만소니 감염에서 육아종 반응을 크게 제어했음을 입증하여 이 결합 치료 접근법의 이점을 강조했습니다. 본 프로토콜은 무균 질환 하에서 부고환 지방 조직을 꼼꼼하게 채취하는 과정에서 발생하는 물리적 처리와 격렬한 흔들림을 수반하는 연구 격차를 해결하고, 지방 유래 줄기세포를 활용하기 위한 최적의 시기를 식별합니다.당사의 프로토콜은 상당한 이점을 제공하며, 특히 중간엽 줄기 세포의 분리를 위한 풍부한 가용성과 잠재적 강도를 제공합니다. 또한 중간엽 줄기 세포는 항염증 및 조직 재생 능력을 포함한 강력한 치료 특성을 나타냅니다.

isolation-vitro-expansion-characterization-mesenchymal-stem-cells

Research

JoVE Journal

Biology

Isolation, In Vitro Expansion, and Characterization of Mesenchymal Stem Cells from Mouse Epididymal Adipose Tissue

370 조회수.  State University of Santa Cruz - UESC. 제 연구는 여러 질병의 염증을 개선하기 위해 다른 치료법과 연관되거나 연관되지 않은 새로운 접근법 치료법으로 지방 유래 중간엽 줄기 세포에 중점을 둡니다. 우리는 지방 중간엽 줄기 세포를 프라지콴텔과 함께 사용하여 주혈흡충 만소니 감염에서 육아종 반응을 크게 제어했음을 입증하여 이 결합 치료 접근법의 이점을 강조했습니다. 본 프로토콜은 무균 질환 하에서 ?...

비디오: 저자 스포트라이트: Advancing Treatment Approaches with Adipose-Derived Stem Cells(지방 유래 줄기세포를 이용한 치료 접근법 발전)

Mesenchymal stem cells (MSCs) have been extensively studied as a new therapeutic approach, mainly to stop exacerbated inflammation due to their potential to modulate the immune response. The MSCs are immune-privileged cells capable of surviving in immunologically incompatible allogeneic transplant recipients based on low expression of class I major histocompatibility complex (MHC) molecules and in the use of cell-based therapy for allogeneic transplant. These cells can be isolated from several tissues, the most commonly used being the bone marrow and adipose tissues. We provide an easy protocol to isolate, culture, and characterize MSCs from epididymal adipose tissue of mice. The epididymal adipose tissue is surgically excised, physically fragmented, and digested with 0.15% collagenase type II solution. Then, primary adipose tissue-derived stem (ADSCs) cells are cultured and expanded in vitro, and the phenotypic characterization is performed by flow cytometry. We also provide the steps to differentiate the ADSCs into osteogenic, adipogenic, and chondrogenic cells, followed by functional characterization of each cell lineage. The protocol provided here can be used for in vivo and ex vivo experiments, and as an alternative, the adipose-derived stem cells can be used to generate MSCs-like immortalized cells.

The adipose tissue is an excellent source of mesenchymal stem cells. Here, we bring the step-by-step extraction, cultivation, and characterization of adipose tissue-derived stem cells (ADSCs) from Swiss mice epididymal adipose tissue.