우리는 굉장히 자신의 기술 지원에 대한 자신의 중요한 원반 씨 모하마드 레자 Khadem 샤리프에 박사 라일라 Shakeri 및 박사 Aref Dournaei을 인정합니다.

1. 효소 솔루션 및 확산 매체 (PM)를 준비 <ol><li> Collagenase 종류 나 솔루션을 만들기 : collagenase 유형 I (12 MG / ML)을 무게 0.2 μm의 주사기 필터를 사용하여 1 ML PBS와 필터에 용해. 그런 다음 15 ML 튜브를 삽입하고 필요 할 때까지 -20 ° C에 보관하십시오. </li><li> dispase 솔루션을 확인합니다 dispase을 펴라 (16 MG / ML) 1 ML PBS 및 필터는 0.2 μm의 주사기 필터를 사용하여 분해. 그런 다음 15 ML 튜브를 삽입하고 필요한 ° C까지 4에 보관하십시오. </li><li> 효소 솔루션을 확인하십시오 : 2 ML 멸균 PBS 100 MG / ML 페니실린, 100 MG / ML의 스트렙토 마이신을 포함하는으로 한 ML collagenase 유형 I 솔루션 (12 MG / ML) 1 ML dispase 솔루션을 (16 MG / ML)를 추가합니다. 최종 볼륨의 dispase과 Collagenase I의 총 농도는 receptively 4 MG / ML, 3 MG / ML,해야합니다. 그런 다음, 네 15 ML 튜브, 각 포함 1 ML 효소 솔루션에에 나누어지는이 볼륨입니다. 각 관은 한 펄프 소화에 사용될 수. </li><li> 솔루션을 세척하십시오 : 100 밀리그램 / ML 페니실린, PBS로 100 MG / ML의 스트렙토 마이신을 추가합니다. </li><li> 기본 미디어를 만들기 : 이글의 매체의 알파 수정은 (α-가상) 10 % FBS 및 100 단위 / ML 페니실린, 100 MG / ML 스트렙토 마이신과 보충. </li><li> 10% FBS, 100 μM L-아스코르브 산 2 - 인산염, 2 MM L-글루타민, 100 단위 / ML 페니실린, 100 MG / ML로 보충 이글 배지 (α-가상)의 알파 수정 : 확산 미디어 (PM)를 확인 스트렙토 마이신, 0.25 MG / ML amphotericin B. </li><li> Odontogenic 미디어를 만들기 : 이글의 매체의 알파 수정은 (α-가상) 10 % FBS, 100 μM L-아스코르브 산 2 - 인산염, 2 MM L-글루타민, 100 단위 / ML 페니실린, 100 MG / ML의 스트렙토 마이신, 0.01 μM로 보충 Dexamethasone, 5 MM β-글리세롤 인산 1.8 밀리미터 Monopotassium 인산. </li></ol> 2. 치과 펄프 줄기 세포 Isolatio에 대한 인간 치과 펄프 조직을 준비N <ol><li> 일반 인간의 셋째 어금니가 승인 기관 검토위원회 (IRB)에서 이맘 알리의 치과 클리닉의 성인 (세 21-29년)에서 수집되었다. </li><li> 치아는 기본 매체 (α-가상 10 % FBS와 보충)에 배치 된, 4 ° C.에서 실험실로 이송되었다 </li><li> 무균 상태에서 생물학적 층류 후드 내에서 작업, 세트 업을 처리 할 수​​ 있도록 각 치아에 대해 하나의 100mm 배양 접시를 완료했다. 치아 표면은 70 % 에탄올에 의해 청소했다. </li><li> 페트리 - 요리 중 하나에서 작업하는 동안, 치아는 치아 포셉 보관되었으며, 메스 블레이드는 파편과 뿌리에 치주 인대를 닦아하는 데 사용되었다. </li><li> Cemento - 에나멜 접합은 천천히 펄프 챔버을 나타 내기 위해 살균 치과 디스크를 사용하여 절단되었습니다. 이것은 절단 과정은 치과 조직의 과열 줄이기 위해 천천히 수행해야하는 것으로 간주합니다. </li><li> 펄프 조직은 부드럽게 왕관과 루트에서 분리되었다. </리&gt; <li> 펄프 조직은 메스 블레이드의 도움으로 작은 조각으로 절단되었다. </li></ol> 3. 인간 치과 펄프 절연 <ol><li> 펄프 조직은 hDPSCs의 절연을위한 펄프 조직 explants에서 펄프 조직 또는 직접 휴대 가지 중 하나 효소 분해를 받았습니다. </li><li> 펄프 조직의 효소 분해 : <ol><li> 펄프 조직의 작은 조각이 37 ° C.에 1 시간을위한 효소 솔루션으로 이동되었습니다 조직을 파괴하는 데 도움이 소용돌이 매 30 분. </li><li> 그 후 70 μM 셀 스트레이너를 통해 세포를 통과하여 큰 셀을 합산이 삭제되었습니다 및 단일 세포 현탁액을 얻었다. </li><li> 단일 셀 중지는 상온에서 5 분에 1,200 rpm으로 centrifuged했다. </li><li> Supernatants 신중하게 해제 pipetted되었고 펠릿은 1 ML 확산 중간 (PM)에 다시 중지되었습니다. (참고 : 확산 매체에 FBS를 종료 깔 효소sociation.) </li><li> 치과 펄프의 단일 셀 중지는 PM과 25cm 두 문화 플라스크에 놓는 후 5 % CO 2에서 37 ° C에서 incubated되었습니다. </li><li> 셀 confluency가 달성 될 때까지 문화 매체는 매 3 일마다 교체되었습니다. </li></ol></li><li> 펄프 조직 explants에서 직접 세포 가지 : <ol><li> 펄프 조직이 1-2-mm 조각으로 다진되었고, 각 조각은 PM 25-cm 2 문화 플라스크에 배치 된 후 37 incubated ° C 5 % CO 2인치 그것은 세포의 가지에 대한 PM의 총 양이 더 셀 가지에 대한 펄프 조각 (플라스크 당 2 ~ 3 개의 ML)의 첨부 파일을 지원해야 고려되어야한다. </li><li> 가지가 관찰 된 후 보통이 변경되었습니다. </li><li> 합류에 능가 셀 1시 4분의 비율 (통로 1)의 하위 양식이었다. </li></ol></li></ol> 4. Immunophenotyping <ol><li> / 250000 셀통로 3 세포의 두 종류의 튜브는 4 ° C 어두운 곳에서 30 분 동안 PE 또는 isotypes과 FITC-복합 항 인간 항체에 의해 incubated되었습니다. (참고 :. 항체의 농도가 제조 프로토콜에 따라 적용) </li><li> 부화 시간 후, 100 μl PBS 또는 FACS 희석 솔루션은 각 튜브에 추가되었습니다하고 어두운 장소에서 5 분 동안 1,200 rpm으로 centrifuged. </li><li> Supernatants이 삭제되었습니다 &amp; 펠릿는 100 μl PBS 또는 어두운 장소에서 FACS 희석에 다시 일시 중지되었습니다. </li><li> 타겟 표면 마커 (CD 마커)가 BD FACSCalibur에 의해 평가되었다. </li></ol> 5. Mineralized 셀에 DPSCs의 차별화 유도 및은 알리자린 레드 S 분석을 정량화 <ol><li> 펄프 조직 explants (DPSC-OG)에서 펄프 조직의 효소 분해 (DPSC-ED라고도 함) 또는 직접 휴대 가지에 의해 획득 된 DPSCs 3 구절에 대한 하위 양식이었다. </li><li>t 항로 3, 세포는 5000 셀 /도에서 6도 판에 씨앗이되었다. </li><li> 60 % 합류에 odontogenic 매체는 기존의 PM에 의해 대체되었다. 세 우물은 성장 매체를 추가하여 대조군으로 남아 있었다. </li><li> 보통 하루에 21까지 삼일마다 변경합니다. </li><li> 일 21시, 셀 PBS로 세척 한 1 ML / 잘 실온에서 15 분 10 % paraformaldehyde에 의해 수정되었습니다. </li><li> 그런 다음 paraformaldehyde는 조심스럽게 해제 pipetted되었고 세포는 소주 H 2 O.으로 3 회 (각 시간 5-10 분)을 씻어되었습니다 (참고 : monolayer을 방해하지 마십시오.) </li><li> 세척 후, 1 ML / 잘 알리자린 레드 S는 실온에서 20 분에 추가되었습니다. </li><li> 셀은 추가 알리자린의 붉은 색을 제거 할 deionized H 2 O 네 번 (부드러운 요동 각 시간 5 분)에 의해 세척되었다. </li><li> 1 ML / 잘 H 2 O는 건조에서 세포를 방지하기 위해 추가되었습니다. </li><li> 플레이트는 시각적 인 기능으로 간주되었다pection 및 / 또는 이미지 수집. </li><li> 알리자린 레드 S 정량화 분석의 경우, H 2 O가 흔들리고있는 실온에서 30 분 6 - 잘 접시 및 배양의 각 잘 1 ML 10% 아세트산에 의해 대체되었다. </li><li> 그런 다음 잘 세포 monolayer / 털 깎는 &amp; 아세트산 및 세포 모두 별도의 1.5 ML 마이크로 원심 분리기 튜브로 전송 있었다. (참고 :. 각도 각 ED &amp; OG 그룹에 대한 별도의 튜브 즉 세 테스트 우물 및 세 제어 우물에 전송해야합니다) </li><li> 튜브는 30 초에 힘차게 vortexed했다. </li><li> 85 열 ° C 10 분에. (참고 : 증발을 방지하기 위해 튜브는 parafilm으로 밀봉되었다.) </li><li> 난방 후, 튜브는 5 분에 얼음으로 전송되었습니다. (참고 : 냉각 될 때까지 튜브를 열 수 없습니다.) </li><li> 슬러리는 15 분에 20,000 XG에서 centrifuged했다. centrifuging 있지만, 알리자린 레드 표준은 만들어졌다까지. </li></ol> 알리자린 레드 S 표준은 먼저 희석 버퍼를 diluting 및 테이블을 기반으로 2 배 시리얼 dilutions을 사용하여 준비 하였다. <table border="1"><tbody><tr><td colspan="7"> 염료의 높은 범위 농도 </td><td colspan="7"> 염료의 낮은 범위 농도 </td></tr><tr><td> 이 MM </td><td> 1 ㎜ </td><td> 500 μM </td><td> 250 μM </td><td> 125 μM </td><td> 62.5 μM </td><td> 31.3 μM </td><td> 15.6 μM </td><td> 7.8 μM </td><td> 3.9 μM </td><td> 1.9 μM </td><td> 0.9 μM </td><td> 0.4 μM </td><td> 공백 </td></tr></tbody></table><ol start="16"><li> 원심 분리 한 후, 표면에 뜨는 / 튜브의 한 ML은 새로운 별도의 1.5 ML 마이크로 원심 분리기 튜브로 전송되었습니다. </li><li> 산도는 ~ 375 μl 10을 무력화되었다튜브 당 %의 암모니아 수산화. (참고 : pH의 분노 4.1-4.5해야합니다.) </li><li> 표준 및 샘플 (별도 테스트 및 컨트롤을 포함)의 150 μl를 96 - 웰 플레이트 불투명 벽, 투명 하단에 추가되었습니다. </li><li> 흡광도는 405 nm의에서 읽고 있던 샘플의 OD 값은 추가 분석을 위해 표준 계획과 비교되었다. </li></ol>

<ol>
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Res. 88, 792-806 (2009).</li><li>Graziano, 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=Scaffold's+surface+geometry+significantly+affects+human+stem+cell+bone+tissue+engineering.">Scaffold's surface geometry significantly affects human stem cell bone tissue engineering.</a> J. Cell Physiol. 214, 166-172 (2008).</li><li>d'Aquino, R., 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+dental+pulp+stem+cells:+from+biology+to+clinical+applications.">Human dental pulp stem cells: from biology to clinical applications.</a> J. Exp. Zool. B. Mol. Dev. Evol. 312, 408-415 (2009).</li><li>Mitsiadis, T. 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Res. 18, 696-704 (2003).</li><li>Tomic, 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=Immunomodulatory+properties+of+mesenchymal+stem+cells+derived+from+dental+pulp+and+dental+follicle+are+susceptible+to+activation+by+toll-like+receptor+agonists.">Immunomodulatory properties of mesenchymal stem cells derived from dental pulp and dental follicle are susceptible to activation by toll-like receptor agonists.</a> Stem Cells Dev. 20, 695-708 (2011).</li><li>Tsukamoto, 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=Mineralized+nodule+formation+by+cultures+of+human+dental+pulp-derived+fibroblasts.">Mineralized nodule formation by cultures of human dental pulp-derived fibroblasts.</a> Arch. Oral Biol. 37, 1045-1055 (1992).</li><li>About, I., 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+dentin+production+in+vitro.">Human dentin production in vitro.</a> Exp. Cell Res. 258, 33-41 (2000).</li><li>Couble, M. 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=Odontoblast+differentiation+of+human+dental+pulp+cells+in+explant+cultures.">Odontoblast differentiation of human dental pulp cells in explant cultures.</a> Calcif. Tissue Int. 66, 129-138 (2000).</li><li>Onishi, T., Kinoshita, S., Shintani, S., Sobue, S., Ooshima, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stimulation+of+proliferation+and+differentiation+of+dog+dental+pulp+cells+in+serum-free+culture+medium+by+insulin-like+growth+factor.">Stimulation of proliferation and differentiation of dog dental pulp cells in serum-free culture medium by insulin-like growth factor.</a> Arch. Oral Biol. 44 (99), 361-371 (1999).</li><li>Huang, G. T., Sonoyama, W., Chen, J., Park, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+characterization+of+human+dental+pulp+cells:+various+isolation+methods+and+culturing+environments.">In vitro characterization of human dental pulp cells: various isolation methods and culturing environments.</a> Cell Tissue Res. 324, 225-236 (2006).</li><li>Bakopoulou, 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=Assessment+of+the+impact+of+two+different+isolation+methods+on+the+osteo/odontogenic+differentiation+potential+of+human+dental+stem+cells+derived+from+deciduous+teeth.">Assessment of the impact of two different isolation methods on the osteo/odontogenic differentiation potential of human dental stem cells derived from deciduous teeth.</a> Calcif. Tissue Int. 88, 130-141 (2011).</li><li>Curtis, K. 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=EF1alpha+and+RPL13a+represent+normalization+genes+suitable+for+RT-qPCR+analysis+of+bone+marrow+derived+mesenchymal+stem+cells.">EF1alpha and RPL13a represent normalization genes suitable for RT-qPCR analysis of bone marrow derived mesenchymal stem cells.</a> BMC Mol. Biol. 11, 61 (2010).</li><li>Suchanek, 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=Dental+pulp+stem+cells+and+their+characterization.">Dental pulp stem cells and their characterization.</a> Biomed. Pap. Med. Fac. Univ. Palacky Olomouc Czech Repub. 153, 31-35 (2009).</li><li>d'Aquino, R., 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+postnatal+dental+pulp+cells+co-differentiate+into+osteoblasts+and+endotheliocytes:+a+pivotal+synergy+leading+to+adult+bone+tissue+formation.">Human postnatal dental pulp cells co-differentiate into osteoblasts and endotheliocytes: a pivotal synergy leading to adult bone tissue formation.</a> Cell Death Differ. 14, 1162-1171 (2007).</li><li>Atari, 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=Isolation+of+pluripotent+stem+cells+from+human+third+molar+dental+pulp.">Isolation of pluripotent stem cells from human third molar dental pulp.</a> Histol. Histopathol. 26, 1057-1070 (2011).</li></ol>

관심 없음 충돌이 선언 없습니다.

이 프로토콜은 두 가지 방법, 효소 분해 및 펄프 조직 explants에서 줄기 세포를 직접 가지를 사용하여 치과 펄프에서 hDPSCs의 절연 및 특성화 과정을 설명합니다. 또한, odontoblasts에 이러한 세포의 체외 차별화에 정량 알리자린 레드 S 검정 &amp; QPCR에 의해 평가되었다. 인간의 치아에서 펄프 조직을 분리에 대한 기존 프로토콜은 이러한 플라이어 (뼈 포셉) 9 근절 바늘 2...

줄기 세포는 멀티 효력과 자기 갱신 15 두명의 놀라운 기능을 가지고 clonogenic 세포입니다. 다른 replicative의 potencies 모든 줄기 세포 중에서 출생 후의 줄기 세포와 같은 치과 줄기 세포 때문에 자신의 접근성, 소성 및 기타 성인 줄기 세포 16에 비해 높은 proliferative 능력의 최근 관심을 모으고있다. 특질 상, 중간 엽 줄기 세포와 유사한, 치과 펄프의 줄기 세포는 여러 차별화를 mesenchyme로 용량 및 / 또는 체외 및 생체에서 모두 비 mesenchyme lineages을 가지고 자기편 clonogenic 세포입니다. 1-8,17,18 DPSCs에 의해 식별됩니다 자신의 부정적인 조혈 항원의 표현 (예를 들어, CD45, CD34, CD14) 및 CD90의 긍정적 인 표현, CD29, CD73, CD105, CD44 및 STRO-1. 19,20 최소 통증 및 병적 쉽게 얻을 가능성이 valu로 인간 DPSCs을MSCS의 수 출처는 골수 중간 엽 줄기 세포 21로 보통 소스에 비해. 일반적으로 DPSCs는 두 가지 방법, 즉 펄프 조직 explants (DPSC-OG) 22-24과 /이나 효소 소화 (DPSC-ED) 4,6,25에 의한 줄기 세포의 이동에 의해 격리되었습니다. 이전 연구는 격리 방법과 문화 조건 체외 통로 26,27의 아래에 다른 인구 나 lineages을 일으킬 수 것으로 나타났습니다. 영구 치아 (pDPSCs)의 경우, 황 외이 효소 소화 pDPSCs이 강해 DPSCs에 비해 높은 확산 가능성이있는 것으로 밝혀졌습니다. 또한 26, 낙엽 치아 (dDPSCs)의 경우,이 입증 된 그 STRO-1 &amp; CD34 마커 dDPSC-OG에 비해 dDPSC-ED에 더 많은 표현. 또한, dDPSC-ED는 정의 osteo / odonto 매체에 높은 mineralization 속도를 표시. 따라서 27으로 인해 연구에서 DPSCs의 뛰어난 잠재력egenerative 약을 더 연구는 서로 다른 분리 방법에서 파생됩니다 가능한 다양한 인구를 더 잘 이해해야합니다. 여기, 펄프 추출의 과정을 촉진 한 단계 치과 다이아몬드 디스크를 사용하여, 펄프 추출의 쉬운 방법을 소개하려고했습니다. 또한, ED 또는 OG 방법을 적용하여 인간의 펄프 - 파생 줄기 세포의 분리 후, 두 그룹 사이의 일반적인 특성 및 차별화 용량도 조사했다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 또는 로트. 수 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> α-가상 </td><td> GIBCO </td><td> 11900-073 </td><td></td></tr><tr><td> Collagenase 유형 I </td><td> 시그마 - 알드리치 </td><td> C0130-100MG </td><td></td></tr><tr><td> Dispase </td><td> GIBCO </td><td> 17105-041 </td><td></td></tr><tr><td> 페니실린 / 스트렙토 마이신 </td><td> GIBCO </td><td> 15140-122 </td><td></td></tr><tr><td> Amphotericin B </td><td> GIBCO </td><td> 15290-018 </td><td></td></tr><tr><td> 태아 소 혈청 정의 (FBS) </td><td> HyClone </td><td> SH30070.03 </td><td></td></tr><tr><td> 2 인산 L-아스코르브 산 </td><td> 시그마 </td><td> A8960-5G </td><td></td></tr><tr><td> L-글루타민 </td><td> GIBCO </td><td> 25030-024 </td><td></td></tr><tr><td> Dexamethasone </td><td> 시그마 </td><td> D4902 </td><td></td></tr><tr><td> β-글리세롤 인산 나트륨 소금 하이드레이트, BioUltra </td><td> 시그마 </td><td> G9422-100G </td><td></td></tr><tr><td> 인산 칼륨 일 염기의 </td><td> 시그마 - 알드리치 </td><td> P5655 </td><td></td></tr><tr><td> Osteogenesis 분석 키트 </td><td> Millipore </td><td> PS01802031 </td><td></td></tr><tr><td> 마우스 IgG2b-PE isotype 제어 </td><td> BD pharmingen </td><td> 50808088029 </td><td></td></tr><tr><td> FITC 마우스 IgG2b isotype 제어 </td><td> BD pharmingen </td><td> 559532 </td><td></td></tr><tr><td> FITC 마우스 IgG1 κ isotype </td><td> BD pharmingen </td><td> 11471471 </td><td></td></tr><tr><td> FITC / PE 마우스 안티 - 인간 CD34/CD45 </td><td> BD pharmingen </td><td>341071 </td><td></td></tr><tr><td> PE 안티 - 인간 CD146 </td><td> BD pharmingen </td><td> 550315 </td><td></td></tr><tr><td> 단클론 마우스 반 인간 CD90/FITC </td><td> Daka </td><td> 00034418 </td><td></td></tr><tr><td> PE 마우스 반 인간 CD73 </td><td> BD pharmingen </td><td> 550257 </td><td></td></tr><tr><td> 안티-H CD105/Endoglin PE </td><td> BD pharmingen </td><td> FAB10971P </td><td></td></tr><tr><td> PE 마우스 반 인간 CD11b/Mac1 </td><td> BD pharmingen </td><td> 5553888 </td><td></td></tr><tr><td> CD44 PE 마우스 반 인간 </td><td> BD pharmingen </td><td> 555479 </td><td></td></tr><tr><td> 인산염 완충 용액 (PBS) </td><td> GIBCO </td><td> 003000 </td><td></td></tr><tr><td> 70 μm 세포 스트레이너 </td><td> 매 </td><td> 352360 </td><td></td></tr><tr><td&gt; 0.2 μm 주사기 필터 </td><td> Millex-GV </td><td> SLGV033RB </td><td></td></tr><tr><td> 25cm 두 문화 플라스크 </td><td> 시그마 - 알드리치 </td><td> Z707481 </td><td></td></tr><tr><td></td><td></td><td></td><td> 장비 </td></tr><tr><td> BD FACSCalibur </td><td> BD </td><td> 342975 </td><td></td></tr><tr><td> multiskan microplate 분광 광도계 </td><td> 열 과학 </td><td> 51119200 </td><td></td></tr><tr><td> Fleurcense 현미경 </td><td> 하늘 </td><td></td><td></td></tr><tr><td> 소프트웨어 버전 2.3.1을 흐르는 </td><td></td><td></td><td></td></tr></tbody></table>

isolation, characterization and comparative differentiation of human dental pulp stem cells derived from permanent teeth by using two different methods

<p class="jove_content"&gt; 개발 사랑니는 일상적인 교정 치료를하여 얻을 수있는 성인 중 줄기 세포의 쉽게 액세스 할 수있는 소스입니다. 인간 펄프 유래 줄기 세포는 (hDPSCs) 성인 줄기 세포의 일반 소스로 비교 멀티 혈통 차별화 용량 높은 확산 가능성을 가지고<sup&gt; 1-8</sup&gt;, 따라서 hDPSCs는 조직 공학 및 재생 의학에서 autologous 이식을위한 좋은 후보자가 될 수 있습니다. 이러한 혜택과 함께, 이러한 immunolodulatory 효과로 중간 엽 줄기 세포 (MSC) 기능을 가진, 심지어 allograft 이식의 경우, hDPSCs 더 가치가<sup&gt; 6,9,10</sup&gt;. 따라서 줄기 세포의 소스를 사용하기위한 기본 단계는 펄프 조직에서 hDPSCs를 분리에 가장 적합한 프로토콜을 선택하는 것입니다. 이러한 목표를 달성하기 위해서는, 이러한 공통의 표면 마커 및 또한 차별화 능력 등 다양한 세포 행동에 다양한 절연 조건의 효과를 조사하기 위해 매우 중요합니다.</p&gt;<p class="jove_content"&gt; 따라서, 우리가 영향을 셋째 어금니 치아에서 인간의 펄프 조직을 분리하고 분리 hDPSCs을위한 문학을 바탕으로 기존의 프로토콜 모두를 사용<sup&gt; 11-13</sup&gt;<em&gt; 즉,</em조직 explants에서&gt; 효소 펄프 조직의 분리 (DPSC-ED) 또는 가지 (DPSC-OG). 이있어서, 우리는 치과 다이아몬드 디스크를 사용하여 절연 방법을 용이하게하려고했습니다. 그런 다음,이 세포는 CD146 있으며, STRO-1과 같은 stromal - 관련 마커 (CD73, CD90, CD105 및 CD44), 조혈 / 내피 마커 (CD34, CD45 및 CD11b), perivascular 마커의 측면에서 특징. 그 후,이 두 프로토콜은 정량 중합 효소 연쇄 반응 (QPCR) 및 알리자린 레드 얼룩 모두에 의해 odontoblasts에 차별화의 효력에 따라 비교했다. QPCR은 mineralization 관련 유전자의 표현 (; 알프, 매트릭스 세포 phosphoglycoprotein, MEPE 및 상아질 sialophosphoprotein, DSPP 알칼리성 인산 가수 분해 효소)의 평가를 위해 사용되었습니다.<sup&gt; 14</sup</p

<ol><li> 효소 분해 (DPSC-ED)에 의해 획득 된 DPSCs는 하루에 10 15,18 (그림 1)에서 여기에 표시됩니다. 분리 한 후 거의 3-5일에 형성 시작 식민지가. </li><li> 능가 DPSCs (DPSC-OG)는 5 일, 10, 13 &amp; 18 일 그림 2에 표시됩니다. 세포가 병렬 심는 후 약 5 일까지 주문에 의해 펄프 조직에서 병으로 마이그레이션하기 시작 섬유 아세포과 같은. </li><li> 두 방법을 사용하여 통로 3 DPSCs은 그림 3에 표시됩니...

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Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

방법 중 하나를 사용하여 인간의 치과 펄프의 줄기 세포 (hDPSCs)의 분리 및 특성 설명<strong펄프&gt; 효소 분해 (DPSC-ED)</strong&gt; 또는<strong펄프 조직 explants에서 줄기 세포&gt; 직접 가지 (DPSC-OG). 그런 다음</strong&gt;<em&gt; 체외에서</emodontoblasts에 hDPSCs 두 종류의&gt; 비교 차별화.

인간 치과 펄프의 줄기 세포의 분리, 특성화 및 비교 차별화 두 가지 방법을 사용하여 영구 치아에서 파생

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

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26.2K 조회수.  Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran. 방법 중 하나를 사용하여 인간의 치과 펄프의 줄기 세포 (hDPSCs)의 분리 및 특성 설명 효소 분해 (DPSC-ED) 또는 직접 가지 (DPSC-OG). 그런 다음 체외에서 비교 차별화.

비디오: Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

환자 유래 세포 배양 및 CD133의 절연<sup&gt; +</sup흑종에서&gt; Putative 암 줄기 세포

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

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Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

신선한 수술 샘플에서 분리 및 인간의 심실 심근 기능 특성

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

인간의 전립선 암 샘플에서 암 줄기 세포의 분리

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Two Methods for Establishing Primary Human Endometrial Stromal Cells from Hysterectomy Specimens

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell culture systems arising from human endometrial samples are no exception. Applications range from normal cyclic physiological processes to endometrial pathologies such as gynecological cancers, infectious diseases, and reproductive deficiencies. Here, we provide two methods for establishing primary endometrial stromal cells from surgically resected endometrial hysterectomy specimens. The first method is referred to as &#8220;the scraping method&#8221; and incorporates mechanical scraping using surgical or razor blades whereas the second method is termed &#8220;the trypsin method.&#8221; This latter method uses the enzymatic activity of trypsin to promote the separation of cells and primary cell outgrowth. We illustrate step-by-step methodology through digital images and microscopy. We also provide examples for validating endometrial stromal cell lines via quantitative real time polymerase chain reactions (qPCR) and immunofluorescence (IF).

Establishing primary endometrial stromal cell culture systems from hysterectomy specimens is a valuable biological technique and a crucial step prior to pursuing a vast array of research aims. Here, we describe two methods used to establish stromal cultures from surgically resected endometrial tissues of human patients.&#160;

자궁 적출술 검체에서 차 인간의 자궁 내막 기질 세포를 구축하는 두 가지 방법

Many efforts have been devoted to establish in vitro cell culture systems. These systems are designed to model a vast number of in vivo processes. Cell ...

Isolation and Cellular Phenotyping of Mesenchymal Stem Cells Derived from Synovial Fluid and Bone Marrow of Minipigs

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, synovial-fluid-derived MSCs were reported to have multi-lineage differentiation potential and immunomodulatory features, which indicates that these cells can be used for tissue engineering and systemic treatments. This study presents a protocol for simple and non-invasive isolation of MSCs derived from the bone marrow and synovial fluid of minipigs to analyze cell surface markers for cell phenotyping and in vitro culturing. Using sexually mature six-month-old minipigs, bone marrow was extracted from the iliac crest bone using a bone marrow extractor, and the synovial fluid was aspirated from the femorotibial joint. Procedures for the collection of samples from both sources were non-invasive. The protocols for effective isolation of MSCs from harvested cell sources and for creating in vitro culture conditions to expand stable MSCs from minipigs and the application of systemic autologous treatments are provided. For cell phenotyping, the cell surface markers of both cells were analyzed using flow cytometry. In the results, the MSCs were isolated from the synovial fluid of the minipigs and showed that synovial-fluid-derived MSCs have a similar morphology and cell phenotype to bone-marrow-derived MSCs. Therefore, non-invasively obtained synovial fluid is a valuable source of MSCs.

A protocol establishing mesenchymal stem cells (MSCs) isolated from the synovial fluid and bone marrow of minipigs and non-invasively collected using syringe aspiration is presented. Cellular phenotyping was performed using flow cytometry after cell isolation and in vitro cultivation of bone marrow and synovial fluids derived from MSCs.

Minipigs의 활액과 골수 유래 중간 엽 줄기 세포의 분리 및 세포 표현형

Mesenchymal stem cells (MSCs) have been established after isolation from various tissue sources, including bone marrow and synovial fluid. Recently, ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

Cytometry 사용 하 여 인간 지방 조직에서 면역 세포의 특성

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Oral Biofilm Formation on Different Materials for Dental Implants

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low microbial adhesion may reduce the prevalence and progression of peri-implant diseases. In view of the oral environment complexity and oral biofilm heterogeneity, microscopy techniques are needed that can enable a biofilm analysis of the surfaces of teeth and dental materials. This article describes a series of protocols implemented for comparing oral biofilm formation on titanium and ceramic materials for prosthetic abutments, as well as the methods involved in oral biofilms analyses at the morphological and cellular levels. The in situ model to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments as described in this study provides a satisfactory preservation of the 48 h biofilm, thereby demonstrating methodological adequacy. Multiphoton microscopy allows the analysis of an area representative of the biofilm formed on the test materials. In addition, the use of fluorophores and the processing of the images using multiphoton microscopy allows the analysis of the bacterial viability in a very heterogeneous population of microorganisms. The preparation of biological specimens for electron microscopy promotes the structural preservation of biofilm, images with good resolution, and no artifacts.

Here, we present a protocol to evaluate oral biofilm formation on titanium and zirconia materials for dental prosthesis abutments, including the analysis of bacterial cells viability and morphological characteristics. An in situ model associated with powerful microscopy techniques is used for the oral biofilm analysis.

치과 임 플 란 트에 대 한 다른 자료에 구두 Biofilm 형성

Dental implants and their prosthetic components are prone to bacterial colonization and biofilm formation. The use of materials that provides low ...

An Enzyme-free Method for Isolation and Expansion of Human Adipose-derived Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. As a clinically relevant cell type, optimal methods are needed to isolate and expand these cells in vitro. Most methods to isolate adipose-derived MSCs (ADSCs) rely on harsh enzymes, such as collagenase, to digest the adipose tissue. However, while effective at breaking down the adipose tissue and yielding a high ADSC recovery, these enzymes are expensive and can have detrimental effect on the ADSCs &#8212; including the risks of using xenogeneic components in clinical applications. This protocol details a method to isolate ADSCs from fresh lipoaspirate and abdominoplasty samples without enzymes. Briefly, this method relies on mechanical disassociation of any bulk tissue followed by an explant-type culture system. The ADSCs are permitted to migrate out of tissue and onto the tissue culture plate, after which the ADSCs can be cultured and expanded in vitro for any number of research and/or clinical applications.

This protocol provides an enzyme-free method for isolating mesenchymal stem cells from abdominoplasty and lipoaspirate samples using an explant method. The absence of harsh enzymes or centrifugation steps provides for clinically relevant stem cells that can be used for studies in vitro or transferred back to the clinic.

인간 지방분지 유래 중간엽 줄기 세포의 분리 및 확장을 위한 효소 없는 방법

Mesenchymal stem cells (MSCs) are a population of multipotent cells that can be isolated from various adult and fetal tissues, including adipose tissue. ...

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow transplantation. While UCB has several unique advantages, the limited numbers of HSCs within each UCB unit limits their use in regenerative medicine and HSC transplantation in adults. Efficient expansion of functional human HSCs can be achieved by ex vivo culturing of CD34+ cells isolated from UCBs and treated with a deacetylase inhibitor, valproic acid (VPA). The protocol detailed here describes the culture conditions and methodology to rapidly isolate CD34+ cells and expand to a high degree a pool of primitive HSCs. The expanded HSCs are capable of establishing both short-term and long-term engraftment and are able to give rise to all types of differentiated hematopoietic cells. This method also holds potential for clinical application in autologous HSC gene therapy and provides an attractive approach to overcome the loss of functional HSCs associated with gene editing.

Ex Vivo Expansion of Hematopoietic Stem Cells from Human Umbilical Cord Blood-derived CD34+ Cells Using Valproic Acid

Here, we describe the ex vivo expansion of hematopoietic stem cells from CD34+ cells derived from umbilical cord blood treated with a combination of a cytokine cocktail and VPA. This method leads to a significant degree of ex vivo expansion of primitive HSCs for either clinical or laboratory applications.

인간 탯 줄 혈액 파생 CD34에서 조 혈 줄기 세포의 생체 내 확장 전+ 세포 Valproic 산 사용 하 여

Umbilical cord blood (UCB) units provide an alternative source of human hematopoietic stem cells (HSCs) for patients who require allogeneic bone marrow ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

다른 균질화 방법을 통해 마우스 뇌 / 척수에서 검색 된 여러 세포 유형의 동시 유량 세포 측정 특성 분석

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...