이 작품은 왕립 학회 URF 부여 UF051616, 영국 및 BS에 대한 유럽 연구 협의회 StG 보조금 243,261에 의해 지원되었다. MZ 연구실의 작업은 또한 재생 의료 보조금 RB1-01417의 캘리포니아 공과 대학에서 지원됩니다.

1. 신경 전구 세포 격리 <ol><li> 추운 DMEM/F12 기초 중간에 E14-16 생쥐와 장소에서 전체 뇌를 해부. 35mm 배양 접시에 해부 현미경 및 전송 두뇌 아래의 모든 meninges를 제​​거합니다. </li><li> 기계적 조직 파편에 머리를 해리 및 15 ML 관에게 전송하는 미세 집게를 사용하여 다음 파편을 제거하기 위해 3 분 동안 800 rpm으로 샘플을 원심 분리기. </li><li> DMEM/F12 포함된 bFGF와 EGF를 추가하고 1 ML의 피펫으로 씹다. </li><li> 단일 세포 현탁액을 얻기 위해 셀 스트레이너를 통해 세포 현탁액을 전달합니다. </li><li> 2-5에서 flasks로 플레이트 전지 X 10 4 세포 / ML, 그때 전체 매체가 3 일 통과 세포에게 매 6 일 모든 변경 수행합니다. </li><li> 적어도 5 구절 후에 다이제스트 neurospheres 단일 세포에 트립신과 EDTA (에틸렌 다이아 민 테트라 초산)을 사용하고 poly-D-lysine/laminin-coated electrotactic의 챔버 (2 아래에 설명된대로 준비)에서 성장. , N2 - 보충을 포함하는 성장 매체를 사용하여bFGF와 EGF는 항상 NPCs의 속성을 유지합니다. </li></ol> 2. electrotactic 챔버의 준비 <ol><li> 다이아몬드 펜을 사용하여 반 22 X 22mm 1 호 두께 coverslips을 autoclaved 나누어 유리의 22 X 11mm 스트립을 준비합니다. </li><li> gluing하여 내무부 크기 22 × 10 mm의 자유 서 유리를 만들기 함께 네 세로로 높은 진공 실리콘 기름 22 X 11mm 스트립 서. 완전히 마르도록 우물과 강화가 하룻밤 허용합니다. </li><li> 다음 날, 실리콘 그리스를 사용하는 100mm 문화 요리의 기본에, 10 mm의 간격을두고 서로 평행하게 두 개의 22 X 11mm 유리 스트립을 첨부합니다. 중심에 가장 가까운 것이 22 X 22mm 커버 삼면이 기름과 함께 접시의 바닥에 부착된 각 끝에 슬립,지만을 배치하여 이러한 스트립 사이의 지역을 봉쇄하라. </li><li> 잘 커버로 올라서 2.2에서 준비 유리 장소는 실내 벽이 퍼뜨리고을위한 제한된 공간을 만들 수 있도록 실수할접시의 바닥쪽으로 세포. 실리콘 기름과 방수 모두 관절. 코트 폴리-D-리신 후 laminin과 순차적이 협소한 지역 : 챔버로 한 ML는 폴리-D-라이신을 추가하고 폴리-D-라이신 판의 하단에 바인딩 수 있도록 5 분 동안 떠나, 챔버를 씻어 멸균 PBS로 두 번, 그리고 20 μg / ML을 양보하고 접시의 바닥을 충당하기 위해 사용하는 멸균 PBS에서 laminin을 희석. 하룻밤 동안 실온에 둡니다. </li><li> 다음 날, 추수 세포 및 1 X 104 세포를 포함하는 정지 1 ML을 준비합니다. 완전히 건조 공기와 세포 현탁액 1 ML로 대체할 수 있도록 잘 유리에서 laminin을 제거합니다. 첨부를 허용하기 위해 4 시간 최소 37 ° C에서 인큐베이터에서 접시를 놓습니다. </li><li> 세포가 충분히 합류가되면 챔버에서 모든 매체를 제거합니다. 조심스럽게 잘 유리를 제거합니다. 실리콘 그리스, autoclaved 22 X 22mm 유리 coverslip으로 신중하게 부착하여 세포를 통해 지붕을 형성두 22 X 11mm 스트립 사이에 브리지가. 밖으로 건조하지 않도록 매체의 몇 방울과 세포를 커버. </li><li> 챔버 지붕 위에 접시 한 ​​가장자리에서 다른 실행이 방수 실리콘 그리스 장벽을 만들어 챔버의 각 끝부분에 절연 매체 저수지를 형성하고 있습니다. 흐름을 관통 중간 저수지에서 서로에 대한 확보, 신선한 매체와 챔버를 채우십시오. 세포 회복을 할 수 있도록 12 시간 동안 인큐베이터에 요리를 반환합니다. </li></ol> 3. electrotactic 챔버에 대한 전기장의 응용 <ol><li> 구멍, 마이 그 레이션 챔버의 각 저수지 위에 위치 하나를 뚫는하여 요리를 충당하기 위해 뚜껑을 준비합니다. </li><li> 25 MM HEPES 버퍼를 포함하는 배지와 챔버에있는 모든 매체를 교체하고 온도 제어 영상 시스템으로 요리를 전송합니다. 시간이 경과 및 멀티 포지션 레코딩을위한 실험 매개 변수를 설정합니다. 음극과 양극이되도록 챔버 정렬오른쪽 각각 왼쪽에서 EF 벡터가 실행되도록 수평으로 현미경을 조회 및 이미징 시스템에 기록. </li><li> 스테인 버그의 솔루션 (58 MM NaCl, 0.67 MM KCl, 0.44 MM 칼슘 (NO 3) 2 0.4 H 2 0, 1.3 MM MgSO 4 0.7 H 2 0 및 4.6 밀리미터 Trizma 기지, 산도 7.4)으로 두 비커를 채웁니다. 2퍼센트 (W / 가득 미리 준비된 유리 다리를 (~ 13cm 길이 ~ 3mm 직경 및 분젠 화염에 가열하여 U 자형으로 구부 러 유리 튜브)를 사용하여 각 매체 저수지로 별도의 비커를 연결 뚜껑에있는 구멍을 통과 V) Steinberg&#39;s-한천 솔루션. 스테인 버그의 솔루션의 각 비커에 DC 전원 공급 장치에 연결된 자세 / AgCl 전극을 배치하여 전기 회로를 완료합니다. </li><li> 0과에서 전환 전원 공급 장치의 전압을 다이얼을 설정합니다. 전압 측정기를 사용하여 전압을 다이얼을 돌리면서 electrotactic 챔버를 통해 전압을 측정하고, 실험 요구 사항에 맞게 조정할 수 있습니다. </li><li> 촬영된 녹화를 시작합니다. 매체 변경을 수행하고 매 시간마다 필요에 따라, 전압을 정리하다. 필요에 따라 신선한 매체, 약물 또는 화학 약품은 저수지에 추가할 수 있습니다. 매체 변경을 수행하면 두 가지 옵션은 다음과 같이 생각할 수 : <ol><li> 옵션 1 호 - 일시적으로 정지 시간 저속도 녹음을 위하여는, 신중하게 커버 뚜껑을 perturbing 피하기 위해 챔버에서 유리 다리를 제거, 신선한 매체와 모든 매체를 대체할 부드럽게 멸균 파스퇴르 피펫을 사용하여 유리 다리를 다시 넣어 다음 녹음을 재개하십시오. </li><li> 옵션 2 호는 - 또는, 사용자는 또한 매체 변경 목적으로 사용할 수있는 4 홀 (두 마이 그 레이션 챔버의 각 저수지 위에 위치)로 커버 뚜껑을했다. 유리 교량 연결을위한 두, 다른 두 매체를 변경하기위한. 옵션 2는 매체 변화하는 동안 간섭없이 연속 녹화 가능합니다. </li></ol></li></ol> 4. organotypic 척수 슬라이스의 작성 <ol><li> lumb 해부이주 오래된 C57 BL / 6 생쥐의 AR 척수가 나란히있어. </li><li> McIlwain 조직 헬기와 500 μm의 두께 부분으로 척수 코드를 썰어. </li><li> 별도의 해부 현미경으로 조각하고 그대로 축 / 화살 척수의 구조와 조각을 선택합니다. </li><li> Matrigel 단백질 생산 자체 조립까지 30 μl Matrigel 그리고 그것을 배치를 포함하는 35mm 배양 접시에있는 플레이트 조각 가능한 한 중심가에 가까이하고 적어도 30 분 동안 37 ° C에서 5 % CO 2 배양기에서 그들을 유지 척수의 단면의 표면을 커버 박막. 그것은 완전히 다른 30 ​​분 필요한 경우에 대해 37 ° C에서 페트리 접시를 품어 Matrigel이 조립되어 있는지 확인하는 것이 매우 중요합니다. </li><li> 슬라이스에 직접 매체 흐름을 피하기 위해 25 밀리미터 HEPES 버퍼와 매우 부드럽게하므로 같은 15-20% 소태아 혈청을 포함하는 4-6 ML DMEM/F-12 매체를 추가하십시오. 슬라이스가 완전히 잘 노출 explants의 표면을두고, 중간에 빠져들되지 않았음을 확인공기. 주 2 회 매체를 변경합니다. </li></ol> 5. organotypic 척수의 슬라이스에 Hoechst 33342로 분류 NPCs의 분사 <ol><li> 하나의 NPC 서스펜션 × 10 6 세포 / μl를 준비합니다. </li><li> 30 분 5 μm의 Hoechst 33342와 함께 중간에 세포 현탁액을 사전 품어. </li><li> 현미경으로 천천히 척수의 슬라이스로 정지 2 μl를 microinject하는 모세관 유리관을 사용합니다. 모세 유리관이 matrigel (현미경 빨간 부분)을 통과하고 척수의 단면 (현미경 회색 조직)의 내부에 도달했는지 확인, 달려든 세포 현탁액을 피하기 위해 최소 30 초 동안 척수 슬라이스 안에 남아 있습니다. 인큐베이터로 척수의 슬라이스를 포함하는 배양 접시 (37 ° C에서 5 % CO 2)를 넣고 하룻밤 동안 그것을 두십시오. </li><li> 다음날, (에 설명된 방법을 사용 electrotactic 챔버에 33,342-라벨 NPCs Hoechst를 포함한 척수 슬라이스 500 MV / mm의 EF를 적용3). </li></ol> 6. 대표 결과 NPCs들은 음극쪽으로 높은 감독 셀 마이 그 레이션 (그림 1)을 보였다 생리 EFS의 범위에 노출되었을 때. 동일한 관측도 organotypic 척수의 슬라이스 전직의 생체내 모델, 생체내 조건에서 3D 환경 흉내낸 (그림 2)에서 단일 세포 수준에서 만들어졌습니다. <img alt="그림 1" src="/files/ftp_upload/3453/3453fig1.jpg" /> 1 그림. NPCs은 EFS의 감독이 마이 그 레이션을 보여줍니다. EFS에 노출되면 PC는 음극쪽으로 높은 감독이 마이 그 레이션을 보여줬, 빨간색 라인과 파란색 화살표는 세포 운동의 궤도와 방향 (A) 나타냅니다. B는 NPCs의 마이 그 레이션 경로를 보여줍니다. 바 : 50 μm의. <img alt="그림 2" src="/files/ftp_upload/3453/3453fig2.jpg" /> 그림 2. 이식 NPCs는 organotypic 척수의 단면에서 음극쪽으로 이동 마이 그 레이션을 보여 </st룽&gt;. () Hoechst 33342로 표시된 NPCs는 EF 치료의 시작 시점에서 organotypic 척수의 슬라이스로 이식되었다. NPCs은 EF의 극성은 (B) 반대하는 시점에서, 2.5 시간 동안 음극쪽으로 directionally 마이 그 레이션. EF 극성을 변경하면 새로운 음극 (C) 향해 electrotaxis의 날카로운 반전을 촉발시켰다. 시간 경과 기록의 끝에 척수 슬라이스 내에 이식된 NPCs의 (D) 이미지. 척수의 슬라이스 내에 이식된 NPCs의 (E) 3D 재건. 3D 스캔 섹션 슬라이스의 하단에 중간부터 시작 및 종료, 두께 300 μm의했다. 점선은 초기, 반전, 그리고 EF 처리 끝 지점 (- C, 각각)에서 이식된 세포의 동일한 인구의 상대적 위치를 나타냅니다. 애로우 헤드 Hoechst 33342 레이블 NPCs 같은 인구를 나타냅니다. 바 : 50 μm의.

<ol>
	<li>Huttenlocher, A., Horwitz, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wound+healing+with+electric+potential.">Wound healing with electric potential.</a> N. Engl. J. Med. 356, 303-303 (2007).</li><li>McCaig, C. D., Rajnicek, A. M., Song, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+cell+behavior+electrically:+current+views+and+future+potential.">Controlling cell behavior electrically: current views and future potential.</a> Physiol. Rev. 85, 943-943 (2005).</li><li>Borgens, R. B., Jaffe, L. F., Cohen, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large+and+persistent+electrical+currents+enter+the+transected+lamprey+spinal+cord.">Large and persistent electrical currents enter the transected lamprey spinal cord.</a> PNAS. 77, 1209-1209 (1980).</li><li>Shapiro, S., Borgens, R., Pascuzzi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oscillating+field+stimulation+for+complete+spinal+cord+injury+in+humans:+a+phase+1+trial.">Oscillating field stimulation for complete spinal cord injury in humans: a phase 1 trial.</a> J. Neurosurg. Spine. 2, 3-3 (2005).</li><li>Zhao, M., Song, B., Pu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+signals+control+wound+healing+through+phosphatidylinositol-3-OH+kinase-gamma+and+PTEN.">Electrical signals control wound healing through phosphatidylinositol-3-OH kinase-gamma and PTEN.</a> Nature. 442, 457-457 (2006).</li><li>Yao, L., Shanley, L., McCaig, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+applied+electric+fields+guide+migration+of+hippocampal+neurons.">Small applied electric fields guide migration of hippocampal neurons.</a> J. Cell. Physiol. 216, 527-527 (2008).</li><li>Li, L., El-Hayek, Y. H., Liu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct-current+electrical+field+guides+neuronal+stem&#47;progenitor+cell+migration.">Direct-current electrical field guides neuronal stem&#47;progenitor cell migration.</a> Stem Cells. 26, 2193-2193 (2008).</li><li>Meng, X., Arocena, M., Penninger, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K+mediated+electrotaxis+of+embryonic+and+adult+neural+progenitor+cells+in+the+presence+of+growth+factors.">PI3K mediated electrotaxis of embryonic and adult neural progenitor cells in the presence of growth factors.</a> Exp. Neurol. 227, 210-210 (2011).</li><li>Bonnici, B., Kapfhammer, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cells+promote+neurite+extension+in+organotypic+spinal+cord+slice:+significance+for+cell+transplantation+therapy.">Bone marrow stromal cells promote neurite extension in organotypic spinal cord slice: significance for cell transplantation therapy.</a> Neurorehabil. Neural Repair. 22, 447-447 (2008).</li><li>Shichinohe, H., Kuroda, S., Tsuji, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cells+promote+neurite+extension+in+organotypic+spinal+cord+slice:+significance+for+cell+transplantation+therapy.">Bone marrow stromal cells promote neurite extension in organotypic spinal cord slice: significance for cell transplantation therapy.</a> Neurorehabil. Neural Repair. 22, 447-447 (2008).</li><li>Song, B., Gu, Y., Pu, j. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+direct+current+electric+fields+to+cells+and+tissues+in+vitro+and+modulation+of+wound+electric+field+in+vivo.">Application of direct current electric fields to cells and tissues in vitro and modulation of wound electric field in vivo.</a> Nature Protocol. 2, 1479-1479 (2007).</li></ol>

우리는 공개 할게 없다.

우리가 사용하는 프로토콜은 이전 연구 5,11에 바탕을두고 있습니다. 표준화와 정확한 차원 맞춤 디자인 electrotactic의 실에서 배양해 세포 또는 조각으로 한천 교량, 스테인 버그의 솔루션 및 자세 / AgCl 전극를 통해 EF을 적용하는 동안 이러한 방법을 사용하여 안정적인 문화와 전류 조건이 유지됩니다. 챔버의 깊이는 다른 예제는 11 두께에 대한 수용하도록 조정될 수 있으며, 세포?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 </td></tr><tr><td> FGF-기본적인 재조합 인간 </td><td> Invitrogen </td><td> PHG0026 </td><td> 20 NG / ML </td></tr><tr><td> EGF 재조합 인간 </td><td> Invitrogen </td><td> PHG0311 </td><td> 20 NG / ML </td></tr><tr><td> N2 - 보충 (100X) 액체 </td><td> Invitrogen </td><td> 02,048 </td><td></td></tr><tr><td> DMEM/F12 매체 (높은 혈당) </td><td> Invitrogen </td><td> 31330-095 </td><td></td></tr><tr><td> 폴리-D-라이신 </td><td> Millipore </td><td> -003-E </td><td></td></tr><tr><td> 자연 마우스 Laminin </td><td> Invitrogen </td><td> 23017-015 </td><td></td></tr><tr><td> 성장 요인은 지하 분리막 매트릭스 (Matrigel)을 감소 </td><td> BD Biosciences </TD&gt; <td> 354,230 </td><td></td></tr><tr><td> HEPES 버퍼 </td><td> Gibco </td><td> 15,630 </td><td></td></tr><tr><td> McIlwain 조직 헬기 </td><td> Mickle 연구소 엔지니어링 주식 회사 </td><td> TC752-PD </td><td></td></tr><tr><td> 다우 코닝 높은 진공 실리콘 그리스 </td><td> 시그마 - 올드 리치 </td><td> Z273554 </td><td></td></tr></tbody></table>

electric field-controlled directed migration of neural progenitor cells in 2d and 3d environments

내생 전기 분야 (EFS)는 생체내 자연적으로 발생하며 중추 신경계의 1,2의 것을 포함한 조직 / 기관이 개발하고 재생하는 동안 중요한 역할을 재생합니다. 이러한 내생 EFS는 세포와 조직의 전기 저항과 결합 이온 수송의 셀룰러 규정에 의해 생성됩니다. 이것은 적용된 EF 치료는 동물과 인간 3,4의 척수 부상의 기능 복구를 홍보할 수 있다고보고되었습니다. 특히, EF의 감독의 세포 이주는 신경 전구 세포 (NPCs) 7,8 포함한 세포 유형 5,6의 다양한에서 입증되었습니다. 직류 (DC) EFS의 응용은 대부분의 실험실에서 일반적으로 사용 가능한 기법이 아닙니다. 우리는 세포와 조직 문화를 이전 5,11에 직류 EFS의 응용 프로그램에 대한 자세한 절차를 설명했습니다. 여기 2D를 설정하는 계산 필드 강도를 기반 표준 방식의 비디오 데모를 소개합니다NPCs에 대한 D 3D 환경, 그리고 2D에서 단일 세포 성장 조건에서 EF의 자극, 그리고 3D에서 organotypic 척수의 슬라이스에 세포 반응을 조사합니다. 척추 cordslice는 cytoarchitectonic 조직 조직도 이러한 문화 9,10 내에 보존되기 때문에 전직 NPC의 생체내 행동, 사후 이식을 공부하는 데 이상적인받는 조직이다. 또한이 전 생체내 모델은 또한 단일 세포 수준에서 촬영된 기록을 사용하여 생체내에서 세포를 추적할 기술적으로 가능하지 않습니다 수속을하실 수 있습니다. 그것은 비판적으로 2 차원 환경뿐만 아니라에서 셀 행동을 평가하는 것이 필수적뿐만 아니라 생체내 환경에서 mimicks 3D organotypic 상태입니다. 이 시스템은 수 체외 및 생체내 전직의 단세포 이주의 3D 추적과 조직 또는 장기의 문화에 커버 유리 기반 요리를 사용하고 월이면쪽으로 이동하기 전에 중간 단계가 될 수 고해상도 이미징조종의 패러다임.

Watch this Scientific Journal Video about Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments at JoVE.com

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

이 프로토콜은 세포를 추적할 수 있습니다 맞춤 디자인 electrotactic의 실에서 2D 및 3D 환경을 설정하는 데 사용 방법을 보여줍니다<em&gt; 생체내 / 전직 생체내에</em&gt; 전류 (DC) 전기 분야 (EFS)를 지시하는 galvanotaxis / electrotaxis 및 기타 세포 반응을 조사하기 위해, 단일 세포 수준에서 촬영된 녹화를 사용합니다.

2D 및 3D 환경에서 신경 전구 세포의 전기장 제어 감독 마이 그 레이션

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

electric-field-controlled-directed-migration-neural-progenitor-cells

Research

JoVE Journal

Bioengineering

11.6K 조회수.  Cardiff University. 이 프로토콜은 세포를 추적할 수 있습니다 맞춤 디자인 electrotactic의 실에서 2D 및 3D 환경을 설정하는 데 사용 방법을 보여줍니다 생체내 / 전직 생체내에 전류 (DC) 전기 분야 (EFS)를 지시하는 galvanotaxis / electrotaxis 및 기타 세포 반응을 조사하기 위해, 단일 세포 수준에서 촬영된 녹화를 사용합니다.

비디오: Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Cultivation of Human Neural Progenitor Cells in a 3-dimensional Self-assembling Peptide Hydrogel

The influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally neuronal differentiation is of great interest in order to find new methods for cell-based and standardised therapies in neurological disorders or neurodegenerative diseases. 3D structures are expected to provide an environment much closer to the in vivo situation than 2D cultures. In the context of regenerative medicine, the combination of biomaterial scaffolds with neural stem and progenitor cells holds great promise as a therapeutic tool.1-5 Culture systems emulating a three dimensional environment have been shown to influence proliferation and differentiation in different types of stem and progenitor cells. Herein, the formation and functionalisation of the 3D-microenviroment is important to determine the survival and fate of the embedded cells.6-8 Here we used PuraMatrix9,10 (RADA16, PM), a peptide based hydrogel scaffold, which is well described and used to study the influence of a 3D-environment on different cell types.7,11-14 PuraMatrix can be customised easily and the synthetic fabrication of the nano-fibers provides a 3D-culture system of high reliability, which is in addition xeno-free.
Recently we have studied the influence of the PM-concentration on the formation of the scaffold.13 In this study the used concentrations of PM had a direct impact on the formation of the 3D-structure, which was demonstrated by atomic force microscopy. A subsequent analysis of the survival and differentiation of the hNPCs revealed an influence of the used concentrations of PM on the fate of the embedded cells. However, the analysis of survival or neuronal differentiation by means of immunofluorescence techniques posses some hurdles. To gain reliable data, one has to determine the total number of cells within a matrix to obtain the relative number of e.g. neuronal cells marked by &beta;III-tubulin. This prerequisites a technique to analyse the scaffolds in all 3-dimensions by a confocal microscope or a comparable technique like fluorescence microscopes able to take z-stacks of the specimen. Furthermore this kind of analysis is extremely time consuming. 
Here we demonstrate a method to release cells from the 3D-scaffolds for the later analysis e.g. by flow cytometry. In this protocol human neural progenitor cells (hNPCs) of the ReNcell VM cell line (Millipore USA) were cultured and differentiated in 3D-scaffolds consisting of PuraMatrix (PM) or PuraMatrix supplemented with laminin (PML). In our hands a PM-concentration of 0.25% was optimal for the cultivation of the cells13, however the concentration might be adapted to other cell types.12 The released cells can be used for e.g. immunocytochemical studies and subsequently analysed by flow cytometry. This speeds up the analysis and more over, the obtained data rest upon a wider base, improving the reliability of the data.

Here we describe the use of a self-assembling 3-dimensional scaffold to culture human neural progenitor cells. We present a protocol to release the cells from the scaffolds to be analysed subsequently e.g. by flow cytometry. This protocol might be adapted to other cell types to perform detailed mechanistically studies.

3 차원 자기 조립 펩티드 히드로겔에서 인간 신경 전구 세포의 배양

The  influence of 3-dimensional (3D) scaffolds on growth, proliferation and finally  neuronal differentiation is of great interest in order to find new ...

연구

생체공학

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

단일 세포 및 3D 감금 단일 배아 주문 : 하이 콘텐츠 스크리닝을위한 새로운 장치

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Electrotaxis Studies of Lung Cancer Cells using a Multichannel Dual-electric-field Microfluidic Chip

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of physiological dcEF in guiding cell movement during embryo development, cell differentiation, and wound healing has been demonstrated in many studies. By applying microfluidic chips to an electrotaxis assay, the investigation process is shortened and experimental errors are minimized. In recent years, microfluidic devices made of polymeric substances (e.g., polymethylmethacrylate, PMMA, or acrylic) or polydimethylsiloxane (PDMS) have been widely used in studying the responses of cells to electrical stimulation. However, unlike the numerous steps required to fabricate a PDMS device, the simple and rapid construction of the acrylic micro&#64258;uidic chip makes it suitable for both device prototyping and production. Yet none of the reported devices facilitate the efficient study of the simultaneous chemical and dcEF effects on cells. In this report, we describe our design and fabrication of an acrylic-based multichannel dual-electric-field (MDF) chip to investigate the concurrent effect of chemical and electrical stimulation on lung cancer cells. The MDF chip provides eight combinations of electrical/chemical stimulations in a single test. The chip not only greatly shortens the required experimental time but also increases accuracy in electrotaxis studies.

Many microfluidic devices have been developed for use in the study of electrotaxis. Yet, none of these chips allows the efficient study of the simultaneous chemical and electric-field (EF) effects on cells. We developed a polymethylmethacrylate-based device that offers better-controlled coexisting EF and chemical stimulation for use in electrotaxis research.

멀티 채널 듀얼 전기장 미세 유체 칩을 이용하여 폐암 세포의 Electrotaxis 연구

The behavior of directional cell migration under a direct current electric-field (dcEF) is referred to as electrotaxis. The significant role of ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

개선 2D 및 3D 스킨<em&gt; 체외</em거대 분자의 크라우 딩 사용&gt; 모델

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

2D and 3D Matrices to Study Linear Invadosome Formation and Activity

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor cells have to cross anatomical barriers to invade and migrate through the surrounding tissue in order to reach blood or lymphatic vessels. This requires the interaction between cells and the extracellular matrix (ECM). At the cellular level, many cells, including the majority of cancer cells, are able to form invadosomes, which are F-actin-based structures capable of degrading ECM. Invadosomes are protrusive actin structures that recruit and activate matrix metalloproteinases (MMPs). The molecular composition, density, organization, and stiffness of the ECM are crucial in regulating invadosome formation and activation. In vitro, a gelatin assay is the standard assay used to observe and quantify invadosome degradation activity. However, gelatin, which is denatured collagen I, is not a physiological matrix element. A novel assay using type I collagen fibrils was developed and used to demonstrate that this physiological matrix is a potent inducer of invadosomes. Invadosomes that form along the collagen fibrils are known as linear invadosomes due to their linear organization on the fibers. Moreover, molecular analysis of linear invadosomes showed that the discoidin domain receptor 1 (DDR1) is the receptor involved in their formation. These data clearly demonstrate the importance of using a physiologically relevant matrix in order to understand the complex interactions between cells and the ECM.

This protocol describes how to prepare a 2D mixed matrix, consisting of gelatin and collagen I, and a 3D collagen I plug to study linear invadosomes. These protocols allow for the study of linear invadosome formation, matrix degradation activity, and the invasion capabilities of primary cells and cancer cell lines.

선형 Invadosome 형성과 활동을 연구하기위한 2D와 3D 매트릭스

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

환경 상처 3 차원 생체 외에서 내 셀 마이그레이션 특성화

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance function result in the same outcome: excess inflammation leads to gliosis, cytotoxicity, and/or formation of a glial scar that collectively exacerbate injury or prevent healthy recovery. With the intent of creating a system to model glial scar formation and study inflammatory processes, we have generated a 3D cell scaffold capable of housing primary cultured glial cells: microglia that regulate the foreign body response and initiate the inflammatory event, astrocytes that respond to form a fibrous scar, and oligodendrocytes that are typically vulnerable to inflammatory injury. The present work provides a detailed step-by-step method for the fabrication, culture, and microscopic characterization of a hyaluronic acid-based 3D hydrogel scaffold with encapsulated rat brain-derived glial cells. Further, protocols for characterization of cell encapsulation and the hydrogel scaffold by confocal immunofluorescence and scanning electron microscopy are demonstrated, as well as the capacity to modify the scaffold with bioactive substrates, with incorporation of a commercial basal lamina mixture to improved cell integration.

Improved 3D Hydrogel Cultures of Primary Glial Cells for In Vitro Modelling of Neuroinflammation

Herein, we present a protocol for the 3D culture of rat brain-derived glia cells, including astrocytes, microglia, and oligodendrocytes. We demonstrate primary cell culture, methacrylated hyaluronic acid (HAMA) hydrogel synthesis, HAMAphoto-polymerization and cell encapsulation, and sample processing for confocal and scanning electron microscopic imaging.

생체 외에서 Neuroinflammation의 모델링에 대 한 기본 Glial 세포의 3 차원 하이드로 겔 문화 개선

In the central nervous system, numerous acute injuries and neurodegenerative disorders, as well as implanted devices or biomaterials engineered to enhance ...

Manipulation of Single Neural Stem Cells and Neurons in Brain Slices using Robotic Microinjection

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, manipulate, and follow single cells in the brain tissue with high resolution over time. This task is extremely challenging due to the complexity of tissues in the brain. We have recently developed a robot, that guide a microinjection needle into brain tissue upon utilizing images acquired from a microscope to deliver femtoliter volumes of solution into single cells. The robotic operation increases resulting an overall yield that is an order of magnitude greater than manual microinjection and allows for precise labeling and flexible manipulation of single cells in living tissue. With this, one can microinject hundreds of cells within a single organotypic slice. This article demonstrates the use of the microinjection robot for automated microinjection of neural progenitor cells and neurons in the brain tissue slices. More broadly, it can be used on any epithelial tissue featuring a surface that can be reached by the pipette. Once set up, the microinjection robot can execute 15 or more microinjections per minute. The microinjection robot because of its throughput and versality will make microinjection a broadly straightforward high-performance cell manipulation technique to be used in bioengineering, biotechnology, and biophysics for performing single-cell analyses in organotypic brain slices.

This protocol demonstrates the use of a robotic platform for microinjection into single neural stem cells and neurons in brain slices. This technique is versatile and offers a method of tracking cells in tissue with high spatial resolution.

로봇 미세 주입을 사용하여 뇌 조각의 단일 신경 줄기 세포 및 뉴런 조작

A central question in developmental neurobiology is how neural stem and progenitor cells form the brain. To answer this question, one needs to label, ...

Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell culture system that was used for a long-term cell differentiation study using microscopy. The microfluidic system consists of the following major components: an optically transparent electrotactic chip, a transparent indium-tin-oxide (ITO) heater, a culture media-filling pump, an electrical power supply, a high-frequency power amplifier, an EF multiplexer, a programmable X-Y-Z motorized stage, and an inverted phase-contrast microscope equipped with a digital camera. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This work involves the differentiation of neural stem and progenitor cells (NPCs) induced by direct current (DC) pulse stimulation. In the stem cell maintenance medium, the mouse NPCs (mNPCs) differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. The results suggest that simple DC pulse treatment could control the fate of mNPCs and could be used to develop therapeutic strategies for nervous system disorders. The system can be used for cell culture in multiple channels, for long-term EF stimulation, for cell morphological observation, and for automatic time-lapse image acquisition. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.

In this study, we present a protocol for the differentiation of neural stem and progenitor cells (NPCs) solely induced by direct current (DC) pulse stimulation in a microfluidic system.

미세 유체 장치에서 전기장 유발 신경 전구체 세포 분화

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

생물학적 조직의 자극을 위한 전기 및 자기장 장치

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...