This work was funded in part by grant P40RR17447 from the National Institute of Health and award RP150637 from the Cancer Prevention and Research Institute of Texas. We would like to thank Dr. Darwin J. Prockop for his support on the project.

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The authors declare they have no competing financial interests.

일부 연구 및 임상 응용에 사용하기위한 최적의 MSC는 그들의 높은 이득을 최대화하기 위해 작동하고, 우선적으로 이러한 FBS 배지와 같은 이종 성분의 잠재적 인 항원의 전달을 최소화하기 위하여 화학적 XF 조건 하에서 제조한다. 여기에 설명 된 프로토콜에서, 우리는 1) 방법을 도시 한 타원체를 형성하여 3D 배양에서 중간 엽 줄기 세포를 활성화 2) XF 조건에서 중간 엽 줄기 세포의 3D 활성화를 달?...

중간 엽 줄기 / 기질 세포 (중간 엽 줄기 세포는) 다양한 재생 의학 접근 방식에 대한 큰 잠재력을 보여 주었다. 중간 엽 줄기 세포는 초기 골수 간질 성분으로서 단리되었지만 사람의 지방 조직의 1, 2, 3을 포함하여 다수의 다른 성인 조직으로부터 수득 하였다. 흥미롭게도, 메인 분리 방법은 중간 엽 줄기 세포의 현저한 재산권 소 태아 혈청 (FBS)의 존재하에 조직 배양 플라스틱에 단단히 부착 품은. 전통적인 분리 기술은 2 차원 배양의 MSC 쉽고 신속한 팽창을 허용하는 동안, 그것은 오도 매우 인위적인 중요한 세포 특성이 (4)의 전위 손실로 이어지는 기본 3 차원 환경의 중요성을 무시 6. 따라서, 3D 문화에서 중간 엽 줄기 세포의 연구하는전통적인 2D 문화보다 더 생리있다 &quot;손실 / 감소&quot;MSC 특성에 대한 검색에 등장했다. 또한, 관심 임상 응용 프로그램에 대한 세포가 더 순종하게하여 MSC 문화 활성화를 위해 이종없는 (XF) 화학적으로 정의 된 조건을 식별하고 상승했다. 많은 연구는 생체 재료 및 구형 집합체 또는 회전 타원체로 모두 중간 엽 줄기 세포의 3 차원 배양을 보여주는 발표되었다. 중간 엽 줄기 세포의 타원체 배양 전임상 또는 임상 시험에서의 치료를위한 세포의 투여 후 생체 내에서 MSC 동작을 이해하는 방법으로 보였다 반면 생체 재료의 중간 엽 줄기 세포는 초기 셀 시드 지지체에 손상된 조직을 대체하는 조직 공학 방법을 위해 설계된 4, 5, 7. 흥미롭게도, 중간 엽 줄기 세포의 형태 타원체는 자발적으로 조직 배양 플라스틱에 부착이 허용되지 않는 경우8, 9, 10. 전통적으로, 세포 응집은 스피너 플라스크 방법 또는 액체 오버레이 기술, 종양 미세 환경을 모방하려고 노력 암 생물학에서 처음 사용 된 방법에 의해 촉진되었다. 최근 추가 방법은 배양 접시에서 세포 응집 세포 간 접착 수지 4, 5, 6을 방지하기 위해 특정 화학 물질로 미리 코팅 된 표면이 보여왔다. MSC의 타원체를 생성하는 가장 간단하고 경제적 인 방법 중 하나는 종종 배아 줄기 세포로부터 배아 체를 생성하기 위해 사용 된 기법에 매달려 방울 배양 그들이다. 드롭 배양 기술을 걸려, 조직 배양 플라스틱에 대한 세포 부착은 세포 aggreg을 용이하게하기 위해 중력을 조직 배양 접시 뚜껑의 밑면에 중간 방울에 세포를 현탁하고시킴으로써 방지드롭의 정점에 ATION. 회전 타원체 크기 용이 세포 농도 또는 액적 부피 변화를 제어하는 ​​것이 특히 용이 매달려 드롭 배양함으로써 조작 될 수있다. 중간 엽 줄기 세포의 3 차원 배양에 대한 초기 연구는 2D 대응 6, 8, 9에 비해 3 차원 셀의 특성 라디칼 차이를 보였다. 동시에, 보고서는 생체 내에서 중간 엽 줄기 세포의 유익한 효과는, 응답, 마이크로 환경 단서에 의해 활성화 될 항염증제 및 면역 11 요인을 생성하는 능력에 의존하고 있음을 보여 주었다. 흥미롭게도, 예컨대 프로스타글란딘 E2 (PGE2), 종양 괴사 인자 - 자극 된 유전자 6 (TSG6), 및 간세포 성장 인자 (HGF) 등이 요인의 많은위한 방법을 포장 전통적인 2D 엽 줄기 세포보다 MSC의 타원체에 의해 훨씬 더 많은 양으로 생산되었다 아이디어세포를 8, 12, 13를 활성화 차원 배양을 사용. 또한, 3D 배양에서 유전자 활성화는 마우스 (12)에 주입 한 후 세포 활성의 적어도 일부분 메커니즘 요점을 되풀이 나타났다. 생체 내에서 전통적인 MSC 효과는 종종 과도 지연 및 &quot;뺑소니&quot;로 설명 될 수있다 같은 실험들은 사용 전에 중간 엽 줄기 세포를 활성화시킴으로써, 세포의 장기간 효과는보다 두드러 수있다. 지난 몇 년 동안, MSC의 타원체를 사용하는 중요한 기능 연구는 염증 반응을 억제하며 타원체를 준비하는 중간 엽 줄기 세포 (2)의 매력적인 양식을 대 식세포, 수지상 세포, 호중구 및 T 세포와 같은 효과기 세포 영향에 의해 생체 내에서 면역성을 조절할 수 있음을 입증 3. 또한, int와 같은 항암 분자의 제조erleukin-24 (IL-24) 및 종양 괴사 인자 관련 세포 사멸 유도 리간드 (TRAIL)는, 중간 엽 줄기 세포를 단일 층에 중간 엽 줄기 세포의 상대 3D 문화에 표적 암 치료 8, 10, 14 악용 될 수있는 현상을 증가한다. 기존의 MSC 문화 조직 배양 플라스틱뿐만 아니라 FBS의 사용뿐만 아니라 필요에 따라 임상 극복 할 수 있었다위한 또 다른 장애물은 MSC의 회전 타원체보다 의무 확인합니다. 이 장애물을 해결하기 위해 최근에 특정 화학적 XF 조건 MSC의 구 상체의 형성을 보였다 얻어진 MSC의 구 상체는 FBS (14)의 조건에서 발생되는 회전 타원체와 같은 항염증제 및 항암 성 분자를 생성하기 위해 활성화되었는지 세웠다. 여기서, 이러한 결과는 XF를 이용하여 3 차원 배양에 미리 활성화 된 중간 엽 줄기 세포의 생성을 보여주는 몇 가지 상세한 프로토콜에 제시미디어. 또한, 프로토콜은 함께 마우스에 그대로 타원체를 전달하는 실제적인 방법으로 그들의 항 염증, 면역 조절 및 항암 효과에 관해서는 상기 중간 엽 줄기 세포의 활성화 수준을 평가하기위한 효과적인 방법을 설명이 제시된다.

<table><tbody><tr><td>MEM-&amp;alpha; (minimal essential medium alpha)</td><td>ThermoFisher/Gibco</td><td>12561049; 12561056; 12561072</td><td>minimal essential medium for preparation of MSC growth medium (CCM)</td></tr><tr><td>FBS (fetal bovine serum), premium select</td><td>Atlanta Biologicals</td><td>S11595; S11510; S11550; S11595-24</td><td>component of complete culture media for all types of cells</td></tr><tr><td>L-glutamine</td><td>ThermoFisher/Gibco</td><td>25030081; 25030149; 25030164</td><td>component of complete culture media for all types of cells</td></tr><tr><td>Penicillin/Streptomycin</td><td>ThermoFisher/Gibco</td><td>15070063</td><td>component of complete culture media for all types of cells</td></tr><tr><td>Sterilization Filter Units, 0.22 &amp;micro;m PES membrane</td><td>MilliporeSigma</td><td>SCGPU01RE; SCGPU02RE; SCGPU05RE; SCGPU10RE; SCGPU11RE</td><td>media sterilization</td></tr><tr><td>150 mm cell culture dish</td><td>Nunc</td><td>D8554&amp;nbsp;SIGMA</td><td>cell culture</td></tr><tr><td>Thermo Forma water-jacketed CO&lt;sub&gt;2&lt;/sub&gt; humidified incubator</td><td>Thermo Fisher</td><td>Model 3110</td><td>incubation of cultured cells</td></tr><tr><td>Early passage MCSs</td><td>Center for the preparation and Distribution of Adult Stem Cells at The Texas A&amp;amp;M Health Science Center College of Medicine Institute for Regenerative Medicine at Scott &amp;amp; White</td><td>NA</td><td>preparation of 2D and 3D cultures of MSCs</td></tr><tr><td>water bath</td><td>VWR</td><td>89501-468</td><td>warming media to 37 &amp;deg;C</td></tr><tr><td>Pipettes</td><td>Eppendorf</td><td>492000904</td><td>manual liquid handling</td></tr><tr><td>Pipete-Aid</td><td>Drummond Scientific Company</td><td>4-000-300</td><td>handling sereological pipetes</td></tr><tr><td>Costar sterile serological pipet (5, 10, 25 and 50 ml)</td><td>Corning</td><td>4487; 4101; 4251; 4490</td><td>liquid handling</td></tr><tr><td>PBS (phosphate buffered saline), pH 7.4</td><td>ThermoFisher/Gibco</td><td>10010023; 10010072; 10010031; 10010049</td><td>cell culture processing</td></tr><tr><td>0.25% trypsin/EDTA solution</td><td>ThermoFisher/Gibco</td><td>25200056; 25200072; 25200114</td><td>lifting adherent cells and dispersing cell aggregates</td></tr><tr><td>15 ml conical tube</td><td>Corning/BD Falcon</td><td>352097</td><td>cell centrifugation</td></tr><tr><td>50 ml conical tube</td><td>Corning/BD Falcon</td><td>352098</td><td>cell centrifugation</td></tr><tr><td>Eppendor refrigerated centrifuge</td><td>Eppendorf/Fisher Scientific</td><td>Model 5810R</td><td>cell centrifugation</td></tr><tr><td>hemocytometer</td><td>Fisher Scientific</td><td>26716</td><td>cell counting</td></tr><tr><td>trypan blue</td><td>Sigma-Aldrich</td><td>T8154 SIGMA</td><td>dead cell exclusion during cell counting in hemacytometer</td></tr><tr><td>Defined xenofree MSC medium-1 (XFM-1)</td><td>ThermoFisher/Gibco</td><td>A1067501</td><td>Xeno-free media specifically formulated for the growth and expansion of human mesenchymal stem cells</td></tr><tr><td>Defined xenofree MSC medium-2 (XFM-2)</td><td>Stem Cell Technologies</td><td>5420</td><td>Defined, xeno-free medium for human mesenchymal stem cells</td></tr><tr><td>HSA (Human serum albumin)</td><td>Gemini</td><td>800-120</td><td>Component of xeno-free MSC media</td></tr><tr><td>rHSA (recombinant Human serum albumin)</td><td>Sigma-Aldrich</td><td>A9731&amp;nbsp;SIGMA</td><td>Component of xeno-free MSC media</td></tr><tr><td>Total RNA isolation Mini Kit</td><td>Qiagen</td><td>74104</td><td>Total RNA extraction</td></tr><tr><td>Qiashredder</td><td>Qiagen</td><td>79654</td><td>Sample homogenization prior to total RNA extraction</td></tr><tr><td>RNAse-free DNase Set&amp;nbsp;</td><td>Qiagen</td><td>79254</td><td>On-column DNA elimination during total RNA extraction</td></tr><tr><td>&amp;beta;-mercaptoethanol&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M6250&amp;nbsp;ALDRICH</td><td>inhibition of RNAses in RLT buffer</td></tr><tr><td>Vortex</td><td>VWR</td><td>97043-562</td><td>mixing sample</td></tr><tr><td>Spectrophotometer</td><td>Biorad</td><td>NA</td><td>RNA concentration and quality</td></tr><tr><td>High capacity cDNA Reverse Transcription Kit</td><td>ThermoFisher/Applied Biosystems</td><td>4368814</td><td>transcription of total RNA into cDNA</td></tr><tr><td>Gene Expression Assays</td><td>ThermoFisher/Applied Biosystems</td><td>varies</td><td>primer/probe combination for real-time PCR</td></tr><tr><td>Fast Universal PCR Master Mix</td><td>ThermoFisher/Applied Biosystems</td><td>4352042; 4364103; 4366073; 4367846</td><td>master mix for real-time PCR reaction</td></tr><tr><td>Real-time PCR system (ABI Prism 7900 HT Sequence Detection System)</td><td>ABI Prizm</td><td>NA</td><td>real-time PCR</td></tr><tr><td>1.5 ml centrifuge tube</td><td>Eppendorf</td><td>22364111</td><td>cell centrifugation, sample collection and storage</td></tr><tr><td>(-80 &amp;deg;C) freezer</td><td>Thermo Fisher</td><td>Model Thermo Forma 8695</td><td>sample storage</td></tr><tr><td>PGE2 (Prostaglandin E2) ELISA Kit</td><td>R&amp;amp;D Systems</td><td>KGE004B</td><td>estimation of cytokine concentration in the sample</td></tr><tr><td>DMEM (Dulbecco&amp;rsquo;s modified Eagle medium)</td><td>ThermoFisher/Gibco</td><td>10566-016; 10566-024;10566-032</td><td>macrophage culture media</td></tr><tr><td>J774 mouse macrophages</td><td>ATCC</td><td>TIB-67</td><td>mouse macrophage cell line</td></tr><tr><td>12-well plate</td><td>Corning</td><td>3513</td><td>&lt;em&gt;in vitro&lt;/em&gt; macrophage stimulation</td></tr><tr><td>LPS (lipopolysaccharide)</td><td>Sigma-aldrich</td><td>L4130</td><td>&lt;em&gt;in vitro&lt;/em&gt; macrophage stimulation</td></tr><tr><td>Mouse TNF-a ELISA kit</td><td>R&amp;amp;D Systems</td><td>MTA00B</td><td>estimation of cytokine concentration in the sample</td></tr><tr><td>Mouse IL-10 (interleukin 10) ELISA kit</td><td>R&amp;amp;D Systems</td><td>M1000B</td><td>estimation of cytokine concentration in the sample</td></tr><tr><td>RPMI-1640 medium</td><td>ThermoFisher/Gibco</td><td>11875-085</td><td>splenocyte culture media</td></tr><tr><td>BALB/c mice</td><td>The Jackson Laboratory</td><td>651</td><td>&lt;em&gt;in vivo&lt;/em&gt; spheroid delivery; splenocyte preparation</td></tr><tr><td>Anti-Mouse CD3e Functional Grade Purified</td><td>eBioscience</td><td>145-2C11</td><td>&lt;em&gt;in vitro&lt;/em&gt; splenocyte stimulation</td></tr><tr><td>70 &amp;mu;m strainer&amp;nbsp;</td><td>Corning</td><td>352350</td><td>Splenocyte preparation</td></tr><tr><td>Red blood cell lysis solution (1x)</td><td>Affymetrix eBioscience</td><td>00-4333</td><td>removal of red blood cells during splenocyte isolation</td></tr><tr><td>Mouse IFN-&amp;#947; (interferon gamma) ELISA kit</td><td>R&amp;amp;D Systems</td><td>MIF 00</td><td>estimation of cytokine concentration in the sample</td></tr><tr><td>LNCaP prostate cancer cells&amp;nbsp;</td><td>ATCC</td><td>CRL-1740</td><td>study the effect of 3D MSCs on cancer cell lines &lt;em&gt;in vitro&lt;/em&gt;</td></tr><tr><td>DNA-based cell proliferation assay kit</td><td>ThermoFisher</td><td>C7026</td><td>cell number measurement based on DNA content</td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S5150</td><td>component of lysis reagent</td></tr><tr><td>EDTA (ethylenediaminetetraacetic acid)</td><td>ThermoFisher</td><td>FERR1021</td><td>calcium chelator, component of lysis reagent</td></tr><tr><td>Rnase A</td><td>Qiagen</td><td>19101</td><td>RNA degradation for measurement of DNA</td></tr><tr><td>Filter-based multi-mode microplate reader</td><td>BMG Technology</td><td>NA</td><td>Microplate assays (ELISA, cell quantification, &lt;em&gt;etc.&lt;/em&gt;)</td></tr><tr><td>HBSS (Hanks balanced salt solution), no calcium, no magnesium, no phenol red</td><td>ThermoFisher/Gibco</td><td>14175079</td><td>resupsension of MSC spheroids prior to &lt;em&gt;in vivo&lt;/em&gt; injections</td></tr><tr><td>Isoflurane</td><td>MWI Vet Supply</td><td>502017</td><td>Anesthesia for &lt;em&gt;in vivo&lt;/em&gt; injections</td></tr><tr><td>Oxygen, compressed gas</td><td>Praxair</td><td>NA</td><td>For use with isoflurane</td></tr><tr><td>Thermo Forma BSL-2 cabinet</td><td>Thermo Fisher</td><td>Model 1385</td><td>Sterile cell culture</td></tr><tr><td>Safety I.V. catheter/needle stiletto, 20 G , 1 inch</td><td>Terumo</td><td>SR*FNP2025</td><td>Delivery of shperoids into peritoneal cavity</td></tr><tr><td>Sterile micropipette tips</td><td>Eppendorf</td><td>varies</td><td>liquid/cells handling</td></tr></tbody></table>

production and administration of therapeutic mesenchymal stem/stromal cell (msc) spheroids primed in 3-d cultures under xeno-free conditions

중간 엽 줄기 / 간질 세포 (중간 엽 줄기 세포)는 생명 공학 및 재생 의학에 큰 약속을 개최합니다. 중간 엽 줄기 세포는 조직 배양 플라스틱에 강한 접착을 통해 다수의 성인 조직으로부터 분리 한 후, 추가 가장 일반적으로 소 태아 혈청 (FBS)을 사용하여, 시험 관내에서 확장 될 수있다. FBS는 중간 엽 줄기 세포는 면역 원성가 발생할 수 있기 때문에, MSC 문화에서의 존재는 세포의 모두 임상 및 실험 응용 프로그램을 제한합니다. MSC 문화에 대한 따라서 연구는 화학적으로 채용하는 이종없는 (XF) 미디어는 매우 가치가있다. 중간 엽 줄기 세포의 많은 이로운 효과는 종양 괴사 인자 - 자극 된 유전자 6 (TSG6) 및 프로스타글란딘 E2 (PGE2)로 주로 면역 인자의 분비를 통해 염증 및 면역 조절 능력에 기인하고있다. 그러나, 중간 엽 줄기 세포는 이러한 요소를 생성하도록 활성화를 요구하고 중간 엽 줄기 세포의 효과는 종종 일시적이므로, 큰 관심은 세포 프리 오 미리 활성화시키는 방법을 발견하는 등장자신의 사용에 대한 연구, 따라서 생체 내에서 활성화에 대한 지연 시간을 제거. 여기에서 우리는 3 차원 (3D) 문화에서 효율적으로 작동하는 프로토콜이나 주요 중간 엽 줄기 세포를 제시 화학적으로 XF 조건 및 생체 내에서 이러한 사전 활성화 된 중간 엽 줄기 세포를 관리하는. 특히, 우리는 먼저 XF 매체를 이용하여 구형 MSC 마이크로 조직이나 매달려 방울의 &#39;타원체&#39;를 생성하고 분야와 에어컨 매체 (CM)는 다양한 애플리케이션에 수확 할 수있는 방법을 보여주기 위해 방법을 설명합니다. 둘째, 유전자 발현 화면 설명 및 시험 관내 기능 분석을 신속하게 세포의 항 염증 및 항암 가능성을 강조 타원체 MSC의 활성화 수준을 평가. 셋째, 생체 내 효능을 시험하기위한 마우스 복강 내로 그대로 MSC의 구 상체를 분사하기위한 신규 한 방법을 설명한다. 전반적으로, 프로토콜은 여기에 화학적으로 XF 콘에서 미리 활성화 된 중간 엽 줄기 세포를 얻는 주요 문제를 극복ditions 및 치료에 대한 MSC의 타원체를 관리 할 수있는 유연한 시스템을 제공합니다.

현재 작업에 매달려 드롭 문화는 소형 구형 마이크로 조직 또는 XF 조건에서 활성화 된 중간 엽 줄기 세포의 &#39;타원체&#39;을 생성하기 위해 사용되었다. 그림 1의 임상 로드맵은 중간 엽 줄기 세포는 자기 조립에 권장되는 타원체, 또는 CM은 구 유래의 치료 요인로드 한 후 72 시간에 대한 방울 매달려에 현탁 때 타원체로 수집 될 수 있으며, 잠재적으로 모두 사...

Watch this Scientific Journal Video about Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions at JoVE.com

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

제노없는 조건에서 3-D 문화의 생산과 치료 중간 엽 줄기의 관리 / 기질 세포 (MSC) 타원체 뇌관

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

production-administration-therapeutic-mesenchymal-stemstromal-cell

Research

JoVE Journal

Developmental Biology

Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

11.8K 조회수.  University of Mary Hardin-Baylor. The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

비디오: Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

마이크로 웰 플레이트를 사용하여 크기 조절 종양 상체 많은 수의 생산

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically ...

연구

생체공학

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

유 전적으로 변형 된 세포 이식을위한 미세 패턴 배양 플레이트에 회전 타원체 세포를 향해 유전자 형질

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) substrates such as tissue culture polystyrene (TCPS). However, characteristic changes of PDL cells have been observed during in vitro culture. This phenomenon is probably because the 2D TCPS differs from the in vivo three-dimensional (3D) microenvironment. Compared to cells cultured on 2D substrates, cells grown in a 3D microenvironment exhibit more similarities to in vivo cells. Therefore, 3D cell culture models provide a promising alternative for conventional 2D monolayer cell culture. To improve conventional PDL cell culture models, we have recently developed a 3D cell culture method, which is based on spheroid formation of PDL cells on chitosan films. Here, we present detailed cell spheroid culture protocols based on chitosan films. The 3D culture system of PDL cellular spheroids overcome some of the limitations related to conventional 2D monolayer cell culture, and thus may be suitable for producing PDL cells with an enhanced therapeutic efficacy for future periodontal tissue regeneration.

Here, we present protocols of culturing human periodontal ligament (PDL) cell spheroids by chitosan films. The culture of three-dimensional (3D) cellular spheroids provides an alternative to conventional tissue culture polystyrene (TCPS) culture system.

키토산 필름에 인간 치주 인대 세포 스페로이드의 형성

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) ...

발생학

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 ...

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 &#181;m. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and &gt;95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

<meta charset="utf8"></head><body>3D 프린팅된 스탬프와 같은 장치를 통한 조직 스페로이드 생성

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

3D 프린팅된 스탬프와 같은 장치를 통한 조직 스페로이드 생성

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have ...

생화학

A Label-free Xenograft Model for Investigating the Behavior of Human Stem Cell Spheroids in Chick Embryos

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell migration, differentiation, and morphogenesis, with unique instructive signals and germ layer identity that are often absent from adult xenograft models. In addition, embryonic models cannot discriminate self versus non-self tissues, eliminating the risk of rejection of the graft and the need for immune suppression of the host. This paper presents a methodology for transplantation of spheroids of human cells into chicken embryos, which are accessible, amenable to manipulation, and develop at 37 &#176;C. 
Spheroids allow the selection of a specific region of the embryo for transplantation. After being grafted, the cells become integrated into the host tissue, allowing the follow-up of their migration, growth, and differentiation. This model is flexible enough to allow the utilization of different adherent populations, including heterogeneous primary cell populations and cancer cells. To circumvent the need for prior cell labeling, a protocol for the identification of donor cells through hybridization of human-specific Alu probes is also described, which is particularly important when investigating heterogeneous cell populations. Furthermore, DNA probes can be easily adapted to identify other donor species. This protocol will describe the general methods for preparing spheroids, grafting into chicken embryos, fixing and processing tissue for paraffin sectioning, and finally identifying the human cells using DNA in situ hybridization. Suggested controls, examples of interpretation of results and various cell behaviors that can be assayed will be discussed in addition to the limitations of this method.

Here, we describe a method to transplant and identify human cell spheroids into chick embryos. This xenograft model uses the embryonic microenvironment as a source of instructive signals to assay cell migration, differentiation, and tropism and is especially suited for the study of primary and/or heterogeneous cell populations.

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

3D 바이오프린팅을 위한 지방유래 줄기세포 구상체의 대규모, 자동화 생산

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

iPSC에서 효율적인 인간 장 오가노이드 생성을 위한 매트릭스 비교

Comparative Study of Basement-Membrane Matrices for Human Stem Cell Maintenance and Intestinal Organoid Generation

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) are in the spotlight of many research areas. However, the lack of physiological cues in classical cell culture materials hinders efficient iPSC differentiation. Incorporating commercially available ECM into stem cell culture provides physical and chemical cues beneficial for cell maintenance. Animal-derived commercially available basement membrane products are composed of ECM proteins and growth factors that support cell maintenance. Since the ECM holds tissue-specific properties that can modulate cell fate, xeno-free matrices are used to stream up translation to clinical studies. While commercially available matrices are widely used in hiPSC and organoid work, the equivalency of these matrices has not been evaluated yet. Here, a comparative study of hiPSC maintenance and human intestinal organoids (hIO) generation in four different matrices: Matrigel (Matrix 1-AB), Geltrex (Matrix 2-AB), Cultrex (Matrix 3-AB), and VitroGel (Matrix 4-XF) was conducted. Although the colonies lacked a perfectly round shape, there was minimal spontaneous differentiation, with over 85% of the cells expressing the stem cell marker SSEA-4. Matrix 4-XF led to the formation of 3D round clumps. Also, increasing the concentration of supplement and growth factors in the media used to make the Matrix 4-XF hydrogel solution improved hiPSC expression of SSEA-4 by 1.3-fold. Differentiation of Matrix 2-AB -maintained hiPSC led to fewer spheroid releases during the mid-/hindgut stage compared to the other animal-derived basement membranes. Compared to others, the xeno-free organoid matrix (Matrix 4-O3) leads to larger and more mature hIO, suggesting that the physical properties of xeno-free hydrogels can be harnessed to optimize organoid generation. Altogether, the results suggest that variations in the composition of different matrices affect stages of IO differentiation. This study raises awareness about the differences in commercially available matrices and provides a guide for matrix optimization during iPSC and IO work.

저자 스포트라이트: iPSC를 사용한 약물 개발을 위한 오가노이드 생성 발전

Organoids have become valuable tools for disease modeling. The extracellular matrix (ECM)&#160;guides cell fate during organoid generation, and using a system that resembles the native tissue can improve model accuracy. This study compares the generation of induced pluripotent stem cells-derived human intestinal organoids in animal-derived ECM and xeno-free hydrogels.

인간 줄기세포 유지와 장 오가노이드 생성을 위한 기저막 매트릭스의 비교 연구

Extracellular matrix (ECM) plays a critical role in cell behavior and development. Organoids generated from human induced pluripotent stem cells (hiPSCs) ...

제노없는 조건에서 3-D 문화의 생산과 치료 중간 엽 줄기의 관리 / 기질 세포 (MSC) 타원체 뇌관

요약

더 많은 비디오 탐색

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마이크로 웰 플레이트를 사용하여 크기 조절 종양 상체 많은 수의 생산

유 전적으로 변형 된 세포 이식을위한 미세 패턴 배양 플레이트에 회전 타원체 세포를 향해 유전자 형질

키토산 필름에 인간 치주 인대 세포 스페로이드의 형성

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

3D 프린팅된 스탬프와 같은 장치를 통한 조직 스페로이드 생성

3D 프린팅된 스탬프와 같은 장치를 통한 조직 스페로이드 생성

A Label-free Xenograft Model for Investigating the Behavior of Human Stem Cell Spheroids in Chick Embryos

3D 바이오프린팅을 위한 지방유래 줄기세포 구상체의 대규모, 자동화 생산

인간 줄기세포 유지와 장 오가노이드 생성을 위한 기저막 매트릭스의 비교 연구

인간 줄기세포 유지와 장 오가노이드 생성을 위한 기저막 매트릭스의 비교 연구