이 연구는 중국의 자연 과학 재단 (http://www.nsfc.gov.cn/ Grant No. 30971665)의 Li Ka Shing Foundation, 광동 고 수준 대학 프로젝트 &quot;해양 산업을위한 녹색 기술&quot; 81172894, 81370925) 및 광동 교육부 (http://www.gdhed.edu.cn/ 보조금 번호 cxzd1123).

8-12 주된 BALB / c 마우스는 Shanghai Experimental Animal Center (Shanghai, China)에서 구입하여 특정 병원균이없고 공기가없는 동물 시설에서 자랐습니다. 동물 관리 및 실험 절차는 산 터우 대학 (Shantou University)에 의해 설립 된 실험용 동물의 사용 및 관리에 대한 지침을 준수했습니다. 1. 장치의 조립 <ol><li> 무균 상태를 유지하려면 사용하기 전에 30 분 동안 조직 배양 후드의 자외선을 켭니다. </li><li> 일회용 25 mm 필터 유닛의 나사를 풀고 캡과 장치의 바닥을 75 % 에탄올이 채워진 200 mL 유리 컵에 30 분간 담근다. 의료용 폴리 프로필렌으로 만들어졌습니다. </li><li> 포셉을 사용하여 장치의 뚜껑과 바닥을 잡고 남은 에탄올을 손으로 털어 내고 부품을 후드에서 10 분 동안 공기 건조시킵니다. </li><li> PBS에 19mm 폴리 카보네이트 멤브레인을 적신 다음 조심스럽게 보토의지지 매트릭스 위에 올려 놓습니다m입니다. 고분자 필름은 매끄럽고 평평한 표면을 가지고 있으며 3 μm의 공극을 트랙 에칭했습니다. </li><li> 캡을 바닥에 단단히 조여서 장치를 다시 조립하십시오. 멤브레인이 중앙에서 빠지지 않았는지 확인하십시오. </li><li> 1 ML 인슐린 주사기의 바늘을 제거하고 PBS 1 ML을 그립니다. 주사기를 필터 장치에 연결 한 다음 PBS를 장치에 밀어 넣어 어셈블리를 적시고 누출이 있는지 테스트하십시오. </li></ol> 2. 플라즈마 막 소포 (PMVs)의 생성 <ol><li> Nemeth et al. 에 의해보고 된대로 BMSC 문화를 확립 하십시오. 15 , 15 % FBS 및 1 % 페니실린 / 스트렙토 마이신을 함유하는 DMEM 배양 배지에서 배양 하였다. 37 ° C에서 5 % CO 2 가 포함 된 humidified 인큐베이터에서 6 잘 접시에있는 세포를 육성. </li><li> 세포를 증식시키기 위해 배양 배지를 제거하고 칼슘이없는 PBS 0.5 mL로 잘 헹구십시오. </li><li> 0.25 % 트립신 - EDTA 0.5 ML을 추가하고 37에서 세포를 품어 ° C3 분 동안. 손바닥에 대해 접시를 몇 번 두드려 세포 분리를 촉진하십시오. 배양액 1 mL를 넣어 소화를 멈춘다. </li><li> 벤치 상단 원심 분리기에서 2 분 200 XG 15 ML 원뿔 튜브와 회전에서 세포를 수집합니다. 배지 4 ML에 세포를 Resuspend하고 6 - 웰 플레이트의 두 우물에 세포를 나누어지는. 참고 : 세포는 대개 24 시간 내에 합류합니다. </li><li> PMVs를 생성하기 위해 트립신 소화에 의해 6-well plate의 한 well (약 5 x 10 5 cell)에서 세포를 수확하고 0.5 mL의 배양 배지에서 monodisperse시킨다. </li><li> 인슐린 주사기에 세포를 넣고 필터 장치에 연결 한 다음 플런저를 빠르게 밀어 필터를 통과하는 세포를 쥐어 짜십시오. 내부에 몇 개의 PMV 만 있어야하기 때문에 압출 된 매체를 버립니다. </li><li> 주사기에 추가로 0.5 mL의 배지를 넣고 신속하게 필터를 통과시킵니다. 포함 된 두 번째 압출 매체 수집다양한 크기의 PMV. </li><li> 수집 된 매체 150 μL를 35 mm 유리 바닥 접시에 넣고 PMV를 약 10 분 동안 바닥에 고정시킵니다. 20X 대물 렌즈를 사용하여 CCD 카메라가 장착 된 역상 위상차 현미경으로 PMV를 검사합니다. </li></ol> 3. 공 촛점 현미경을 이용한 PMV 내의 내용물 검사 <ol><li> BMSC에서 트랜 스펙 션을 수행하려면 50 μL의 무 혈청 DMEM에 2 μg의 supercoiled plasmid DNA와 6 μL의 양이온 transfection reagent ( 예 : PolyJet)를 희석하십시오. 소용돌이가 잘 섞이고 튜브의 측면에서 모든 액체를 수집하기 위해 잠시 회전합니다. </li><li> 희석 된 DNA 용액에 희석 된 용액 (단계 3.1에서)을 적가하고, 1 분 동안 강하게 와동시키고, 짧게 회전시키고, 실온에서 10 분 동안 인큐베이션한다. </li><li> 하룻밤 동안 약 30 %의 농도로 배양 된 BMSC의 새 배지를 2 mL의 새로운 배양 배지로 교체하고 DN형질 전환 혼합물을 적가합니다. </li><li> 형질 감염 12 시간 후 신선한 매체 2 ML로 바꿉니다. </li><li> 세포질로 현지화 된 단백질을 추적하려면 위에 설명한대로 EGFP 발현 카세트 (pEGFP-N1)를 포함하는 플라스미드로 세포를 형질 감염시킵니다. 위에서 설명한대로 PMV 생성에 대한 transfection 48 시간 후 트립신 소화에 의해 세포를 수확. </li><li> 미토콘드리아를 추적하려면 37 ° C에서 30 분 동안 신선한 배지에서 미토콘드리아 염료 (1 μM, MitoTracker)로 세포를 염색하십시오. </li><li> 미토콘드리아의 막 전위를 검출하기 위해, 시아닌 염료 JC-1 (5,5 &#39;, 6,6&#39;- 테트라 클로로 -1,1&#39;, 3,3&#39;- 테트라 에틸 벤즈 미미 - 다 졸릴 카르 보시 아닌 요오드화물) (10 μg / ML) 37 ° C에서 30 분 신선한 배지에서. <ol><li> 위에서 설명한대로 PBS 0.5 ML의 세 번 세포를 씻어 후 PMV 생성에 대한 트립신 소화에 의해 세포를 수확. </li><li> 150 μL의 수집 된 med로드ium을 35mm 유리 바닥 접시에 넣고 PMV를 약 10 분 동안 바닥에 내려 놓습니다. 100X 오일 객관적인 렌즈를 사용하여 공 촛점 현미경하에 PMVs를 검사하십시오. </li></ol></li><li> EGFP 또는 미토콘드리아 염료를 검출하려면 488 nm 레이저를 켜고 505-530 nm에서 신호를 수집하십시오. JC-1을 감지하려면 488nm 및 543nm 레이저를 켜고 505-530nm 및 560-615nm에서 신호를 수집합니다. 핀홀 값을 약 1.4로 설정하십시오. </li></ol> 4. 원심 분리에 의한 농축 된 PMV의 제조 <ol><li> 트립신 소화에 의해 세포를 수확하고 위에서 설명한대로 PMVs를 0.5 ML 문화 매체에 생성합니다. </li><li> 상온에서 5 분 동안 벤치 탑 원심 분리기에서 1,200 xg로 PMV를 돌립니다. 조심스럽게 상등액을 기음과 부드러운 소용돌이와 문화 매체의 100 μL에 펠렛을 resuspend. </li><li> 35mm 유리 바닥 접시에 50 μL의 PMV를로드하고 약 10 분 동안 실온에서 안정되게하십시오. PMV 검사40X 오일 대물 렌즈를 사용하는 공 촛점 현미경 하에서. </li><li> 1 시간 동안 BMSC 배양 물에 일련의 희석 된 폴리에틸렌 이민 (PEI) 용액을 첨가하고 세포 독성의 징후를 확인하십시오. 참고 : BMSC에서 독성의 징후가 발견되지 않았으므로 여기서 2 μg / mL의 PEI를 선택했습니다. </li><li> 트립신 소화하여 세포를 수확하고 응집을 방지하는 수단으로 세포막을 충전 1 H 2 μg / ML에 PEI를 추가합니다. 위에서 설명한대로 0.5 ML 문화 매체에서 PMVs를 생성합니다. 위에서 설명한대로 PMV를 집중시키고 공 촛점 현미경으로 검사하십시오. </li></ol> 5. 비복근 근육에 PMV 주사 <ol><li> 막을 염색하기 위해 30 분 동안 BMSCs에 신선한 문화 매체 1 ML에 CM - DiI (chloromethyl dialkyl - indocarbocyanine) (10 μm의) 염료를 추가합니다. </li><li> 트립신 소화에 의해 세포 (약 5 x 10 5 )를 수확하고 배양 배지 0.5 mL에 그들을 재현 탁한다. PEI (2 μg / mL)를 1h. 위에 설명한대로 PMV를 생성하고 100 μL의 배지에 집중시킵니다. </li><li> 12 시간의 금식 후 sodium barbital (10 mg / kg)을 주사하여 BALB / c 마우스를 마취시킨다. </li><li> 75 % 에탄올로 위의 근육의 외부를 청소하십시오. 천천히 30 G 인슐린 주사기를 사용하여 비복근 근육에 PMV (100 μL)를 주입합니다. </li><li> 마취를 시행 한 후, 실험 기간 동안 눈 위에 젖은 거즈를 놓고 마우스가 깨어날 때까지 백열등으로 따뜻하게합니다. 개별 환기 케이지에 마우스를 홀로 두십시오. </li><li> 수술 12 시간 후 gastrocnemius 근육을 수확하려면 경추 탈구로 마우스를 죽이십시오. </li><li> gastrocnemius 근육 위에 75 % 에탄올을 뿌리고, 가위로 피부를 열고 양끝에서 전체 gastrocnemius 근육을 잘라내십시오. </li><li> PBS로 근육을 헹구고 20X 객관적인 렌즈를 사용하여 형광 현미경으로 검사하십시오. </li><li> 근육을 손질해라.날카로운 면도날로 형광이없는 부분을 제거합니다. 약 9mm 3의 입방체로 강한 붉은 색 형광으로 부품을 자릅니다. </li><li> 최적의 절삭 온도 매체 (OCT)에 각 큐브를 삽입하고 5 분 동안 액체 질소에 담그고 -20 ° C의 냉동고에 보관하십시오. </li><li> 냉동 섹션을 저온 유지 장치를 사용하여 20 μm 두께로 자르고 각 섹션을 유리 슬라이드에 부착하십시오. PBS로 절편을 한 번 헹굽니다. </li><li> 얼룩 원형 펜으로 섹션 주위에 원을 그려 DAPI (1 μg / ML) 100 μL를 추가합니다. 어둠 속에서 실온에서 10 분 잠복 후 DAPI 솔루션을 대기음과 PBS로 세 번 씻어. </li><li> 시험편 위에 오일을 떨어 뜨리고, 유리 슬립으로 덮고 매니큐어로 슬라이드를 밀봉하십시오. 100X 오일 객관적인 렌즈를 사용하여 공 촛점 현미경하에 섹션을 검사하십시오. </li><li> CM-DiI를 검출하기 위해서는 543 nm 레이저를 켜고 560-615 nm에서 신호를 수집하십시오. DAPI를 감지하려면 405를 켭니다.420-480 nm에서 신호를 수집합니다. 핀홀 값을 약 1.4로 설정하십시오. </li></ol>

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N., 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=Emerging+roles+for+extracellular+vesicles+in+tissue+engineering+and+regenerative+medicine.">Emerging roles for extracellular vesicles in tissue engineering and regenerative medicine.</a> Tissue Eng B. 21 (1), 45-54 (2015).</li><li>Baumgart, 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=Large-scale+fluid/fluid+phase+separation+of+proteins+and+lipids+in+giant+plasma+membrane+vesicles.">Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles.</a> Proc Natl Acad Sci. 104 (9), 3165-3170 (2007).</li><li>Sezgin, 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=Elucidating+membrane+structure+and+protein+behavior+using+giant+plasma+membrane+vesicles.">Elucidating membrane structure and protein behavior using giant plasma membrane vesicles.</a> Nat Protoc. 7 (6), 1042-1051 (2012).</li><li>Lingwood, D., Ries, J., Schwille, P., Simons, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membranes+are+poised+for+activation+of+raft+phase+coalescence+at+physiological+temperature.">Plasma membranes are poised for activation of raft phase coalescence at physiological temperature.</a> Proc Natl Acad Sci. 105 (29), 10005-10010 (2008).</li><li>Pandey, A. P., Sawant, K. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylenimine:+A+versatile,+multifunctional+non-viral+vector+for+nucleic+acid+delivery.">Polyethylenimine: A versatile, multifunctional non-viral vector for nucleic acid delivery.</a> Mater Sci Eng C Mater Biol Appl. 68, 904-918 (2016).</li></ol>

저자는 공개 할 것이 없습니다.

Cytoplasm replacement therapy as proposed in this manuscript has unique advantages over other reported approaches such as gene, molecular, and cell therapy. PMVs generated from BMSCs encapsulate not only the products of stemness genes but also intact cellular organelles, which are essential to remedy the ageing phenotypes associated with senescence. When young cytoplasm is delivered to senescent cells, the malfunctioning mechanisms may gain a brief relief; at the same time, the epigenome could be reprogrammed and invigor...

<html> 유전자, 효소 및 세포 대체 요법에 대한 접근법을 수립하는데 엄청난 노력이 기울여왔다. 이것은 획기적인 발전을 가져 왔고 심지어 임상 적용 1 , 2 , 3을 가져 왔습니다. 최근 핵 전달 기술에 기반을 둔 논란이되고있는 미토콘드리아 보충 요법이 ​​노년기 여성 또는 치명적인 mitochondrial DNA 돌연변이를 가지고있는 체외 수정에 적용되었습니다. 아테롬성 동맥 경화증, 2 형 당뇨병, 알츠하이머 병 및 암을 포함한 연령 관련 질환에서 발견되는 결함은 대개 다각적입니다. 지질 방울의 축적이 문서화되어있다. 아밀로이드 단백질의 침전; 소포체에서 전개되지 않은 단백질의 유지; autophagosome, mitochondria가 이들 질병의 발달 또는 악화에 기여한다.&quot;xref&quot;&gt; 5 , 6 , 7 , 8 , 9 , 10 , 11 . 현재, 노화 및 노화 표현형을 일으키는 세포질 및 세포 기관에서의 오작동의 직접적인 개선을 목표로하는 이용 가능한 메커니즘이 없다. 우리는 이전에 포유 동물 세포의 기계적 압출을 통해 플라즈마 막 소포 (PMVs)의 생성에 대해보고했습니다 12 . 핵을 제외하고는 미토콘드리아와 같은 세포 소기관뿐만 아니라 단백질과 RNA를 비롯한 막 또는 세포질의 구성 요소가 PMV에서 발견되었습니다. 본질적으로, PMV는 소형 핵 세포로 간주 될 수 있습니다. 더 중요한 것은, 미토콘드리아 결핍 Rho0 세포와 PMVs의 융합은 정상적인 문화 조건 하에서 mitotic 활동을 복원했다. 이것은 첫 번째 보고서입니다.세포질 대체 요법을위한 잠재적으로 효율적인 접근 방법을 제시하고있다. 골수 간엽 줄기 세포 (bone marrow mesenchymal stem cells, BMSCs)는 골수에서 일상적으로 생성되는 다 분화 선조 세포 (multipotent progenitor cell)이며 배양 물에서 쉽게 확장됩니다. 배아 줄기 세포 마커 Oct4, Nanog 및 SOX2는 MSCs 13 에서 낮은 수준으로 검출되었다. 텔로 머라 아제 활성도 측정 가능합니다. 또한 MSC에서의 낮은 HLA Class I 발현뿐만 아니라 공동 자극 분자 및 인간 백혈구 항원 (HLA) 클래스 II 분자의 부재는 동종 이성체 또는 &quot;기성품 인&quot;사용에 이상적인 세포로 작용합니다. 재생 의학 및 면역 조절 응용 14 . 여기, 우리는 3 μm의 공극을 가진 폴리 카보 네이트 멤브레인을 통해 압출을 통해 마우스 BMSC에서 PMV를 준비하는 방법, 미토콘드리아의 존재를 결정하는 방법 및 PMV에서 멤브레인 잠재력의 유지를 검사하는 방법을 설명합니다.현미경 검사법으로 원심 분리에 의해 응축 된 PMV가 아닌 농축 된 PMV를 준비하고 PMV를 생쥐의 비복근 내로 주입합니다.

<table><tbody><tr><td>IsoporeTM membranes</td><td>Millipore</td><td>TSTP04700</td><td>3 mm pore</td></tr><tr><td>Disposable filter unit</td><td>Xinya, Shanghai, China</td><td>25 mm</td><td>Medical grade polypropylene</td></tr><tr><td>Insulin syringe</td><td>BD</td><td>328446</td><td>1 mL</td></tr><tr><td>pN1-EGFP</td><td>Clontech</td><td>&amp;nbsp;6085-1</td><td></td></tr><tr><td>MitoTracker</td><td>Molecular Probes</td><td>M7514</td><td>Green FM, 1 &amp;mu;M</td></tr><tr><td>JC-1</td><td>Beyotime, Haimen, China</td><td>C2006</td><td>10 mg/mL</td></tr><tr><td>CM-DiI</td><td>Beyotime, Haimen, China</td><td>C1036</td><td>10 mM</td></tr><tr><td>PEI</td><td>Sigma</td><td>P3143</td><td>Mn = 75,000</td></tr><tr><td>Fluorescence Microscope</td><td>Nikon</td><td>Eclipse TE 2000</td><td>With CCD camera</td></tr><tr><td>Confocol Microscope</td><td>Carl Zeiss</td><td>LSM 510 Meta</td><td></td></tr><tr><td>PolyJet</td><td>SigaGen</td><td>SL100688</td><td>For cell transfection</td></tr></tbody></table>

preparation of plasma membrane vesicles from bone marrow mesenchymal stem cells for potential cytoplasm replacement therapy

우리는 이전에 포유류 세포의 기계적 압출을 통한 원형질막 소포 (PMVs)의 생성에 대해보고했다. 미토콘드리아 결핍 Rho0 세포와 PMVs의 융합은 정상적인 배양 조건 하에서 유사 분열 활성을 회복시켰다. 죽상 동맥 경화증, 제 2 형 당뇨병, 알츠하이머 병 및 암은 다양한 세포 유형의 세포질 및 세포 소기관에서 여러 가지 기계적 및 기능적 결함과 관련이 있다고보고 된 연령 관련 질환입니다. 골수 간엽 줄기 세포 (bone marrow mesenchymal stem cells, BMSCs)는 다 기능성을 유지하면서자가 재생 능력을 보유한 골수에서 유래 된 독특한 세포 집단을 대표합니다. PMVs의 융합을 통한자가 BMSCs로부터의 어린 세포질을 가진 노화 세포의 보충은 노화와 연관된 표현형을 개선하거나 심지어 역전시키는 유망한 접근법을 제공한다. 이 프로토콜은 압출을 통해 BMSC에서 PMV를 준비하는 방법을 설명합니다.1, 공극, 미토콘드리아의 존재를 결정하고 공 촛점 현미경을 사용하여 PMV 내의 막 전위의 유지를 검사하고, 원심 분리에 의해 PMV를 농축하고 생쥐의 비복근 내로 PMV의 생체 내 주입을 수행한다.

PMV의 성공적인 제조의 열쇠는 1 mL의 PBS를 멤브레인을 통과시켜 시험 할 수있는 필터 유닛 ( 그림 1 )의 정확한 조립에 크게 의존합니다. 누출이 발생하면 필터 장치를 다시 조립하고 다시 테스트하십시오. 그러나 누설은 세포가 멤브레인을 통과 할 때만 신뢰성있게 테스트 할 수 있습니다. 단지 10X 대물 렌즈를 사용하는 일반적인 현미경으로 PMV가 몇 개 발견되...

Watch this Scientific Journal Video about Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy at JoVE.com

Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy

연령 관련 질환은 세포질 구성 요소의 여러 결함과 관련이 있습니다. 여기, 우리는 골수 간엽 줄기 세포에서 플라즈마 막 소포를 준비하는 프로토콜을 제시합니다. 이 기법은 연령 관련 phenotypes을 개선하거나 심지어 심지어 세포질 대체 요법의 수단으로 잠재적으로 사용할 수 있습니다.

잠재적 인 세포질 대체 요법을위한 골수 간엽 줄기 세포로부터의 혈장 막 소포의 제조

We have previously reported on the generation of plasma membrane vesicles (PMVs) through the mechanical extrusion of mammalian cells. The fusion of PMVs ...

preparation-plasma-membrane-vesicles-from-bone-marrow-mesenchymal

Research

JoVE Journal

Developmental Biology

7.5K 조회수.  Shantou University.  연령 관련 질환은 세포질 구성 요소의 여러 결함과 관련이 있습니다. 여기, 우리는 골수 간엽 줄기 세포에서 플라즈마 막 소포를 준비하는 프로토콜을 제시합니다. 이 기법은 연령 관련 phenotypes을 개선하거나 심지어 심지어 세포질 대체 요법의 수단으로 잠재적으로 사용할 수 있습니다. 

비디오: Preparation of Plasma Membrane Vesicles from Bone Marrow Mesenchymal Stem Cells for Potential Cytoplasm Replacement Therapy

Isolation of Human Mesenchymal Stem Cells and their Cultivation on the Porous Bone Matrix

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.

인간 중간 엽 줄기 세포의 분리 및 다공성 뼈 매트릭스에 자신의 재배

Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific ...

연구

발생학

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade clots in the bone marrow and release MSCs for further use. This protocol provides a rapid and inexpensive alternative to bone marrow resampling. Bone marrow is a major source of MSCs, which are interesting for tissue engineering and autologous stem cell therapies. Upon withdrawal bone marrow may clot, as it comprises all of the hematopoietic system. The resulting clots contain also MSCs that are lost for expansion culture or direct stem cell therapy. We experienced that 74% of canine bone marrow samples contained clots and yielded less than half of the stem cell number expected from unclotted samples. Thus, we developed a protocol for enzymatic digestion of those clots to avoid labor-intense and costly bone marrow resampling. Urokinase - a clinically approved and readily available thrombolytic drug &#8211; clears away the bone marrow clots almost completely. As a consequence, treated bone marrow aspirates yield similar numbers of MSCs as unclotted samples. Also, after urokinase treatment the cells kept their metabolic activity and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. Our protocol salvages clotted blood and bone marrow samples without affecting the quality of the cells. This obsoletes resampling, considerably reduces sampling costs and enables the use of clotted samples for research or therapy.

Mesenchymal stem cells are usually obtained from bone marrow and require expansion culture. When samples clot before processing, a protocol using the (enzymatic) thrombolytic drug urokinase can be applied to degrade the clot. Thus, cells are released and available for expansion culture. This protocol provides a rapid and inexpensive alternative to resampling.

효소 방법은 응고 골수 샘플에서 중간 엽 줄기 세포를 구출

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

에서 간세포 이식 체외에서 시뮬레이션 국소 빈혈 / Reperfusion 모델

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

의학

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel cell population specifically selected for growth in human serum supplemented medium. These cells are characterized by morphological, phenotypic, and molecular features distinct from MSCs and we named them Mesodermal Progenitor Cells (MPCs). MPCs are round, with a thick highly refringent core region; they show strong, trypsin resistant adherence to plastic. Failure to expand MPCs directly revealed that they are slow in cycling. This is as also suggested by Ki-67 negativity. On the other hand, culturing MPCs in standard medium designed for MSC expansion, gave rise to a population of exponentially growing MSC-like cells. Besides showing mesenchymal differentiation capacity MPCs retained angiogenic potential, confirming their multiple lineage progenitor nature. Here we describe an optimized highly reproducible protocol to isolate and characterize hBM-MPCs by flow cytometry (CD73, CD90, CD31, and CD45), nestin expression, and F-actin organization. Protocols for mesengenic and angiogenic differentiation of MPCs are also provided. Here we also suggest a more appropriate nomenclature for these cells, which has been re-named as "Mesangiogenic Progenitor Cells".

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Here we describe an optimized, highly reproducible protocol to isolate Mesodermal Progenitor Cells (MPCs) from human bone marrow (hBM). MPCs were characterized by flow cytometry and nestin expression. They showed the ability to give rise to exponentially growing MSC-like cell cultures while retaining their angiogenic potential.

사람 골수에서 분리 Mesangiogenic 전구 세포 (MPC와)

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

An experimental technique for the treatment of osteochondral defects in the rabbit's knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.

섬유소 혈전의 동종 간엽 줄기 세포의 주입에 의한 토끼의 무릎 관절의 골 연골 결함의 치료

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular ...

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products.
Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions &#8212;&#160;namely,&#160;patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor&#39;s own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Culturing human Mesenchymal Stromal Cells (hMSCs) with autologous serum, reduces the risks of rejection by xenogeneic material and other negative effects. It also allows for the recovery of a subset of mesodermal progenitors, which can deliver fresh hMSCs. Embedding hMSCs in an autologous fibrin clot enables easy handling and effective surgical implantation.

중간 엽 기질자가 조건에서 세포 배양 배송 : 정형 외과 응용 프로그램에 대한 스마트 접근

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...

생체공학

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

재조합 콜라겐 나 세포 확장과 생물에 셀 납품 시스템으로 그들의 잠재적인 사용에 대 한 펩 티 드 Microcarriers 모델

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

성인 골 수 유래 조 혈 줄기 세포로 세포 공학 자기 대상으로 함께 있도록 예측에 관한 전송을 위한 프로토콜

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Isolation and Identification of Porcine Bone Marrow Mesenchymal Stem Cells and their Derived Extracellular Vesicles

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of pluripotent stem cells, are favored for their instant availability and proven safety. It has been reported that transplantation of BM-MSCs is of great benefit to repairing injured tissues in various diseases, which might be related to modulating the immune and inflammatory responses via paracrine mechanisms. Extracellular vesicles (EVs), featuring a double-layer lipid membrane structure, are considered to be the main mediators of the paracrine effects of stem cells. Recognized for their crucial roles in cell communication and epigenetic regulation, EVs have already been applied in vivo for immunotherapy. However, similar to its maternal cells, most of the studies on the efficacy of transplantation of EVs still remain at the level of small animals, which is not enough to provide essential evidence for clinical translation. Here, we use density-gradient centrifugation to isolate bone marrow cells (BMC) from porcine bone marrow at first, and get porcine BM-MSCs (pBM-MSCs) by cell culture subsequently, identified by the results of observation under the microscope, induced differentiation assay, and flow cytometry. Furthermore, we isolate EVs derived from pBM-MSCs in cell supernatant by ultracentrifugation, proved by the techniques of transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting successfully. Overall, pBM-MSCs and their derived EVs can be isolated and identified effectively by the following protocols, which might be widely used in pre-clinical studies on the transplantation efficacy of BM-MSCs and their derived EVs.

<meta charset="utf8"></head><body>Porcine Bone Marrow Mesenchymal Stem Cells and their derived Extracellular Vesicles의 분리 및 식별(Isolation and Identification of Porcine Bone Marrow Mesenchymal Stem C

This article elaborates a method to isolate and identify porcine bone marrow mesenchymal stem cells (pBM-MSCs) and extracellular vesicles (EVs) derived from them, providing a methodological basis for the pre-clinical evaluation of transplantation efficacy of BM-MSCs and their derived EVs.

Porcine Bone Marrow Mesenchymal Stem Cells and their derived Extracellular Vesicles의 분리 및 식별(Isolation and Identification of Porcine Bone Marrow Mesenchymal Stem Cells and their derived Extracellular Vesicles)

With the development of stem cell therapy in translational research and regenerative medicine, bone marrow mesenchymal stem cells (BM-MSCs), as a kind of ...

잠재적 인 세포질 대체 요법을위한 골수 간엽 줄기 세포로부터의 혈장 막 소포의 제조

요약

더 많은 비디오 탐색

이 비디오의 챕터

인간 중간 엽 줄기 세포의 분리 및 다공성 뼈 매트릭스에 자신의 재배

효소 방법은 응고 골수 샘플에서 중간 엽 줄기 세포를 구출

에서 간세포 이식 체외에서 시뮬레이션 국소 빈혈 / Reperfusion 모델

사람 골수에서 분리 Mesangiogenic 전구 세포 (MPC와)

섬유소 혈전의 동종 간엽 줄기 세포의 주입에 의한 토끼의 무릎 관절의 골 연골 결함의 치료

중간 엽 기질자가 조건에서 세포 배양 배송 : 정형 외과 응용 프로그램에 대한 스마트 접근

재조합 콜라겐 나 세포 확장과 생물에 셀 납품 시스템으로 그들의 잠재적인 사용에 대 한 펩 티 드 Microcarriers 모델

성인 골 수 유래 조 혈 줄기 세포로 세포 공학 자기 대상으로 함께 있도록 예측에 관한 전송을 위한 프로토콜

Porcine Bone Marrow Mesenchymal Stem Cells and their derived Extracellular Vesicles의 분리 및 식별(Isolation and Identification of Porcine Bone Marrow Mesenchymal Stem Cells and their derived Extracellular Vesicles)

Porcine Bone Marrow Mesenchymal Stem Cells and their derived Extracellular Vesicles의 분리 및 식별(Isolation and Identification of Porcine Bone Marrow Mesenchymal Stem Cells and their derived Extracellular Vesicles)