C.D.M. was supported by the American College of Surgeons (ACS) Resident Research Scholarship. M.S.H. was supported by the California Institute for Regenerative Medicine (CIRM) Clinical Fellow training grant TG2-01159. M.S.H., H.P.L., and M.T.L. were supported by the American Society of Maxillofacial Surgeons (ASMS)/Maxillofacial Surgeons Foundation (MSF) Research Grant Award. H.P.L. was supported by NIH grant R01 GM087609 and a gift from Ingrid Lai and Bill Shu in honor of Anthony Shu. H.P.L. and M.T.L. were supported by the Hagey Laboratory for Pediatric Regenerative Medicine and The Oak Foundation. M.T.L. was supported by NIH grants U01 HL099776, R01 DE021683-01, and RC2 DE020771. D.C.W. was supported by NIH grant 1K08DE024269, the Hagey Laboratory for Pediatric Regenerative Medicine, and the Stanford University Child Health Research Institute Faculty Scholar Award.

참고 : 샘플 동의를 주었다 환자를 얻었다. 모든 프로토콜을 검토하고 적절한 스탠포드 대학의 임상 시험 심사위원회의 승인을했다. 인간의 조직과 세포를 처리하는 동안, 항상 당신의 기관에 의해 지정된, 바이오 안전성 레벨 2 (BSL2)주의 사항을 준수합니다. 시약 1. 준비 <ol><li> FACS 버퍼를 준비 : 10 ML의 FBS, 188 5 ㎖ 폴록 사머 500 mL의 멸균 인산 완충 생리 식염수 5 mL의 펜 연쇄상 구균 (PBS)를 추가합니다. </li><li> 혼합물을 소화 준비 : 0.375 g C.의 hemolyticum의 콜라게나 제 II 형 분말과 5 ㎖ 폴록 사머 멸균 199 / EBSS 매체 (188) 500 mL를 넣고. </li><li> 표준 매체를 준비 : 50 ML의 FBS 500 mL를 둘 베코의 변형 이글 중간에 5 mL의 펜 연쇄상 구균을 추가합니다. </li></ol> 2. 수확과의 ASC의 분리 참고 : 적절한 기관 승인이 인체 조직을 사용하는 및 분리 시간 동안 제자리에 있는지 확인잃어버린 줄기 세포. 인간의 복부를 확보 측면에, 또는 선택 과목 지방 흡입 수술을받은 건강한 기증자로부터 피하 지방을 허벅지. 플라스틱 흡입 용기에 지방을 유지합니다. <ol><li> 원래 플라스틱 용기에 지방에 : 1 비율로 1 멸균 PBS를 추가합니다. (500 ㎖ 지방이 존재하는 경우, 500 ㎖의 PBS를 추가). 30 초 동안 부드러운 교반 혼합. 수층 흡인에 부착 된 10 mL의 플라스틱 피펫 수층을 하부 (도 1), 그리고, 흡에 정착 폐기 할 수있다. </li><li> 대형 플라스틱 용기에 지방을 가만히 따르다하고 1 혼합물을 소화 추가 : 1의 비율. 용기를 닫고 70 % 에탄올로 외부를 청소합니다. 파라핀 필름으로 뚜껑을 밀봉. </li><li> 30 분 동안 37 ℃에서 180 rpm에서 오비탈 진탕 컨테이너 선동. </li><li> : 1 비율로 1 표준 매체를 추가하여 소화를 중화. (1,000 mL의 지방 혼합물이있는 경우, 1,000 mL로에게 표준 매체를 추가). </li><li> 동일하게 (25)의 짝수로 액을 분배0 mL의 플라스틱 원뿔 원심 분리기 튜브와 원심 분리기 4 ℃에서 20 분 동안 300 XG에 혼합물 </li><li> 뜨는을 대기음 그대로 펠렛을 떠날주의하십시오. 각 원뿔 튜브 5 mL의 표준 매체를 Resuspend 펠릿. </li><li> 한 50ml의 원추형 원심 분리 튜브에 100 미크론 필터를 통해 현탁액을 필터. 그런 다음, 4 ℃에서 15 분 동안 300 XG에 원심 분리기. </li><li> 상등액을 흡인하고 5 ㎖ 실온 RBC 용해 버퍼에 펠렛을 재현 탁. 혼합물을 실온에서 15 분 (RT) 300 XG에서 원심 분리하여 실온에서 5 분 동안 방치시키고. </li><li> 뜨는을 대기음 15 mL의 표준 매체에 펠렛을 재현 탁. 다시 한 50㎖ 원추형 원심 분리 튜브에 100 미크론 필터를 통해 필터링. </li><li> 새로운 50 ML 원뿔에서, 밀도 기반의 분리 (시약의 목록 참조)을 위해 설계된 15 ML의 polysucrose 솔루션을 추가 할 수 있습니다. 다음에, 45 ° 각도로 튜브를 들고 신중 측에 세포를 피펫튜브 polysucrose 솔루션의 상단에있는 세포 현탁액은 부드럽게 층 (그림 2) 그래서. 주 : 용액의 표면에 피펫 세포에 중요하다. 이 돌이킬 수없는 혼합물을 생성하므로 세포의 현탁액에 polysucrose 솔루션을 추가하지 마십시오. 마찬가지로, polysucrose 솔루션의 중심에 세포를 피펫하지 않습니다. </li><li> 실온에서 10 분 동안 300 XG에이 혼합물을 원심 분리기. 낮은 가속 사용 (2 - 10 명 중 3)이 단계 0으로 원심 분리기의 브레이크 기능을 설정합니다. 참고 : 중간에 흐림, 바닥에 분명, 연어 색의 상단에 : 세 가지 레이어가 표시됩니다. 중간 계층은 관심의 세포가 포함되어 있습니다. </li><li> 10 mL를 피펫을 사용하여 조심스럽게 중간 층을 제거하고 새로운 50 ML 원뿔 튜브에 전송할 수 있습니다. 주 :이 완전히 중간층의 일부를 남긴다보다 상부 및 하부 층의 일부를 가지고 중간층 전송 과정에서하는 것이 좋다. </li> &lt;리&gt; 혈구를 사용하여 세포를 세어 총 생균 수를 결정합니다. 참고 : 일반적으로 사용 트리 판 블루 세포 계산을 지원하기 위해 0.8 %의 최종 농도로 세포 혼합액에 첨가 하였다. 유력한 살아있는 세포는 파란색 염료를 차지하지 않습니다과 흰색이 나타납니다. </li></ol> BMPR-IB 양성 세포와 세포 함유 비계의 준비를 위해 정렬 3. FACS <ol><li> 4 ℃에서 5 분 동안 300 XG에 세포 현탁액을 원심 분리기. (2.13 단계에서 수행 된 세포 수에 기초하여) 100 μL 당 백만 세포의 농도로 FACS 완충액에 세포를 재현 탁. </li><li> 라벨 플라스틱 원심 관에 적어도 1 천만 셀로 이동 &quot;BMPR-IB.&quot; 별도로 표시된 별도의 플라스틱 원심 분리기 튜브에 100 μL FACS 버퍼에 백만 세포를 전송 &quot;흠.&quot; 은 &quot;흠&quot;튜브에 1 mL의 FACS 버퍼를 추가합니다. FACS는 EXPER 부분을 정렬하는 동안 얼음에서 FACS 완충액 세포의 나머지 유지iment가 수행되고있다. 이 세포 레이블 &quot;정렬되지 않은합니다.&quot; 컨트롤 나중에 실험에서 이러한를 사용합니다. </li><li> 제조업체의 지시에 따라 &quot;BMPR-IB &#39;관 형광 항 인간 BMPR-IB 항체의 적절한 양을 추가한다. 항체를 배포 부드럽게 상하 혼합물을 피펫. 참고 : 우리는 1:10 희석에 인간 BMPR-IB / ALK-6 APC - 복합 항체를 사용합니다 (자세한 내용은 시약의 목록 참조). 그러나, 다른 제조업체의 항체는 다른 권장 농도를해야합니다. </li><li> 빛을 유지하는 방법으로 얼음 양동이를 커버. 셀 / 항체 혼합물을 30 분 (또는 항체를 제조사의 지시에 따라) 동안 앉아 할 수있다. 주 : 안티 BMPR-IB 항체는 이차 항체를 필요로하는 차 접합 항체 오면, 제조업체의 지시에 따라 이차 항체와 얼음의 별도의 염색 공정을 수행한다. </li><li> 양쪽 튜브를 원심 분리기4 ℃에서 5 분 동안 300 XG. 펠렛을 대기음하지 않도록주의, 뜨는을 대기음. 1 ㎖의 FACS 완충액으로 펠렛 세포를 재현 탁. 또 다시 5 분 동안 300 XG에서 튜브를 원심 분리. 조심스럽게 상층 액을 흡인 및 1 mL의 1 백만 세포의 농도로 FACS 완충액에 세포를 재현 탁. </li><li> 새로운 레이블이 유리 FACS 튜브로 40 마이크론 셀 스트레이너를 통해 &quot;BMPR-IB&quot;, &quot;흠&quot;및 &quot;정렬되지 않은&quot;세포를 필터링합니다. 세포가 필터에 남아되지 않도록 500 μL FACS 버퍼와 필터를 씻어. 또한 &quot;BMPR-IB 양성&quot;을 표시 두 개의 플라스틱 원심 분리기 튜브에 2 mL의 표준 매체를 추가 &quot;BMPR-IB 음성.&quot; FACS 기계에 정렬 된 세포를 수집하기 위해이 두 가지를 사용합니다. </li><li> FACS 기계에서 형광 마커 음의 게이트를 정의하기 위해 흠없는 세포를 사용합니다. </li><li> 100 미크론 노즐 각각 콘 튜브로 정렬 BMPR-IB 양 및 음의 세포를 사용하여표준 매체 이닝. FACS 기계가이 능력이있는 경우 정렬 동안 4 ° C에서 냉각이 튜브를 유지합니다. 참고 : 샤론 외 알에 의해 조브 제 13 조를 참조하십시오. FACS 정렬을위한 우수한 프로토콜. </li><li> 혈구를 사용하여, 각각의 튜브에 세포를 계산. 4 ° C에서 5 분 동안 300 XG에서 &quot;&quot;BMPR-IB 양성 &quot;BMPR-IB 음성&quot;및 &quot;정렬되지 않은&quot;튜브를 원심 분리기와 세포 펠렛을 방해하지 않도록주의, 뜨는을 대기음. 그 후 250 μL 당 백만 세포의 농도를 표준 배지에서 세포를 재현 탁. </li><li> 미리 잘라 4mm PLGA (폴리 [락트산 - 코 - 글리콜 산]) 하이드 록시 아파타이트로 코팅 된 지지체를 얻습니다. 24 웰 플레이트의 우물에 각 발판을 놓습니다. BMPR-IB 양성 정렬되지 않은, 그리고 BMPR-IB 음 (그림 3) : (. 내가합니다 .e, 20 셀) 세 그룹 중 하나에서 세포 현탁액 50 μL 각 발판을 커버. 참고 : 제발소호 등의 알에 의해 조브 제 14 조를 참조하십시오. PLGA 인공 지지체의 구성에 대한 자세한 내용은. </li><li> 부분 (골격의 탈수를 방지하기 위해)의 뚜껑 플레이트를 커버하고, 30 분 동안 37 ℃ 세포 배양 인큐베이터에서 배양한다 표준 배지 주변 빈 웰 채운다. </li></ol> 두개골 결함 및 ACS 함유 비계의 적용 4. 작성 참고 : 적절한 기관 승인이 살아있는 생쥐의 두개골 결함의 생성을 제자리에 있는지 확인합니다. 이 프로토콜은 스탠포드 대학 기관 동물 관리 및 사용위원회에 의해 승인되었습니다. <ol><li> 이소 플루 란 가스 흡입 또는 주입 지속성 칵테일 마취제를 사용하여 CD-한 누드 마우스를 마취. <ol><li> 단독으로 가스를 사용하기 위해, 마취 실에 마우스를 위치시키고이 산소의 흐름을 시작 - 이소 플루 란 2 % 농도 3 L / 분. 마우스는 f를 일단ully 의식, absorbant 패드 따뜻한 물 순환 담요 위에이 발생하기 쉬운 배치하고 산소와 이소 플루 란의 동일한 농도와 마취 코 콘에 그 주둥이를 놓습니다. 조심스럽게 호흡을 모니터링하고 필요에 따라 이소 플루 란의 농도를 조정합니다. </li><li> 전술 한 바와 같이 마취 챔버 내에 마우스를 마취, 주입 된 지속성 마취 칵테일을 사용하고 마취 칵테일 주입. 마취 실에서 마우스를 제거하고 그 행동을 관찰. 참고 : 마우스가 잠시 마취에서하지만 몇 분 내에 나타날 수있다 다시 완전히 주입 마취의 효과에 따라 마취해야합니다. absorbant 패드로 덮여 따뜻한 물 순환 담요에 마우스를 전송합니다. 참고 : 마취 칵테일을 위해, 우리는 생리 식염수에 케타민 10 ㎎ / ㎖과 자일 라진 1 ㎎ / ㎖를 포함하는 혼합물, 복강 전달하는 것이 좋습니다. 이 혼합물의 표준 용량은300 μL 100 ㎎ / ㎏과 자일 라진 10 ㎎ / ㎏을 케타민와 동일합니다 30 g을 마우스합니다. 주 사용 마취제에 대한 자세한 내용은 토론 섹션을 참조하십시오. </li><li> 마취의 깊이를 결정하기 위해 발가락 핀치 책략을 사용합니다. 이 가볍게 외과 의사에 의해 핀치 때 적절하게 마취 마우스는 발을 철회하지 않습니다. </li><li> 각막의 탈수를 방지하기 위해 눈 연고를 적용합니다. </li><li> 1 ㎎ / ㎏ 부 프레 노르 핀의 SR 피하을 관리 할 수 ​​있습니다. 참고 : 우수한 통증 완화의 수술 결과 이전에 진통을 제공. </li><li> 전체 절차 동안 마우스의 호흡 속도를 모니터링한다. 참고 : - / 분 (100) 숨을 제대로 이소 플루 란에서 마취 마우스 (50)의 호흡이 있어야합니다. 증가 된 호흡은 감소 호흡 속도는 마취가 너무 깊은 나타낼 수 있습니다 동안 마취가 너무 빛을 나타냅니다. </li></ol></li><li> 포비돈 - IOD와 두개골의 지느러미 측면의 피부를 준비오프라인 용액을 70 % 에탄올 세번 하였다. 노출 수술 부위를 떠나, 멸균 커튼으로 드레이프. 주 : 멸균 수술 장갑을 착용하고 절차를 수행하는 동안 무균 기술을 유지한다. 그러한 멸균 드레이프와 멸균 도구로 멸균 표면과 객체를 터치합니다. 보조 조명, 오픈 봉합 패킷 등을 조정 한 </li><li> 15 블레이드 메스를 사용하여 지느러미 두개골의 대부분에 걸쳐 연장 시상 중간 선 절개를합니다. 미세 톱니 집게를 사용하여 오른쪽 마루 뼈를 노출시키는 절개 오른쪽의 피부를 철회. </li><li> 멸균 4mm 다이아몬드 코팅 트레 드릴 비트와 드릴을 이용하여 마루 뼈 통해 결함 드릴. 경막 층에 뼈 과거의 결함을 연장하지 마십시오. (그림 4) </li><li> 결함에 발판을 놓은 다음 나일론 봉합사를 실행하는 피부 절개를 닫습니다. </li><li> 마우스를 모니터링하고 표준 수술 후 치료 a를 제공제도적 지침에 ccording. <ol><li> 그것을 복구하는 동안 깨끗한 케이지 내부에 깨끗한 종이 타월에 마우스를 유지합니다. 케이지의 절반은 따뜻한 물 순환 담요 위에 배치되어야하고, 마우스는 처음이 반으로 배치해야합니다. </li><li> 이 ambulate하기에 충분한 의식을 회복 할 때까지 마우스 방치하지 마십시오. 완전히 복구 될 때까지 다른 마우스와 케이지에 수술을 시행 한 마우스를 반환하지 않습니다. </li></ol></li><li> 테스트 할 각 지지체의 새로운 마우스로 위의 단계를 반복합니다. 뜨거운 구슬 살균기 또는 다른 적절한 방법을 사용하여 수술 사이에 수술 도구를 소독. </li><li> 얼마 후 절차 후 2, 4, 6 및 8 주째에, 두개골 폐쇄 결함 비율을 분석하는 마이크로 CT 스캔을 사용한다. 참고 : 리바이스 등의 알에 의해 문서를 참조하십시오. 두개골 결함의 마이크로 CT 스캔에 대한 설명 (6). </li><li> 경우 실험 공단 인TE 클린 안락사 챔버에 넣어서 2 L / min의 속도로 챔버 내로 탄산 가스의 유동을 시작하여 마우스를 안락사. 호흡이 완전히 (후 약 10 분)을 중단 한 후 안락사를 확인하기 위해 자궁 경부 전위를 수행합니다. </li><li> 실험 완료 후 선택적으로, 사용 단면 조직 학적 염색 결함 내 골 형성을 평가. 참고 : McArdle 등의 알에 의해 문서를 참조하십시오. 뼈를 준비 자세한 내용은 12 조직학에 대한 표본. 조직 학적 및 염색 기술 검토를 들어, 외과 병리학 (15)의 수동으로 조직 학적 설명서를 참조하십시오. </li></ol>

<ol>
	<li>Silber, J. 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=Donor+site+morbidity+after+anterior+iliac+crest+bone+harvest+for+single-level+anterior+cervical+discectomy+and+fusion.">Donor site morbidity after anterior iliac crest bone harvest for single-level anterior cervical discectomy and fusion.</a> Spine (Phila Pa 1976). 28 (2), 134-139 (2003).</li><li>Giannoudis, P. V., Dinopoulos, H., Tsiridis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+substitutes:+an+update.">Bone substitutes: an update.</a> Injury. 36, S20-S27 (2005).</li><li>Laurencin, C., Khan, Y., El-Amin, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+graft+substitutes.">Bone graft substitutes.</a> Expert Rev Med Devices. 3 (1), 49-57 (2006).</li><li>Walmsley, G. G., 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=Nanotechnology+in+bone+tissue+engineering.">Nanotechnology in bone tissue engineering.</a> Nanomedicine. 11 (5), 1253-1263 (2015).</li><li>Chapekar, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering:+challenges+and+opportunities.">Tissue engineering: challenges and opportunities.</a> J Biomed Mater Res. 53 (6), 617-620 (2000).</li><li>Levi, B., 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+derived+stromal+cells+heal+critical+size+mouse+calvarial+defects.">Human adipose derived stromal cells heal critical size mouse calvarial defects.</a> PLoS One. 5 (6), e11177 (2010).</li><li>Gimble, J. M., Katz, A. J., 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-derived+stem+cells+for+regenerative+medicine.">Adipose-derived stem cells for regenerative medicine.</a> Circ Res. 100 (9), 1249-1260 (2007).</li><li>Levi, B., 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=CD105+protein+depletion+enhances+human+adipose-derived+stromal+cell+osteogenesis+through+reduction+of+transforming+growth+factor+beta1+(TGF-beta1)+signaling.">CD105 protein depletion enhances human adipose-derived stromal cell osteogenesis through reduction of transforming growth factor beta1 (TGF-beta1) signaling.</a> J Biol Chem. 286 (45), 39497-39509 (2011).</li><li>Chung, M. T., 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=CD90+(Thy-1)-positive+selection+enhances+osteogenic+capacity+of+human+adipose-derived+stromal+cells.">CD90 (Thy-1)-positive selection enhances osteogenic capacity of human adipose-derived stromal cells.</a> Tissue Eng Part A. 19 (7-8), 989-997 (2013).</li><li>Wozney, J. 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=Novel+regulators+of+bone+formation:+molecular+clones+and+activities.">Novel regulators of bone formation: molecular clones and activities.</a> Science. 242 (4885), 1528-1534 (1988).</li><li>Wan, D. 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=Osteogenic+differentiation+of+mouse+adipose-derived+adult+stromal+cells+requires+retinoic+acid+and+bone+morphogenetic+protein+receptor+type+IB+signaling.">Osteogenic differentiation of mouse adipose-derived adult stromal cells requires retinoic acid and bone morphogenetic protein receptor type IB signaling.</a> Proc Natl Acad Sci U S A. 103 (33), 12335-12340 (2006).</li><li>McArdle, 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=Positive+selection+for+bone+morphogenetic+protein+receptor+type-IB+promotes+differentiation+and+specification+of+human+adipose-derived+stromal+cells+toward+an+osteogenic+lineage.">Positive selection for bone morphogenetic protein receptor type-IB promotes differentiation and specification of human adipose-derived stromal cells toward an osteogenic lineage.</a> Tissue Eng Part A. 20 (21-22), 3031-3040 (2014).</li><li>Sharon, Y., Alon, L., Glanz, S., Servais, C., Erez, N. <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+normal+and+cancer-associated+fibroblasts+from+fresh+tissues+by+Fluorescence+Activated+Cell+Sorting+(FACS).">Isolation of normal and cancer-associated fibroblasts from fresh tissues by Fluorescence Activated Cell Sorting (FACS).</a> J Vis Exp. (71), e4425 (2013).</li><li>Lo, D. 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=Repair+of+a+critical-sized+calvarial+defect+model+using+adipose-derived+stromal+cells+harvested+from+lipoaspirate.">Repair of a critical-sized calvarial defect model using adipose-derived stromal cells harvested from lipoaspirate.</a> J Vis Exp. (68), (2012).</li><li>Lester, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual of surgical pathology. , (2010).</li><li>Bunnell, B. A., Flaat, M., Gagliardi, C., Patel, B., Ripoll, C. <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+stem+cells:+isolation,+expansion+and+differentiation.">Adipose-derived stem cells: isolation, expansion and differentiation.</a> Methods. 45 (2), 115-120 (2008).</li></ol>

The authors have no disclosures to make.

프로토콜 내에서 중요한 단계 의 ASC의 수확하는 동안, 중요한 단계는 콜라겐과 지방의 적절한 소화합니다. 부적절한 소화의 ASC의 낮은 수율 발생합니다. BMPR-IB + 세포를 FACS 정렬 동안 신중 양성하는 게이트를 정의하는 것이 중요하다. 너무 느슨하게 게이트를 정의하는 것은 순수하지 정렬 인구의 원인이 될 수 있습니다. 두개골 결함의 생성 동안, 두개골의 뼈를 통해 결함을 드...

손상, 감염 또는 경부암 인한 주요 골 결손 환자의 회복 및 삶의 질에 큰 영향을 미친다. 다른 기술은 환자의 몸으로부터 뼈 건강 이러한 결함을 채우기 위해 존재하지만,이 이동은 자체 이환율과 합병증 1, 2, 3의 위험도를 전달한다. 또한, 일부 결함이 충분한 기증자 뼈 결함을 채우기 위해 사용할 수없는 너무 크거나 복잡하다. 보철 장치는 뼈 결함을 작성을위한 잠재적 인 옵션이 있지만 이러한 감염 위험, 하드웨어 오류 및 이물 반응 (4) 등 여러 가지 단점과 연결되어 있습니다. 이 때문에, 환자 자신의 세포를 이용한 생물학적 5 골 대체재 공학 가능성에 관심이있다. 지방 유래 기질 세포 (의 ASC)이들은 환자 자신의 지방 조직에서 풍부하게 가능하고 새로운 뼈 조직 (6, 7)를 생성하여 뼈의 결함을 치유 할 수있는 능력을 입증하기 때문에이 애플리케이션에 대한 가능성을 갖는다. 세포의 ASC는 여러 다양한 연구 집단은 특정 세포 표면 마커에 대한 선택하는 향상된 골 형성 활성 (8, 9)와 세포 집단을 생산할 수 있음을 보여 주었다이다. 가장 조골 전위의 ASC를 선택하면 이들 세포 시드 발판 큰 골 결손을 재생할 수있는 가능성을 증가시킬 것이다. 뼈 형태 형성 단백질 (BMP) 시그널링은 골 형성 분화의 ASC (10) 및 (11)에서 골 형성에 중요한 것으로 알려진 BMP 수용체 타입 IB (BMPR-IB)를 조절하는 매우 중요하다. 최근에, 우리는 수 b BMPR-IB의 표현을 보여 주었다전자는 강화 된 골 형성 활동 (12)의 ASC를 위해 선택하는 데 사용됩니다. 여기에서 우리는 생체 내 두개골 결손 모델을 사용하여 골 형성 활동의 분석 뒤에 인간 지방에서의 ASC를 BMPR이-IB가 발현의 분리를위한 프로토콜을 보여줍니다.

<table border="0" cellpadding="0" cellspacing="0" width="612">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:152px;">100 micron cell strainer</td>
			<td style="width:136px;">Falcon</td>
			<td style="width:116px;">352360</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:152px;">15 blade scalpel</td>
			<td style="width:136px;">Miltex</td>
			<td>4-515</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:152px;">24 well plate</td>
			<td style="width:136px;">Corning</td>
			<td style="width:116px;">3524</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:152px;">40 micron cell strainer</td>
			<td style="width:136px;">Falcon</td>
			<td style="width:116px;">352340</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:152px;">50 mL conical centrifuge tubes</td>
			<td style="width:136px;">Falcon</td>
			<td style="width:116px;">352098</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:152px;">6-0 Ethilon nylon suture, 18&#34;, P-3 needle,&#160;</td>
			<td style="width:136px;">Ethicon</td>
			<td style="width:116px;">1698G</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:152px;">Anti-BMPR-IB primary antibody</td>
			<td style="width:136px;">R&#38;D systems</td>
			<td>FAB5051A</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:152px;">BioGel PI surgical gloves</td>
			<td style="width:136px;">M&#246;lnlycke Health Care</td>
			<td style="width:116px;">ALA42675Z</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:152px;">Buprenorphine SR</td>
			<td style="width:136px;">ZooPharm</td>
			<td style="width:116px;"></td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:152px;">Castro-Viejo needle driver</td>
			<td style="width:136px;">Fine Science Tools</td>
			<td style="width:116px;">12565-14</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:152px;">CD1 nude mouse</td>
			<td style="width:136px;">Charles River</td>
			<td style="width:116px;">086</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:152px;">Collagenase Type II powder</td>
			<td style="width:136px;">Gibco</td>
			<td style="width:116px;">17101-015</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:152px;">DMEM medium</td>
			<td style="width:136px;">Gibco</td>
			<td style="width:116px;">10564-011</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:152px;">Drill: Circular knife 4.0 mm</td>
			<td style="width:136px;">Xemax Surgical</td>
			<td style="width:116px;">CK40</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:152px;">Drill: Z500 Brushless Micromotor</td>
			<td style="width:136px;">NSK</td>
			<td style="width:116px;">NSKZ500</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:152px;">FBS</td>
			<td style="width:136px;">Gicbo</td>
			<td>10437-077</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:152px;">Fisherbrand Absorbent Underpads, 20&#34; x 24&#34;</td>
			<td style="width:136px;">Fisher Scientific</td>
			<td style="width:116px;">14-206-62</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:152px;">Fisherbrand Sterile cotton gauze pad, 4&#34; x 4&#34;</td>
			<td style="width:136px;">Fisher Scientific</td>
			<td style="width:116px;">22-415-469</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:152px;">Heating pad</td>
			<td style="width:136px;">Kent Scientific</td>
			<td style="width:116px;">DCT-20</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:152px;">Hyclone 199/EBSS medium</td>
			<td style="width:136px;">GE&#160; Life Sciences</td>
			<td style="width:116px;">SH30253.01</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:152px;">Isothesia isoflurane</td>
			<td style="width:136px;">Henry Schein&#160;</td>
			<td style="width:116px;">050033</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:152px;">Micro Forceps with teeth</td>
			<td style="width:136px;">Roboz</td>
			<td style="width:116px;">RS-5150</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:152px;">Micro Forceps with teeth</td>
			<td style="width:136px;">Roboz</td>
			<td style="width:116px;">RS-5150</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:152px;">Paraffin film (Parafilm)</td>
			<td style="width:136px;">Bemis</td>
			<td style="width:116px;">PM996</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:152px;">PBS</td>
			<td style="width:136px;">Gibco</td>
			<td style="width:116px;">10010-023</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:152px;">Pen-Strep</td>
			<td style="width:136px;">Gibco</td>
			<td style="width:116px;">15140-122</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:152px;">PLGA scaffolds</td>
			<td style="width:136px;">Proprietary Formulation</td>
			<td style="width:116px;"></td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:152px;">Poloxamer 188, 10%</td>
			<td style="width:136px;">Sigma</td>
			<td>P5556-100ML</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:152px;">Polylined Sterile Field, 18&#34; x 24&#34;</td>
			<td style="width:136px;">Busse Hospital Disposables</td>
			<td style="width:116px;">696</td>
			<td style="width:208px;">Cut a rectangular hole of the appropriate size</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:152px;">Polysucrose Solution: Histopaque 1119</td>
			<td style="width:136px;">Sigma</td>
			<td>11191</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:152px;">Povidone Iodine Prep Solution</td>
			<td style="width:136px;">Medline</td>
			<td style="width:116px;">MDS093944H</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:152px;">Puralube petrolatum ophthalmic ointment, 1/8 oz. tube</td>
			<td style="width:136px;">Dechra Veterinary Products</td>
			<td style="width:116px;"></td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:152px;">RBC lysis buffer</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:116px;">11814389001</td>
			<td style="width:208px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:152px;">Webcol alcohol prep swabs</td>
			<td style="width:136px;">Covidien</td>
			<td style="width:116px;">6818</td>
			<td style="width:208px;"></td>
		</tr>
	</tbody>
</table>

rapid isolation of bmpr-ib+ adipose-derived stromal cells for use in a calvarial defect healing model

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own body. The ability to grow bone de novo using a patient's own cells would allow bony defects to be filled with adequate tissue without the morbidity of harvesting native bone. There is interest in the use of adipose-derived stromal cells (ASCs) as a source for tissue engineering because these are obtained from an abundant source: the patient's own adipose tissue. However, ASCs are a heterogeneous population and some subpopulations may be more effective in this application than others. Isolation of the most osteogenic population of ASCs could improve the efficiency and effectiveness of a bone engineering process. In this protocol, ASCs are obtained from subcutaneous fat tissue from a human donor. The subpopulation of ASCs expressing the marker BMPR-IB is isolated using FACS. These cells are then applied to an in vivo calvarial defect healing assay and are found to have improved osteogenic regenerative potential compared with unsorted cells.

마이크로 CT 명확하게 두개골 결함을 보여줍니다 수술 당일에 이루어 스캔. 이 때 4mm 결함에 더 증식가 없을 것입니다. 후속 검사는 기준에 비하여 시간 경과에 따른 결함의 크기를 정량화하는 시간에 걸쳐 얻어진다. BMPR-IB-및 정렬되지 않은 세포 (그림 5)과 비교했을 때 BMPR-IB + 세포와 시드 결함은 결함의보다 신속한 폐쇄를 입증해야한다. 또한, 결함을 포함 ?...

Watch this Scientific Journal Video about Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model at JoVE.com

Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

BMPR-IB +의 신속한 격리 두개골 결함 치유 모델에서 사용을위한 기질 세포를 지방이는 유래

Invasive cancers, major injuries, and infection can cause bone defects that are too large to be reconstructed with preexisting bone from the patient's own ...

rapid-isolation-bmpr-ib-adipose-derived-stromal-cells-for-use

Research

JoVE Journal

Developmental Biology

6.6K 조회수.  Stanford University School of Medicine. Adipose-derived stromal cells may be useful for engineering new tissue from a patient's own cells. We present a protocol for the isolation of a subpopulation of human adipose-derived stromal cells (ASCs) with increased osteogenic potential, followed by application of the cells in an in vivo calvarial healing assay.

비디오: Rapid Isolation of BMPR-IB+ Adipose-Derived Stromal Cells for Use in a Calvarial Defect Healing Model

Isolation and Enrichment of Human Adipose-derived Stromal Cells for Enhanced Osteogenesis

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the process by which they must be harvested can be associated with significant donor site morbidity. In contrast, adipose-derived stromal cells (ASCs) are more readily abundant and more easily harvested, making them an appealing alternative to BM-MSCs. Like BM-MSCs, ASCs can differentiate into osteogenic lineage cells and can be used in tissue engineering applications, such as seeding onto scaffolds for use in craniofacial skeletal defects. ASCs are obtained from the stromal vascular fraction (SVF) of digested adipose tissue, which is a heterogeneous mixture of ASCs, vascular endothelial and mural cells, smooth muscle cells, pericytes, fibroblasts, and circulating cells. Flow cytometric analysis has shown that the surface marker profile for ASCs is similar to that for BM-MSCs. Despite several published reports establishing markers for the ASC phenotype, there is still a lack of consensus over profiles identifying osteoprogenitor cells in this heterogeneous population. This protocol describes how to isolate and use a subpopulation of ASCs with enhanced osteogenic capacity to repair critical-sized calvarial defects.

The transcriptional heterogeneity within human adipose-derived stromal cells can be defined on the single cell level using cell surface markers and osteogenic genes. We describe a protocol utilizing flow cytometry for the isolation of cell subpopulations with increased osteogenic potential, which may be used to enhance craniofacial skeletal reconstruction.

분리 및 인간의 농축 향상된 골 연장술에 대한 기질 세포를 지방이 유래

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are considered the gold standard for stem cell-based tissue engineering applications. However, the ...

연구

발생학

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. However, the analysis and quantification of cellular localization is difficult, as in many cases it relies on manual counting, thus bearing the risk of introducing a rater-dependent bias and reducing interrater reliability. Moreover, it is often difficult to judge whether the co-localization between two cells results from random positioning, especially when cell types differ strongly in the frequency of their occurrence. Here, a method for unbiased quantification of cellular co-localization in the bone marrow is introduced. The protocol describes the sample preparation used to obtain histological sections of whole murine long bones including the bone marrow, as well as the staining protocol and the acquisition of high-resolution images. An analysis workflow spanning from the recognition of hematopoietic and non-hematopoietic cell types in 2-dimensional (2D) bone marrow images to the quantification of the direct contacts between those cells is presented. This also includes a neighborhood analysis, to obtain information about the cellular microenvironment surrounding a certain cell type. In order to evaluate whether co-localization of two cell types is the mere result of random cell positioning or reflects preferential associations between the cells, a simulation tool which is suitable for testing this hypothesis in the case of hematopoietic as well as stromal cells, is used. This approach is not limited to the bone marrow, and can be extended to other tissues to permit reproducible, quantitative analysis of histological data.

A strategy to quantitatively analyze histological data in the bone marrow is presented. Confocal microscopy of fluorescently labeled cells in tissue sections results in 2-dimensional images, which are automatically analyzed. Co-localization analyses of different cell types are compared to data from simulated images, giving quantitative information about cellular interactions.

조혈 세포의 자동화 된 정량화 - Undecalcified 뼈의 조직 학적 이미지에서 기질 세포 상호 작용

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

돼지 피하 지방이 조직에서 분리 및 지방이 유래 줄기 세포의 분화

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells (ASCs)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix (ECM) remodeling. In vivo, ECM-rich environment consisting of fibrillar collagens provides a structural support to adipose tissues during the progression and regression of obesity. Physiological ECM remodeling mediated by matrix metalloproteinases (MMPs) plays a major role in regulating adipose tissue size and function1,2. The loss of physiological collagenolytic ECM remodeling may lead to excessive collagen accumulation (tissue fibrosis), macrophage infiltration, and ultimately, a loss of metabolic homeostasis including insulin resistance3,4. When a phenotypic change of the adipose tissue is observed in gene-targeted mouse models, isolating primary ASCs from fat depots for in vitro studies is an effective approach to define the role of the specific gene in regulating the function of ASCs. In the following, we define an immunomagnetic separation of Sca1high ASCs.

Immunomagnetic Separation of Fat Depot-specific Sca1high Adipose-derived Stem Cells ASCs

We present the techniques required to isolate the stromal vascular fraction (SVF) from mouse inguinal (subcutaneous) and perigonadal (visceral) adipose tissue depots to assess their gene expression and collagenolytic activity. This method includes the enrichment of Sca1high adipose-derived stem cells (ASCs) using immunomagnetic cell separation.

지방 창고 별 SCA1의 면역 자기 분리<sup&gt; 높은</sup&gt; 지방이 유래 줄기 세포 (의 ASC)

The isolation of adipose-derived stem cells (ASCs) is an important method in the field of adipose tissue biology, adipogenesis, and extracellular matrix ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

인간 태반 조직에서 추출 분리 및 중간 엽 줄기의 확장 / 기질 세포

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes differentiated from stem cells using cytokines. Unfortunately, cytokine-induced chondrogenic differentiation is costly, time-consuming, and associated with a high risk of contamination during in vitro differentiation. However, biomechanical stimuli also serve as crucial regulatory factors for chondrogenesis. For example, mechanical stress can induce chondrogenic differentiation of stem cells, suggesting a potential therapeutic approach for the repair of impaired cartilage. In this study, we demonstrated that centrifugal gravity (CG, 2,400 &#215; g), a mechanical stress easily applied by centrifugation, induced the upregulation of sex determining region Y (SRY)-box 9 (SOX9) in adipose-derived stem cells (ASCs), causing them to express chondrogenic phenotypes. The centrifuged ASCs expressed higher levels of chondrogenic differentiation markers, such as aggrecan (ACAN), collagen type 2 alpha 1 (COL2A1), and collagen type 1 (COL1), but lower levels of collagen type 10 (COL10), a marker of hypertrophic chondrocytes. In addition, chondrogenic aggregate formation, a prerequisite for chondrogenesis, was observed in centrifuged ASCs.

Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

원심 중력에 의해 지방이 유래 줄기 세포의 연골 분화 유도

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

마우스 자궁 내막 상피와 기질 세포의 분리에 대한<em&gt; 체외</em&gt; 탈락

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

인간 제대혈 및 태아 태반에서 중간 엽 기질 세포의 분리 및 특성

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the efficacy of cell persistence and biodistribution of haMSCs in animal models. We demonstrated a method to label the cell membrane of haMSCs with lipophilic fluorescent dye. Subsequently, intra-articular injection of the labeled cells in rats with surgically induced KOA was monitored dynamically by an in vivo imaging system. We employed the lipophilic carbocyanines DiD (DilC18 (5)), a far-red fluorescent Dil (dialkylcarbocyanines) analog, which utilized a red laser to avoid excitation of the natural green autofluorescence from surrounding tissues. Furthermore, the red-shifted emission spectra of DiD allowed deep-tissue imaging in live animals and the labeling procedure caused no cytotoxic effects or functional damage to haMSCs. This approach has been shown to be an efficient tracking method for haMSCs in a rat KOA model. The application of this method could also be used to determine the optimal administration route and dosage of MSCs from other sources in pre-clinical studies.

In Vivo Tracking of Human Adipose-derived Mesenchymal Stem Cells in a Rat Knee Osteoarthritis Model with Fluorescent Lipophilic Membrane Dye

This protocol describes an efficient way to monitor the cell persistence and biodistribution of human adipose-derived mesenchymal stem cells (haMSCs) by far-red fluorescence labeling in a rat knee osteoarthritis (KOA) model via intra-articular (IA) injection.

Vivo에서 인간 지방 유래 중간 엽 줄기 세포 형광 질 성 막 염료와 쥐 무릎 관절염 모델에서의 추적

In order to support the clinical application of human adipose-derived mesenchymal stem cell (haMSC) therapy for knee osteoarthritis (KOA), we examined the ...

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for embryo implantation into the uterine lining of the maternal womb. Improper decidualization has been established as a root cause for implantation failure and subsequent early embryo miscarriage. Therefore, understanding the molecular mechanisms underlying decidualization is advantageous to improving the rate of successful births. In vivo based studies of artificial decidualization are often limiting due to ethical dilemmas associated with human research, as well as translational complications within animal models. As a result, in vitro assays through primary cell culture are often utilized to explore the modulation of decidualization via hormones. This study provides a detailed protocol for the isolation of HESC and subsequent artificial decidualization via the supplementation of hormones to the culturing medium. Further, this study provides a well-designed method to knockdown any gene of interest by utilizing lipid-based siRNA transfections. This protocol permits the optimization of culture purity as well as product yield, thereby maximizing the ability to utilize this model as a reliable method to understand the molecular mechanisms underlying decidualization, and the subsequent quantification of secreted agents by decidualized endometrial stromal cells.

Isolation of Human Endometrial Stromal Cells for In Vitro Decidualization

This study presents a validated and optimized procedure for the isolation and culture of human endometrial stromal cells to conduct in vitro decidualization assay. Further, this study provides a detailed method to efficiently knockdown a specific gene using siRNAs in human endometrial stromal cells.

생체 외에서 Decidualization에 대 한 인간의 자궁내 막 기질 세포의 고립

The differentiation of human endometrial stromal cells (HESC) from fibroblast-like appearance into secretory decidua is a transformation required for ...