저자는 생물 반응기와의 도움을 QuinXell 기술 및 CellD, 특히 로타르 그래너만 도미니크 Ghozlan을 인정하고 싶습니다. 우리는 풀루 란 / 덱스 트란 다당류 발판으로 우리를 제공 캐서린 르 화장을, 감사합니다. 이 작품은 유럽 연합 (ERC-2014-COG 프로젝트 마티스 648779)에 의해 그리고 AgenceNationalede 라 공들인 (ANR), 프랑스 (MagStem 프로젝트 ANR-11 SVSE5)에 의해 지원되었다.

마그네틱 장치 1. 건설 주 : 세포 접종에 사용하는 장치는 애플리케이션에 따라서 변경 (도 1). 자기 팁 (지름 750 μm의) 매우 얇아 야하므로 응집체를 형성하는 셀의 수는 2.5 × 105 / 집합으로 제한된다. 1.8 / ㎠ 7mm 두께 발판 종자, 자석은 크게 (직경 3mm)이어야하고, 지지체의 세공을 세포 이동을 보장한다. <ol><li> 골재 형성을위한 미세 자석 장치의 구성 (도 1a) <ol><li> 알루미늄 판을 통해 0.8 mm의 드릴로 미세 구멍 (직경 3cm, 6 mm 두께)을 만든다. </li><li> 플레이트의 각 구멍에 자기 팁 (지름 750 μm의)을 삽입한다. </li><li> 포화 자화를 보장하는 영구 네오디뮴 자석, 이상이 디스크를 넣습니다. </li></ol></li><li> 비계 파종위한 장치의 건설 ( <str옹&gt;도 1b) <ol><li> 2.4 cm 2 제곱에 하드 컷 폴리스티렌. </li><li> 1.6 cm (2)의 면적에 걸쳐 동일한 간격으로 9 개 작은 자석 (직경 3mm, 길이 6mm)을 삽입한다. </li><li> 영구 네오디뮴 자석을 통해이 장치를 놓습니다. </li></ol></li></ol> 2. 줄기 세포의 라벨링 주 : 30 분 (5 ± 0.4 PG 철 / 셀) 시드 0.2 mM의 자성 나노 입자로 표지 동안 줄기 세포를 30 분 (2.6 ± 0.2 PG 철 / 셀) 집합체를 형성하기 위해 0.1 mM의 자성 나노 입자로 표지 된 비계. 이들 나노 입자의 농도 및 배양 시간은 이전에 사용하고 MSC 및 다른 셀 (18, 19)으로 제작하고,이 나노 입자는 세포 생존도 MSC이나 분화 능력에 영향을 판단되어왔다. 줄기 세포에 의해 혼입 된 철 질량 단일 CE 통해 측정LL의 자기 19, 20. <ol><li> 근처까지 37 ° C, 5 % CO 2에서 완전한 중간 엽 줄기 세포 성장 매체 (MSCGM) 문화 인간 중간 엽 줄기 세포 (MSC)는 (~ 90 %)을 합류한다. </li><li> 글루타민없이 로스웰 파크 메모리얼 연구소 (RMPI)에서 변성 무 혈청 맥코이의 5A 배지 및 5를 포함하는 : 0.1 또는 0.2 밀리미터 마그 헤 마이트, 시트르산 피복 산화철 (8 nm의 직경 코어 γFe 2 O 3)을 혼합하여, 자기 라벨 솔루션을 준비 mM의 시트르산 나트륨. </li><li> 상기 배지를 폐기 글루타민없는 무 혈청 RPMI 배지로 세포를 린스하고, 150 cm 2 배양 플라스크, 전체 셀을 커버하기 위해 필요한 최소 체적 당의 철 산화물 나노 입자 용액 10 mL를 넣고. </li><li> 37 ° C에서 30 분, 5 % CO 2 동안 배양 한 다음 나노 입자 용액을 버린다. 유모를 내면화하는 글루타민없이 무 혈청 RPMI 배지 5 분간 씻어oparticles 여전히 세포막에 부착. </li><li> RPMI 매체를 취소하고 플라스크 당 전체 MSCGM 매체의 25 mL를 넣어. 37 ℃에서 밤새 인큐베이션하고, 5 % CO 2. </li></ol> 3. 자기 셀 시드 <ol><li> 갓 50 μM L- 아스코르브 산 2 인산, 0.1 μM 덱사메타손 (dexamethasone), 1 mM 피루브산 나트륨, 0.35 밀리미터 L 프롤린, 1 % 범용 배양을 첨가하여 둘 베코 변형 이글 중간 (DMEM) L 글루타민 높은 포도당을 이용하여 연골 매체를 준비 인슐린, 인간 트랜스페린 및 selenous 산 (ITS-프리믹스) 10 NG / ㎖ 형질 전환 성장 인자 베타 3 (TGF-β3)을 포함하는 보충. </li><li> 5 분 동안 260 × g에서 0.05 % 150 cm-2 배양 플라스크 당 트립신 EDTA 및 원심 분리기의 해리 된 세포를 8 mL로하여, 자기 세포 분리. 배지를 흡인하고, 세포를 재현 탁 계산. </li><li> 양 자기 소자의 상부에 유리 바닥 세포 배양 페트리 접시 (35mm)를 놓는다. </li><li> m으로agnetically 집합체 당 (증착 될 수있다 (16) 집계까지) 2.5 × 105 개 표지화 된 세포를 포함하는 가능한 가장 작은 양 (더 8보다 μL)을 증착 부드럽게 페트리 접시에 연골 매체 3 mL를 첨가하고, 응집물을 형성한다. 이 구 상체를 형성 할 수 있도록하여 이동하지 않고 20-30 분 동안 배양 접시 두었다가 37 ° C, 5 % CO 2의 인큐베이터에, 16 응집체를 함유하는 페트리 접시를 포함하여 전체 장치를 배치했다. </li><li> 양식 제어 집계는 동일한 프로토콜을 다음과 TGF-β3없이 연골 매체 전체 매체를 교체합니다. <ol><li> , 3 차원 집계 구조를 생성 8 이중선을 형성하고 융합을 시작 8 일째에 접촉이 집계를 놓습니다. 하루 11, 4 네 쌍둥이를 형성하기 위해 2 개의 이중 병합합니다. 마지막으로, 최종 구조물을 얻기 위해 15 일에 4 네 쌍둥이 퓨즈. </li><li> 동시에 × 105 2.5 원심 분리하여 응집체를 형성 (260)에서 줄기 세포를 표지(15); 또는 TGF-β3없이 연골 배지 1.5 mL를 15-㎖의 튜브에 5 분간 g (샘플 및 제어를위한 각각). </li></ol></li><li> 종자 발판 자기 적하려면 페트리 접시에 각각 건조 발판을 놓습니다. 풀루 란 / 덱스 트란 (21)로 이루어진 다당류 다공성 지지체를 사용합니다. 각 지지체를 들어, TGF-β3없이 연골 배지 350 μL, 2 × 106 표지 줄기 세포를 희석 신중 지지체 상에 세포를 피펫. <ol><li> 골격 내의 전체 세포 침투를 허용하기 위해 37 ℃에서 5 분 동안 인큐베이션 한 다음 부드럽게 또는 페트리 접시 (각각 시료 또는 제어를위한) TGF-β3없이 연골 배지 3 ㎖을 추가한다. </li><li> 골격 기공 한정 통해 세포 이동을 허용하도록 2 내지 4 일 동안 37 ° C CO에서의 자기 장치의 발판 cellularized 부화 5 %. </li></ol></li><li> 동시에, 함께 발판을 시드 2 × 10 (6) </s&gt;까지 표지 줄기 세포는 동일한 방법을 다음과 같이 양성 대조군 균일 시드 발판을 얻었다 마그넷없이 배양한다. </li></ol> 연골 세포로의 분화 (4) 참고 : 배양 4 일 후, 자석을 제거하고 연골 성숙 중 하나를 페트리 접시 (정적 상태) 또는 생물 반응기 (동적 조건)에서에 계속. 대조군 샘플은 TGF-β3없이 연골 매체 정적 조건에서 숙성된다. <ol><li> 정적 조건에서, 같은 배양 접시에서 cellularized 비계 또는 집계를 유지한다. 이십일일을 위해 일주일에 두 번 연골 매체를 변경합니다. </li><li> 동적 상태에서 생물 반응기를 준비한다. <ol><li> 제조 업체의 프로토콜에 따라 적절한 길이로 실리콘 튜브를 잘라. </li><li> 500 ㎖의 배양 챔버, 튜브, 2 웨이 회전기 및 케이지 모든 재료를 압력솥. </li><li> 멸균 microbio로 생물 반응기의 부품 배치논리적 안전 역. 제조업체의 지침에 따라 2 방향 회전기와 문화 챔버에 튜브를 연결합니다. </li><li> 조심 멸균 주걱을 이용하여 살균 케이지로 cellularized 발판 옮긴다. 케이지 당 2 발판을 놓습니다. 새장 준비가되면, 더 회전시 이동을 유지하기 위해 뚜껑의 바늘에 삽입. 연골 배지로 배양 챔버를 채우고 케이지를 함유하는 뚜껑을 닫는다. </li></ol></li><li> 연골 매체와 튜브를 채우기 위해 기포를 제거하기 위해 연동 펌프를 켭니다. </li><li> 두고 바이오 리액터의 모터로 채워진 챔버를 고정하고 암과 챔버 모두 회전을 제어하는 ​​컴퓨터를 켜. </li><li> 암부와 챔버 모두에 5 분당 회전 수 (rpm)의 회전을 적용한다. cellularized 비계의 연속 급지 10 rpm의 유속으로 연동 펌프를 조정한다. </li></ol> 참고 : RNA 추출에 앞서, 효소 솔루션으로 발판을 소화. <ol><li> 풀루 100 μL (40 U / ㎖) 및 무 혈청 DMEM 배지에 850 mL의 dextranase 50 μL (60 ㎎ / ㎖)을 첨가하여 효소 용액 1 ㎖를 준비한다. </li><li> , 무 혈청 DMEM 배지로 두 번 발판을 씻어 매체를 폐기하고, 비계 당 효소 용액 800 μL를 추가합니다. 부드러운 교반하에 37 ℃에서 15-30 분 동안 인큐베이션. </li><li> 발판이 완전히 용해 될 때 신중 매체 대기음, 10 분 동안 300 × g에서 ㄱ 1.5mL 용량 튜브에 세포를 포함하는 용액을 원심 분리 전송 × 1 멸균 인산 완충 식염수 (PBS), 원심 분리기의 회 씻어 300 × 10 분간 g 및 RNA 분리 용액으로 세포를 재 - 보류. </li><li> , 집계에서 RNA를 추출 RNA 분리 용액에 회전 타원체를 배치하고완전히 RNA 추출을 수행하기 전에 호모 게 나이저를 사용하여 분쇄. </li><li> 제조업체의 지침에 따라 총 RNA 추출을위한 키트를 사용하여 RNA를 분리합니다. </li><li> ,의 dNTP 믹스 1 μL (10 mM의 각), 40 U / ㎖의 RNase 억제제 랜덤 프라이머 250-ng를 사용하여 제조사의 지침에 따라 역전사 효소를 이용하여 총 RNA의 400 ng의 보완적인 DNA 합성; 반응의 최종 부피는 20 μL이다. 반응의 끝에서 100 μL의 최종 용적을 수득 증류수 80 μL를 추가한다. </li><li> 정량적 폴리머 라제 연쇄 반응 (PCR)의 경우, 10 배 희석 된 cDNA와 같은 aggrecan (AGC) 및 콜라겐 II (골 II) 등의 관심의 유전자의 상대적인 발현을 정량화하는 형광 시약을 포함하는 PCR 혼합물을 사용한다. 기준 유전자 리보솜 단백질 대형 P0 (RPLP0)으로 유전자 발현의 수준을 정상화. ΔΔCT 식 - 2로 계산을 수행, ΔΔCT = ΔCT 차별화 조건 곳 - 제어 ΔCT 조건을 의미하고, 각 ΔCT 관심 유전자의 CT 나타냄 - 참조 유전자 (RPLP0)의 CT. </li><li> 평균 값은 평균 (SEM)의 표준 오차를 ± 통계적 측정치를 결정한다. n 개의 ≥ 2 독립적 인 실험과 분석을 수행합니다. 원심 펠렛 자기 융합 (* p &lt;0.05)의 통계적 차이를 분석하기 위해 스튜던트 t 검정을 사용한다. 제어 지지체 차별화 발판 사이와 통계적 차이 (* p &lt;0.05)을 분석하는 크루스 칼 - 월리스 시험 (일방향 ANOVA의 비모수)와 중요도를 결정한다. </li></ol> 6. 조직 학적 분석 <ol><li> 멸균 1 × PBS로 cellularized 지지체 또는 응집체 린스 1 시간 동안 실온에서 10 % 포르말린 용액에 이들을 고정, 1 × PBS로 헹군다. </li><li> 의 PBS를 제거 최적의 해부 할에 샘플을 포함g 온도 화합물 OCT () 및 액체 질소에 침지 펜탄 욕이를 동결. -20 ° C에서 샘플을 저장합니다. 응집체 또는 cellularized 골격 12 μm의 8 μm의 저온부를 획득하기 위하여 저온 유지 장치로 샘플을 잘라. </li><li> 톨루엔을 사용하여 명확히, 100 % 에탄올로 탈수시키고, 수돗물로 헹구고, 2 분 동안 0.5 % 톨루이딘 블루 용액으로 저온부를 염색하고 광학 현미경위한 장착 매체 슬라이드 마운트. </li></ol>

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저자가 공개하는 게 없다.

여기에 제시된 기술은 자성 나노 입자의 국제화에 의존하기 때문에 그들이 세포 내 지역화하면 첫째, 하나의 중요한 문제는 나노 입자의 결과이다. 이는 철 나노 입자 잠재 독성 또는 노광 (19), (22)의 크기, 코팅, 및 시간에 따라 손상 분화 능력을 트리거 할 수 있다는 사실이다. 캡슐화 철 나노 입자의 형태 magnetoferritin 생물학적 자성 나노 입자 <sup class="...

자기 나노 입자는 이미 자기 공명 영상 (MRI)을위한 조영제로 병원에서 사용하고 있으며, 자신의 치료 응용 프로그램을 확대 유지. 예를 들어, 최근 표지 세포 주입 1, 2, 3의 정의 된 부위에서 외부 자기장을 이용하여 생체 내에서 조작 될 수 있고, 지시 된 수 및 / 또는 유지하는 것이 밝혀졌다. 재생 의학, 그들은 혈관 조직 5, 6, 7, 골 8, 연골 (9)를 포함하여 체외 4 조직 조직, 엔지니어링 할 수 있습니다. 관절 연골이 손상이 발생할 때 매우 제한된 세포 외 기질 성분의 수리를, 무혈성 환경 내에 침지된다. 이러한 이유로, researc 위해H는 현재 결함 부위에 이식 할 수 유리질 연골 대체의 기술에 집중된다. 다른 연골 (12, 13)로 분화하는 중간 엽 줄기 세포 (MSC)의 용량을 강조하면서자가 여분을 생성하기 위해, 몇몇 연구 그룹은 셀 소스 (10), (11)자가 연골 세포의 사용을 연구하고있다. 자신의 골수 채취가 매우 간단하고 자신의 표현형 (14)를 잃을 위험이 건강한 연골 세포의 희생을 필요로하지 않기 때문에 여기에 효과적으로 요약 이전의 연구에서, 우리는 MSC를 선택했습니다. 줄기 세포의 연골 분화를 시작하기에 필수적인 초기 단계는 자신의 응축이다. 셀 집계는 일반적으로 원심 분리 또는 micromass 문화 (15)을 사용하여 형성된다; 그러나, 이러한 방법은 축합 neitheR 두꺼운 발판이나 골재의 융합을 조절하는 세포 내 전위 클러스터를 생성 할 가능성을 제시한다. 본 논문에서는 MSC 자기 라벨 및 자기 인력을 이용하여 줄기 세포를 응축에 대한 혁신적인 접근 방식을 설명합니다. 이 기술은 밀리미터 크기의 연골 조직 (9)을 얻기 위해 서로 골재의 융합을 통해 비계가없는 3 차원 구조를 형성하기 위해 입증되었습니다. 두껍고 큰 비계의 자기 시드 또한 설계 조직의 크기를 증가 이식에 대한 더 쉽게 도움을 모양을 디자인하고, 연골 수리 임상 적용의 가능성을 다양 화의 가능성을 허용했다. 여기서, 천연 다당류, 풀루 란, 덱스 트란으로 이루어지는 다공질 지지체에 MSC 자기 시딩 우리 상세히 프로토콜은 비계 이전 줄기 세포 (16) (17)를 한정하기 위해 사용된다. 연골 분화하면 마지막이었다Y는 세포 고밀도 시드 골격의 매트릭스 코어에 연속 영양소 및 기체 확산을 위해 생물 반응기에서 수행 하였다. 세포에 영양분, 연골 세포 성장 인자, 가스를 제공하는 외에, 생물 반응기는 기계적인 자극을 제공했다. 전반적으로, 생물 반응기의 동적 숙성과 함께 줄기 세포를 한정하는 데 사용되는 자기 기술은 현저 연골 분화를 향상시킬 수있다.

<table><tbody><tr><td>Iron oxide (maghemite) nanoparticules (&amp;gamma;-Fe&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt;)</td><td>PHENIX - University Paris 6</td><td>Made and given by C. M&amp;eacute;nager</td><td>Mean diameter of 8.1 nm and negative surface charge</td></tr><tr><td>Polysaccharide Pullulan/Dextran scaffolds</td><td>LIOAD - University Nantes</td><td>Made and given by C. Le Visage</td><td>Prepared from a 75:25 mixture of pullulan/dextran in alkaline conditions (10&amp;nbsp;M NaOH). Porosity: 185-205&amp;nbsp;&amp;micro;m; Thickness: 7&amp;nbsp;mm; Surface area: 1.8&amp;nbsp;cm&lt;sup&gt;2&lt;/sup&gt;.</td></tr><tr><td>TisXell Regeneration System</td><td>QuinXell Technologies</td><td>QX900-002</td><td>Biaxial bioreactor with 500 mL culture chamber&amp;nbsp;</td></tr><tr><td>Cage for scaffolds: Histosette II M492&amp;nbsp;</td><td>VWR</td><td>720-0909</td><td></td></tr><tr><td>Mesenchymal Stem Cell (MSC)</td><td>Lonza</td><td>PT-2501</td><td>Three independant batches have been used</td></tr><tr><td>MSCGM BulletKit medium</td><td>Lonza</td><td>PT-3001</td><td>For the complete medium, add the provided BulletKit (containing serum, glutamine and antibiotics) to the MSCGM medium</td></tr><tr><td>DMEM with Glutamax I</td><td>Life Technologies</td><td>31966-021</td><td>No sodium pyruvate, no HEPES</td></tr><tr><td>RPMI medium 1640, no Glutamine</td><td>Life Technologies</td><td>31870-025</td><td>No sodium pyruvate, no HEPES</td></tr><tr><td>PBS w/o CaCl&lt;sub&gt;2&lt;/sub&gt; w/o MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Life Technologies</td><td>14190-094</td><td></td></tr><tr><td>0.05% Trypsin-EDTA (1x)</td><td>Life Technologies</td><td>25300-054</td><td></td></tr><tr><td>Penicillin (10,000&amp;nbsp;U/mL) / Streptomycin (10,000&amp;nbsp;&amp;micro;g/mL)</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>ITS Premix Universal Culture Supplement (20x)</td><td>Corning</td><td>354352</td><td></td></tr><tr><td>Sodium pyruvate solution 100 mM</td><td>Sigma</td><td>S8636</td><td></td></tr><tr><td>L-Ascorbic Acid 2-phosphate</td><td>Sigma</td><td>A8960</td><td>Prepare the concentrated solution (25 mM) in distilled water extemporaneously</td></tr><tr><td>L-Proline</td><td>Sigma</td><td>P5607</td><td>Prepare the 175 mM stock solution diluted in distilled water and store at 4&amp;nbsp;&amp;deg;C</td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D4902</td><td>Prepare the 1 mM stock solution diluted in Ethanol 100% and store at -20&amp;nbsp;&amp;deg;C</td></tr><tr><td>TGF-beta 3 protein 10&amp;nbsp;&amp;micro;g</td><td>Interchim</td><td>30R-AT028</td><td></td></tr><tr><td>Tri-sodium citrate</td><td>VWR</td><td>33615.268</td><td>Prepare the 1&amp;nbsp;M stock solution diluted in distilled water and store at 4&amp;nbsp;&amp;deg;C</td></tr><tr><td>Pullulanase from &lt;em&gt;Bacillus acidopullulyticus&lt;/em&gt;</td><td>Sigma</td><td>P2986</td><td></td></tr><tr><td>Dextranase from &lt;em&gt;Chaetomium erraticum&lt;/em&gt;</td><td>Sigma</td><td>D0443</td><td></td></tr><tr><td>NucleoSpin RNA Extraction Kit</td><td>Macherey-Nagel</td><td>740955.5</td><td></td></tr><tr><td>SuperScript II Reverse Transcriptase</td><td>Life Technologies</td><td>18064-014</td><td></td></tr><tr><td>Random Primer - Hexamer&amp;nbsp;</td><td>Promega</td><td>C1181</td><td>500 &amp;micro;g/mL: Use diluted 1/2 and put 1 &amp;micro;L per sample</td></tr><tr><td>Recombinant RNAs in ribonuclease inhibitor</td><td>Promega</td><td>N2511</td><td>40 U/&amp;micro;L: put 1 &amp;micro;L per sample</td></tr><tr><td>PCR nucleotide dNTP mix (10&amp;nbsp;mM each)</td><td>Roche</td><td>10842321</td><td></td></tr><tr><td>SyBr Green PCR Master Mix</td><td>Life Technologies</td><td>4368708</td><td></td></tr><tr><td>Step One Plus Real-Time PCR System</td><td>Life Technologies</td><td>4381792</td><td></td></tr><tr><td>Formalin solution 10% neutral buffered</td><td>Sigma</td><td>HT5012</td><td></td></tr><tr><td>OCT solution</td><td>VWR</td><td>361603E</td><td></td></tr><tr><td>Isopentane</td><td>Sigma</td><td>M32631</td><td></td></tr><tr><td>Toluidine blue O</td><td>VWR</td><td>1.15930.0025</td><td></td></tr><tr><td>Ethanol absolute</td><td>VWR</td><td>20821.310</td><td></td></tr><tr><td>Toluene</td><td>VWR</td><td>1.08323.1000</td><td></td></tr><tr><td>Mounting medium Pertex</td><td>Histolab</td><td>840</td><td></td></tr><tr><td>RPLP0 Primer for qPCR</td><td>Eurogentec</td><td></td><td>5&#039;-TGCATCAGTAC&lt;br/&gt; CCCATTCTATCAT-3&#039;;&lt;br/&gt; 5&#039;-AAGGTGTAATC&lt;br/&gt; CGTCTCCACAGA-3&#039;</td></tr><tr><td>Aggrecan Primer for qPCR</td><td>Eurogentec</td><td></td><td>5&#039;-TCTACCGCTGCGAGGTGAT-3&#039;; 3&#039;-TGTAATGGAACACGATGCCTTT-5&#039;</td></tr><tr><td>Collagen II Primer for qPCR</td><td>Eurogentec</td><td></td><td>5&#039;-ACTGGATTGACCCCAACCAA-3&#039;; 3&#039;-TCCATGTTGCAGAAAACCTTCA-5&#039;</td></tr></tbody></table>

3d magnetic stem cell aggregation and bioreactor maturation for cartilage regeneration

연골 엔지니어링은 기본 조직과 유사한 체외 기능 임플란트를 만드는 어려움으로 인해 도전 남아있다. 최근자가 교체의 개발을위한 탐구 접근 방법은 연골 세포로 줄기 세포의 분화를 포함한다. 이 연골, 필요한 줄기 세포의 압축 정도를 시작하려면; 따라서, 우리는 세포 어 트랙터로 소형화 자장 소스를 사용, 두꺼운 발판과 발판이없는 내 모두 자기 응축 세포의 가능성을 보여 주었다. 이 자기 방식도 집계 융합을 안내하고 비계가없는 조직 구축하는 데 사용 된, 3 차원 (3D)은 크기가 몇 mm를 어떤 조직이. 강화 된 크기를 갖는 외에, 자기 구동에 의해 형성된 융합 조직 콜라겐 II의 발현의 상당한 증가를 제공하고, 유사한 경향 aggrecan 발현이 관찰되었다. 네이티브 연골은 힘 t 하였다으로모자의 3 차원 구조에 영향을 동적 숙성도 수행 하였다. 기계적인 자극을 제공하는 생물 반응기에서는 21 일 동안 배양하여 자기 시드 지지체를 이용 하였다. 생물 반응기의 성숙은 크게 cellularized 지지체에 연골을 개선; 이러한 조건에서 얻어진 세포 외 기질은 콜라겐 II 및 aggrecan 풍부 하였다. 이 작업은 개선 된 연골 세포 분화, 양쪽 발판없고 내의 다당류 골격 용 생물 반응기에 표시된 줄기 세포의 자기 축합 동적 숙성 혁신적인 전위를 설명.

우선, 골재를 개별적으로 2.5 × 105 개 표지 줄기 세포를 (도 2A)을 증착에 의해 미소 자석을 사용하여 형성 될 수있다. 이 하나의 집합체 (~ 크기가 0.8 mm)는 다음 순차적으로, 자기 유도 융합에 더 큰 구조 덕분에 융합 될 수있다. 예를 들어, 연골 성숙의 8 일째에, 집계는 이중선을 형성하는 한 쌍의 접촉에 넣고; 네 쌍둥이 2 이중선을 병합하여 11 일째에 ?...

Watch this Scientific Journal Video about 3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration at JoVE.com

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

줄기 세포에서 연골은 배양 조건 미세 조정이 필요합니다. 여기, 우리는 세포를 응축위한 자기 접근 방식을 제시 필수적인 단계는 연골을 시작합니다. 또, 생물 반응기의 동적 숙성은 셀룰러 구조에 기계적 자극을 적용 연골 세포 외 기질 생산 향상을 보여준다.

연골 재생을위한 3D 자기 줄기 세포 집계 및 생물 반응기 성숙

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

3d-magnetic-stem-cell-aggregation-bioreactor-maturation-for-cartilage

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

9.7K 조회수.  UMR 7057 CNRS and University Paris Diderot.  줄기 세포에서 연골은 배양 조건 미세 조정이 필요합니다. 여기, 우리는 세포를 응축위한 자기 접근 방식을 제시 필수적인 단계는 연골을 시작합니다. 또, 생물 반응기의 동적 숙성은 셀룰러 구조에 기계적 자극을 적용 연골 세포 외 기질 생산 향상을 보여준다. 

비디오: 3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

조직 공학을위한 이축 기계 로딩 생물 반응기의 설계

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

연구

생체공학

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

제대혈 유래 다 능성 줄기 세포로부터 연골 형성 펠렛 형성

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

발생학

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

혈관 조직 재생을위한 엔지니어링 3D Cellularized 콜라겐 젤

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

관절 연골 세포 문화와 연골 생성을위한 3D 하이드로 겔 공사장 공중 발판

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo1, 2.
In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses3.
Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques4.
This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.

Goal of the presentation is to demonstrate a highly reproducible method to generate matrix associated stem cell implants in cartilage defects, which can be visualized with MR imaging. Stem cells are labeled with FDA-approved Ferumoxides, mixed with agarose, implanted into cartilage defects and imaged with a 7T MR scanner.

Ferumoxides의 이식은 연골의 결함에 인간 Mesenchymal 줄기 세포로 표시

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

생물학

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

연골과 피부 조직 아날로그 소설 수동 Bioink Precellularization에 대 한 단위 기술 혼합을 이용 하 여 Bioprinting

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

3 차원 펩 티 드 건설 기계에 포유류 세포 배양

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Construction, Characterization, and Regenerative Application of Self-Assembled Human Mesenchymal Stem Cell Aggregates

Mesenchymal stem cells (MSCs), characterized by their self-renewal ability and multilineage differentiation potential, can be derived from various sources and are emerging as promising candidates for regenerative medicine, especially for regeneration of the tooth, bone, cartilage, and skin. The self-assembled approach of MSC aggregation, which notably constructs cell clusters mimicking the developing mesenchymal condensation, allows high-density stem cell delivery along with preserved cell-cell interactions and extracellular matrix (ECM) as the microenvironment niche. This method has been shown to enable efficient cell engraftment and survival, thus promoting the optimized application of exogenous MSCs in tissue engineering and safeguarding clinical organ regeneration. This paper provides a detailed protocol for the construction and characterization of self-assembled aggregates based on umbilical cord mesenchymal stem cells (UCMSCs), as well as an example of the cranial bone regenerative application. The implementation of this procedure will help guide the establishment of an efficient MSC transplantation strategy for tissue engineering and regenerative medicine.

This study describes a method to construct aggregates based on the self-assembly of human mesenchymal stem cells and identifies the morphological and histological characteristics for the regenerative treatment of cranial bone defects.

Mesenchymal stem cells (MSCs), characterized by their self-renewal ability and multilineage differentiation potential, can be derived from various sources ...

Applying a Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System to Induce Tenogenic Differentiation of Tendon-Derived Stem Cells

Tendinopathy is a common chronic tendon disease relating to inflammation and degeneration in an orthopaedic area. With high morbidity, limited self-repairing capacity and, most importantly, no definitive treatments, tendinopathy still influences patients&#8217; life quality negatively. Tendon-derived stem cells (TDSCs), as primary precursor cells of tendon cells, play an essential role in both the development of tendinopathy, and functional and structural restoration after tendinopathy. Thus, a method that can in vitro mimic the in vivo differentiation of TDSCs into tendon cells would be useful. Here, the present protocol describes a method based on a three-dimensional (3D) uniaxial stretching system to stimulate the TDSCs to differentiate into tendon-like tissues. There are seven stages of the present protocol: isolation of mice TDSCs, culture and expansion of mice TDSCs, preparation of stimulation culture medium for cell sheet formation, cell sheet formation by culturing in stimulation medium, preparation of 3D tendon stem cell construct, assembly of the uniaxial-stretching mechanical stimulation complex, and evaluation of the mechanical stimulated in vitro tendon-like tissue. The effectiveness was demonstrated by histology. The entire procedure takes less than 3 weeks. To promote extracellular matrix deposition, 4.4 mg/mL ascorbic acid was used in the stimulation culture medium. A separated chamber with a linear motor provides accurate mechanical loading and is portable and easily adjusted, which is applied for the bioreactor. The loading regime in the present protocol was 6% strain, 0.25 Hz, 8 h, followed by 16 h rest for 6 days. This protocol could mimic cell differentiation in the tendon, which is helpful for the investigation of the pathological process of tendinopathy. Moreover, the tendon-like tissue is potentially used to promote tendon healing in tendon injury as an engineered autologous graft. To sum up, the present protocol is simple, economic, reproducible and valid.

A three-dimensional uniaxial mechanical stimulation bioreactor system is an ideal bioreactor for tenogenic-specific differentiation of tendon-derived stem cells and neo-tendon formation.

힘줄 유래 줄기 세포의 세포 분화를 유도하기 위해 3차원 동축 기계 자극 생물 반응기 시스템을 적용

Tendinopathy is a common chronic tendon disease relating to inflammation and degeneration in an orthopaedic area. With high morbidity, limited ...

의학

Generating Spheroids from Various Chondrocytes using Low-Adhesive Conditions under Gravity and Homemade Mini-Bioreactors

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a step-by-step method for differentiating induced pluripotent stem cells (iPSCs) into chondrocyte-based spheroids, supporting tissue engineering and cell therapy applications. The differentiation process is carefully structured to promote chondrogenic lineage commitment, beginning with iPSCs cultured in specific media that sequentially guide cells through critical stages of differentiation. Initially, iPSCs are expanded to reach optimal confluency before induction toward chondrogenic lineage using a series of defined media changes. By day 10, cells are transitioned to a chondrogenesis-promoting medium that enhances the formation of chondrocyte-like cells expressing key markers of mature chondrocytes. Further aggregation in 96-well agarose-coated plates leads to the formation of three-dimensional spheroids, which are then cultured in custom mini-bioreactors designed to simulate a microenvironment that encourages extracellular matrix (ECM) deposition. By enabling scalable production of chondrocyte spheroids that mimic native cartilage characteristics, this approach offers a promising, reproducible solution for developing cell-based treatments for cartilage defects, providing broad utility for clinical and research applications in musculoskeletal regenerative medicine.

This study describes a method for producing chondrocytic spheroids by aggregating cells into spheroids under low-adhesion conditions using gravity, followed by culturing the resulting spheroids in mini-bioreactors.

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a ...