Name: a device for performing cell migration/wound healing in a 96-well plate
Uploaded: 2017-03-07T15:00:00.000+00:00
Duration: 5 min 46 s
Description: Watch this Scientific Journal Video about A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate at JoVE.com

저자는이 wounder의 프로토 타입을 만드는 씨 탐 포 렁 씨 웡 치 킨과 기술 능력에 대한 과학 학부 워크샵의 기술 직원, 홍콩 침례 대학과 조언을 감사의 말씀을 전합니다.

Wounder의 부품 1. 소개 (그림 1) <ol><li> 동일한 거리에있는 상처 핀을 고정하여 핀 홀더를 준비합니다. 피펫 팁을 잡고 위아래로 조절 상처 핀을 이동하여 팁 높이를 조정합니다. </li><li> 96- 웰 배양 플레이트의 다른 브랜드의 조절, 안내 막대를 장착하고 상처 면적이 고정 된 위치에있을 것을 보장한다. </li><li> 육각 나사를 조여 조정 상처 핀을 고정합니다. 양단에 육각 소켓 헤드 캡을 조여 위치에 안내 바 수정. </li></ol> 2. 핀 홀더의 폭을 설정한다 (그림 2) <ol><li> M5의 육각 렌치 육각 소켓 헤드 캡을 풉니 다. </li><li> 가이드 바 96 웰 배양 플레이트의 폭 딱 맞도록 상기 핀 홀더의 양측에 고리를 조절하는 적절한 번호를 삽입한다. 참고 : 코닝과 이와키 플레이트가 큰 반지 1 쌍의 작은 반지 1 쌍을 필요로한다. 팔콘과 눈크 (PL)ATES 큰 반지 1 쌍을 필요로한다. </li><li> 육각 소켓 헤드를 체결하면 안내 막대를 해결하기 위해 모자. </li></ol> 3. 안내 바 조정 (그림 3) <ol><li> 멸균 10 μL 플라스틱 피펫 팁으로 부상 핀을 맞 춥니 다. </li><li> M5의 육각 렌치 육각 소켓 헤드 캡을 풉니 다. </li><li> wounder를 잡고 96- 웰 배양 플레이트의 한 컬럼에 상처 핀에 담갔다. </li><li> 간신히 우물의 바닥을 터치 팁의 모든 때까지 안내 바의 높이를 조정합니다. </li><li> 육각 소켓 헤드 캡을 조입니다. wounder 이제 완벽하게 배양 접시에와 핀이 우물의 중간에 잘 배치되어 있는지에 맞는지 확인합니다. </li></ol> 핀 4. 교정 <ol><li> 평면 멸균면 (즉, 페트리 접시)에 수직 wounder를 잡고 M3 육각 렌치로 모든 육각 나사를 풉니 다. </li><li> 균등하게 평면 멸균 표면을 만지지 팁의 모든 때까지 wounder을 누릅니다. &lt;/ 리&gt; <li> 위치에 핀을 고정 다시 육각 나사를 조입니다. </li><li> 평면 멸균 표면에 부드럽게 wounder을 눌러 각 팁의 균일 성을 확인합니다. </li></ol> 5. 긁는 셀 단일 플라이 (그림 4) <ol><li> 0.1 % 젤라틴 - 코팅 된 96- 웰 세포 배양 플레이트에 시드 인간 제대 혈관 내피 세포. 매체 (199)의 배양 세포는 20 % 열 - 불 활성화 소 태아 혈청, 1 % 페니실린 / 스트렙토 마이신 및 0.09 g / L의 헤파린 보충. 밤새 사용하기 전에 37 ºC에서 5 % 이산화탄소 가습 배양기에서 세포를 유지한다. 세포는 상처 전에 100 % 포화 상태에 도달한다. </li><li> 가장 왼쪽 (또는 오른쪽) 문화 판의 같은 열에서 각 웰의 측면의 상처 팁을 놓습니다. 우물의 반대편에 걸쳐 wounder를 밀어; 상처 팁 잘 하단을 터치 있는지 확인합니다. </li><li> 모든 열에 대한 긁힘 (단계 5.2) 반복합니다. </li><li> , 폐기 t 상처 후그는 중간 각 잘 신선한 매체를 포함하는 시험 화합물로 대체합니다. </li></ol> 6. 데이터 수집 및 이미지 분석 <ol><li> 디지털 카메라 이미지 (예 10X 대물 렌즈)와 저전력 현미경을 사용하여 세포 단층에서 기계적 상처와 전체 웰 즉시 (t = 0 시간)을 상처 후. </li><li> 원하는 경우, 시험 화합물을 첨가하고 원하는 시간 동안 배양. 이미지 원하는 배양 시간 (t = ΔH) 후 다시 상처 면적 전체도. </li><li> 이러한 ImageJ에 같은 영상 분석 소프트웨어 (사용 <a href="https://imagej.nih.gov/ij/">https://imagej.nih.gov/ij/</a> )은 자유형 도구를 사용하여 이미지의 상처 면적을 측정한다. </li><li> 상처 봉합의 비율을 계산한다 : <img alt="방정식" src="/files/ftp_upload/55411/55411eq1.jpg" /></li></ol>

<ol>
	<li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-cell+invasion+and+migration:+diversity+and+escape+mechanisms.">Tumour-cell invasion and migration: diversity and escape mechanisms.</a> Nat Rev Cancer. 3 (5), 362-374 (2003).</li><li>Friedl, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prespecification+and+plasticity:+shifting+mechanisms+of+cell+migration.">Prespecification and plasticity: shifting mechanisms of cell migration.</a> Curr Opin Cell Biol. 16 (1), 14-23 (2004).</li><li>Lampugnani, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+migration+into+a+wounded+area+in+vitro.">Cell migration into a wounded area in vitro.</a> Methods Mol Biol. 96, 177-182 (1999).</li><li>Sholley, M. M., Gimbrone, M. A., Cotran, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+migration+and+replication+in+endothelial+regeneration:+a+study+using+irradiated+endothelial+cultures.">Cellular migration and replication in endothelial regeneration: a study using irradiated endothelial cultures.</a> Lab Invest. 36 (1), 18-25 (1977).</li><li>Gotlieb, A. I., Spector, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+into+an+in+vitro+experimental+wound:+a+comparison+of+porcine+aortic+endothelial+and+smooth+muscle+cells+and+the+effect+of+culture+irradiation.">Migration into an in vitro experimental wound: a comparison of porcine aortic endothelial and smooth muscle cells and the effect of culture irradiation.</a> Am J Pathol. 103 (2), 271-282 (1981).</li><li>Chen, Y. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+migration+chip+for+chemotaxis-based+microfluidic+selection+of+heterogeneous+cell+populations.">Single-cell migration chip for chemotaxis-based microfluidic selection of heterogeneous cell populations.</a> Sci Rep. 18 (5), 9980 (2015).</li><li>Yarrow, J. C., Periman, Z. E., Westwood, N. J., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+cell+migration+assay+using+scratch+wound+healing,+a+comparsion+of+image-based+readout+methods.">A high-throughput cell migration assay using scratch wound healing, a comparsion of image-based readout methods.</a> BMC Biotechnol. 4, 21 (2004).</li><li>Lauder, H., Frost, E. E., Hiley, C. R., Fan, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+the+repair+process+involved+in+the+repair+of+a+cell+monolayer+using+an+in+vitro+model+of+mechanical+injury.">Quantification of the repair process involved in the repair of a cell monolayer using an in vitro model of mechanical injury.</a> Angiogenesis. 2 (1), 67-80 (1998).</li><li>Yue, P. Y. K., Leung, E. P. Y., Mak, N. K., Wong, R. N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+method+for+quantifying+cell+migration/+wound+healing+in+96-well+plates.">A simplified method for quantifying cell migration/ wound healing in 96-well plates.</a> J. Biomol Screen. 15 (4), 427-433 (2010).</li><li>Yue, P. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidation+of+the+mechanisms+underlying+the+angiogenic+effects+of+ginsenoside+Rg(1)+in+vivo+and+in+vitro.">Elucidation of the mechanisms underlying the angiogenic effects of ginsenoside Rg(1) in vivo and in vitro.</a> Angiogenesis. 8 (3), 205-216 (2005).</li><li>Kwok, H. H., Chan, L. S., Poon, P. Y., Yue, P. Y., Wong, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ginsenoside-Rg1+induces+angiogenesis+by+the+inverse+regulation+of+MET+tyrosine+kinase+receptor+expression+through+miR-23a.">Ginsenoside-Rg1 induces angiogenesis by the inverse regulation of MET tyrosine kinase receptor expression through miR-23a.</a> Toxicol Appl Pharmacol. 287 (3), 276-283 (2015).</li></ol>

저자는 더 경쟁 재정적 이해 관계가 없음을 선언합니다.

우리의 세포 wounder는 기존의 세포 이동 분석의 문제를 해결하는 몇 가지 독특한 기능을 가지고 있습니다. 8 채널 기계적 셀 wounder은 고압 증기 멸균에 의해 멸균 될 수있는 긴 수명과 높은 등급의 스테인레스 (스틸 304)로 이루어진다. 시판 96- 웰 배양 판의 거의 모든 상업용 브랜드 때문에 조정 안내 막대이 기계적 세포 wounder 함께 사용될 수있다. 조정 안내 바의 설계는 찰 영역을 각 웰의 중앙에있?...

세포 이동은 배아 발생, 신경, 혈관 신생, 창상 치유, 점막 수리, 정상적인 발달 및 질병 상태 하에서 1 상피 간엽 - 천이 같은 세포 과정에서 중요한 역할을한다. 이것은 시그널링 분자 상호 작용, 세포 편광 세포 골격 재구성 매트릭스 리모델링 막 돌기 동적 세포 - 세포 부착 2 변조 등 다양한 인터페이스 및 세포 내 사건의 조정을 포함하는 복잡한 과정이다. 세포 이동을 연구함으로써, 세포 이동과 관련된 생화학 적 경로를 선도 화학 또는 생체 분자의 발견 및 확인을 결정할 수있다. 이러한 기본적인 공정은 이러한 질병 치료 약물 표적 같은 다양한 애플리케이션을위한 프록시로서 유리하게 사용될 수있다. 상처 분석법은 세포 이동 분석 (3)의 하나이다. 여기서,셀들의 각각의 샘플은 부분적으로 셀 영역을 덮는 마이그레이션하는 무 결함 영역을 생성하기 위해 기계적인 방법에 의해 제거 될 것이다. 특정 시점에서 회수의 비율을 모니터링한다. 샘플의 많은 수를 처리 할 때, 이러한 샘플 내에서 실험 비용 및 교차 오염 등의 문제가 문제가 될 수 있습니다. 많은 상용 도구와 분석은 세포 이동 4, 5, 6을 연구 할 수 있지만, 그들 중 많은 사람들이 비싸고 복잡한 장비가 필요하고 개선이 여전히 필요하다. 이러한 이유로, 8 채널 기계적 셀 wounder (도 1)가 개발되고있다. 우리 8 채널 기계적 셀 wounder은 상술 한 문제점을 해결하기에 몇몇의 고유 특징을 포함하고있다. 이것은 96- 웰 배양 플레이트 형식으로 분석 부상 / 세포 이동을 수행 할 수있는 유연성을 제공한다. 조정 번지iding 바 설계는 스크래치 영역을 각 웰의 중앙에 위치되는 것을 보장한다. wounder는 배양 접시의 다양한 브랜드에 적용되도록 또한, 가이드 바의 높이를 조정할 수있다. 마지막으로, 상처 조절 핀 디자인과 동시에 재현성 상처 (7, 8)을 달성하기 위해 배양 접시 표면과 피펫 팁의 짝수 접촉 할 수있다.

<table><tbody><tr><td>96-well cell culture plate</td><td>Nunc</td><td>167008</td><td>Other brands of 96-well cell culture plate can also be used</td></tr><tr><td>P10 pipette tips</td><td>Axygen</td><td>301-03-051</td><td>Short P10 pipette tip is more easy to create a clear wound</td></tr><tr><td>Wounder</td><td>R&amp;amp;P Technology Limited</td><td></td><td></td></tr><tr><td>Medium 199</td><td>Sigma</td><td>M2520-1L</td><td>For cell culture of human umbilical vein endothelial cells.&lt;br/&gt; Use appropirate culture medium and condition for other type of cells.</td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td>26140079</td><td>For cell culture of human umbilical vein endothelial cells.&lt;br/&gt; Use appropirate culture medium and condition for other type of cells.</td></tr><tr><td>Penicillin/Streptomycin</td><td>Gibco</td><td>15140122</td><td>For cell culture of human umbilical vein endothelial cells.&lt;br/&gt; Use appropirate culture medium and condition for other type of cells.</td></tr><tr><td>Heparin sodium salt from porcine intestinal mucosa</td><td>Sigma</td><td>H3393</td><td>For cell culture of human umbilical vein endothelial cells.&lt;br/&gt; Use appropirate culture medium and condition for other type of cells.</td></tr><tr><td>Gelatin from bovine skin</td><td>Sigma</td><td>G9391</td><td>For cell culture of human umbilical vein endothelial cells.&lt;br/&gt; Use appropirate culture medium and condition for other type of cells.</td></tr></tbody></table>

a device for performing cell migration/wound healing in a 96-well plate

셀 마이그레이션 / 분석 부상은 세포 이동 및 혈관 신생 및 종양 전이 등의 생물학적 과정을 공부하기 위해 일반적으로 사용되는 방법이다. 이 분석에서, 세포가 합류 단층을 형성하기 위해 재배 및 기계적 상처는 장치와 긁힘에 의해 생성된다. 다음에 무 결함 영역을 향해 세포의 이동 속도는 촬상에 의해 모니터링 될 수있다. 우리 8 채널 기계적 wounder은 세포 이동 분석법과 관련된 대부분의 문제를 해결하도록 설계된다. 첫째, 우리의 wounder 쉽게 고압 증기 멸균 또는 일반적인 소독제로 살균 할 수있다. 둘째, 개별 조절 핀도 선명하고 재현성 상처가 생성 될 수 있도록, 세포 배양 플레이트에 접촉 허용한다. 셋째, wounder 양측의 안내 바는 각각의 웰에서 일관된 상처 위치를 보장한다. 더 wounder의 더 나은 핸들링을 제공 할뿐만 아니라 크로스 contaminat을 최소화하기 위해 부상에 대한 일회용 플라스틱 피펫 팁의 사용이온. 결론적으로, 우리 셀 wounder은 표준 96 웰 배양 접시를 사용하여 세포 이동 분석을 수행하기위한 사용자 친화적이고 재생 가능한 장치 연구를 제공 할 수있다.

이 8 채널 기계적 wounder은 세포 이동 분석을 수행하기 위해 세포 단층을 긁어 구성된다. 그것은 특별한 교육을받지 않은 분 안에 96 웰 플레이트 세포 긁힘 분석을 수행 할 수있는 사용자 친화적 인 장치입니다. 이 wounder 약 600 ㎛의 균일 한 폭과 날카로운 가장자리 세포 단층에 감긴 영역을 도입 할 수있다 (도 5 및도 6). 상처의 폭은 피펫 팁의 브랜?...

Watch this Scientific Journal Video about A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate at JoVE.com

A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

여기서, 우리는 96 웰 세포 배양 접시에 반 처리량 세포 이동 분석을 수행 할 프로토콜을 제시한다. 이 프로토콜은 셀 단층에 일관성 스크래치 상처를 만들 수있는, 빠르고 간단하고 경제적 인 방법이다.

96 웰 플레이트에 세포 마이그레이션 / 상처 치유를 수행하기위한 장치

The cell migration/wounding assay is a commonly used method to study cell migration and other biological processes, such as angiogenesis and tumor ...

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JoVE Journal

Developmental Biology

10.4K 조회수.  Hong Kong Baptist University.  여기서, 우리는 96 웰 세포 배양 접시에 반 처리량 세포 이동 분석을 수행 할 프로토콜을 제시한다. 이 프로토콜은 셀 단층에 일관성 스크래치 상처를 만들 수있는, 빠르고 간단하고 경제적 인 방법이다. 

비디오: A Device for Performing Cell Migration/Wound Healing in a 96-Well Plate

Evaluation of the Cell Invasion and Migration Process: A Comparison of the Video Microscope-based Scratch Wound Assay and the Boyden Chamber Assay

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration and invasion abilities to understand how cancer cells disseminate, with the aim of developing new treatment strategies. Analysis of the cellular and molecular basis of these abilities has led to the characterization of cell mobility and the physicochemical properties of the cytoskeleton and cellular microenvironment. For many years, the Boyden chamber assay and the scratch wound assay have been the standard techniques to study cell invasion and migration. However, these two techniques have limitations. The Boyden chamber assay is difficult and time consuming, and the scratch wound assay has low reproducibility. Development of modern technologies, especially in microscopy, has increased the reproducibility of the scratch wound assay. Using powerful analysis systems, an &#34;in-incubator&#34; video microscope can be used to provide automatic and real-time analysis of cell migration and invasion. The aim of this paper is to report and compare the two assays used to study cell invasion and migration: the Boyden chamber assay and an optimized in vitro video microscope-based scratch wound assay.

This study reports two different methods for the analysis of cell invasion and migration: the Boyden chamber assay and the in vitro video microscope-based wound-healing assay. The protocols for these two experiments are described, and their benefits and disadvantages are compared.

셀 침공 및 마이그레이션 프로세스의 평가: 비디오 현미경-기반 스크래치의 비교 분석 결과 및 Boyden 챔버 분석 결과 상처

The invasion and migration abilities of tumor cells are main contributors to cancer progression and recurrence. Many studies have explored the migration ...

연구

암 연구학

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of cyclic nucleotides. By modulating intracellular cyclic nucleotide concentration, MRP4 can regulate multiple cyclic nucleotide-dependent cellular events including cell migration. Previously, we demonstrated that in the absence of MRP4, fibroblast cells contain higher levels of intracellular cyclic nucleotides and can migrate faster. To understand the underlying mechanisms of this finding, we adopted a direct yet multifaceted approach. First, we isolated potential interacting protein complexes of MRP4 from a MRP4 over-expression cell system using immunoprecipitation followed by mass-spectrometry. After identifying unique proteins in the MRP4 interactome, we utilized Ingenuity Pathway Analysis (IPA) to explore the role of these protein-protein interactions in the context of signal transduction. We elucidated the potential role of the MRP4 protein complex in cell migration and identified F-actin as a major mediator of the effect of MRP4 on cell migration. This study also emphasized the role of cAMP and cGMP as key players in the migratory phenomena. Using high-content microscopy, we performed cell-migration assays and observed that the effect of MRP4 on fibroblast migration is completely abolished by disruption of the actin cytoskeleton or inhibition of cAMP-dependent kinase A (PKA). To visualize signaling modulations in a migrating cell in real time, we utilized a FRET-based sensor for measuring PKA activity and found, the presence of more polarized PKA activity near the leading edge of migrating Mrp4-/- fibroblast, compared to Mrp4+/+fibroblasts. This in turn increased cortical actin formation and augmented the process of migration. Our approach enables identification of the proteins acting downstream to MRP4 and provides us with an overview of the mechanism involved in MRP4-dependent regulation of fibroblast migration.

MRP4 regulates various cyclic nucleotide-dependent signaling events including a recently elucidated role in cell migration. We describe a direct, but multifaceted approach to unravel the downstream molecular targets of MRP4 resulting in identification of a unique MRP4 interactome that plays key roles in the fine-tuned regulation of fibroblast migration.

방법은 섬유 아세포 마이그레이션의 규정에 Mrp4 함유 Macromolecular의 단지를 공부하기

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of ...

발생학

A Simple Migration/Invasion Workflow Using an Automated Live-cell Imager

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great significance. Migration assays provide basic insight of cell movement at a 2D level, whereas invasion assays are more physiologically relevant, mimicking in vivo cancer cell dislodgment from the original site and invading through the extracellular matrix. The current protocol provides a single workflow for migration and invasion assays. Together with the integrated automated microscopic camera for real-time HD images and built-in analysis module, it gives researchers a time-efficient, simple and reproducible experimental option. This protocol also includes substitutions for the consumables and alternative analysis methods for users to choose from.

The current protocol describes an integrated method investigating cancer cell migration and invasion on a single platform in real-time, providing an easily reproducible and time-efficient option to study cell mobility and morphology.

자동된 라이브 셀 이미징 사용 하 여 간단한 마이그레이션/침공 워크플로

Cancer cell mobility is crucial for the initiation of metastasis. Therefore, investigation of the cell movement and invasive capacity is of great ...

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 ...

생체공학

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to stimulate cell migration. Importantly, cell migration is a rate-limiting event during the wound-healing process to re-establish the integrity and normal function of tissue layers after injury. The advantage of this method over the classical assay, which is based on a manually made scratch in a cell monolayer, is the usage of special silicone culture inserts providing two compartments to create a cell-free pseudo-wound field with a well-defined width (500 &#956;m). In addition, due to an automated image analysis platform, it is possible to rapidly obtain quantitative data on the speed of wound closure and cell migration. More precisely, the effect of two frog-skin AMPs on the migration of bronchial epithelial cells will be shown. Furthermore, pretreatment of these cells with specific inhibitors will provide information on the molecular mechanisms underlying such events.

A Novel In Vitro Wound Healing Assay to Evaluate Cell Migration

Here, we present a protocol to evaluate the effect of peptides on the migration of bronchial epithelial cells. This method allows for the rapid and highly reproducible obtainment of quantitative data on the speed of cell migration and wound closure.

상처 치유 분석 결과 셀 마이그레이션 평가 하는 새로운 체 외에

The aim of this work is to show a novel method to evaluate the ability of some immunomodulatory molecules, such as antimicrobial peptides (AMPs), to ...

면역학 및 감염병학

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During In Vitro Wound Healing

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated experimental procedures, while potentially lacking in precision. In vitro culture of epithelial cell lines provides a suitable platform for researching the cell migratory behavior in wound healing and the impact of treatments on these cells. The physiology of epithelial cells is often studied in non-confluent conditions; however, this approach may not resemble natural wound healing conditions. Disrupting the epithelium integrity by mechanical means generates a realistic model, but may impede the application of molecular techniques. Consequently, microscopy based techniques are optimal for studying epithelial cell migration in vitro. Here we detail two specific methods, the artificial wound scratch assay and the artificial migration front assay, that can obtain quantitative and qualitative data, respectively, on the migratory performance of epithelial cells.

Microscopy Based Methods for the Assessment of Epithelial Cell Migration During  In Vitro Wound Healing

This manuscript describes how incision-like lesions made on cultured epithelial cell monolayers conveniently model wound healing in vitro, allowing for imaging by confocal or laser scanning microscopy, and which can provide high-quality quantitative and qualitative data for studying both cell behavior and the mechanisms involved in migration.

현미경 기반 방법을 생체 외에서 상처 치유 중 상피 세포 이동의 평가 대 한

Cell migration is a mandatory aspect for wound healing. Creating artificial wounds on research animal models often results in costly and complicated ...

의학

In vitro Cell Migration and Invasion Assays

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and inflammation. Methods to examine cell migration are very useful and important for a wide range of biomedical research such as cancer biology, immunology, vascular biology, cell biology and developmental biology. Here we use tumor cell migration and invasion as an example and describe two related assays to illustrate the commonly used, easily accessible methods to measure these processes. The first method is the cell culture wound closure assay in which a scratch is generated on a confluent cell monolayer. The speed of wound closure and cell migration can be quantified by taking snapshot pictures with a regular inverted microscope at several time intervals. More detailed cell migratory behavior can be documented using the time-lapse microscopy system. The second method described in this paper is the transwell cell migration and invasion assay that measures the capacity of cell motility and invasiveness toward a chemo-attractant gradient. It is our goal to describe these methods in a highly accessible manner so that the procedures can be successfully performed in research laboratories even just with basic cell biology setup.

In vitro Cell Migration and Invasion Assays

Commonly used, highly accessible methods for examining cell migration and invasion in vitro are described. The first method is the cell wound closure assay that measures cell motility. The second method is the transwell migration and invasion assay that assesses the chemotactic and invasive capacity of cells.

체외 세포 이동 및 내습 분석 실험에서

Migration is a key property of live cells and critical for normal development, immune response, and disease processes such as cancer metastasis and ...

생물학

Scratch Migration Assay and Dorsal Skinfold Chamber for In Vitro and In Vivo Analysis of Wound Healing

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective treatment. Appropriate in vitro and in vivo approaches are essential for the identification of new target molecules for drug treatments to improve the skin wound healing process. We identified the &#946;3 subunit of voltage-gated calcium channels (Cav&#946;3) as a potential target molecule to influence the wound healing in two independent assays, i.e., the in vitro scratch migration assay and the in vivo dorsal skinfold chamber model. Primary mouse embryonic fibroblasts (MEFs) acutely isolated from wild-type (WT) and Cav&#946;3-deficient mice (Cav&#946;3 KO) or fibroblasts acutely isolated from WT mice treated with siRNA to down-regulate the expression of the Cacnb3 gene, encoding Cav&#946;3, were used. A scratch was applied on a confluent cell monolayer and the gap closure was followed by taking microscopic images at defined time points until complete repopulation of the gap by the migrating cells. These images were analyzed, and the cell migration rate was determined for each condition. In an in vivo assay, we implanted a dorsal skinfold chamber on WT and Cav&#946;3 KO mice, applied a defined circular wound of 2 mm diameter, covered the wound with a glass coverslip to protect it from infections and desiccation, and monitored the macroscopic wound closure over time. Wound closure was significantly faster in Cacnb3-gene-deficient mice. Because the results of the in vivo and the in vitro assays correlate well, the in vitro assay may be useful for the high-throughput screening before validating the in vitro hits by the in vivo wound healing model. What we have shown here for wild-type and Cav&#946;3-deficient mice or cells might also be applicable for specific molecules other than Cav&#946;3.

Here, we present a protocol for an in vitro scratch assay using primary fibroblasts and for an in vivo skin wound healing assay in mice. Both assays are straightforward methods to assess in vitro and in vivo wound healing.

스크래치 마이그레이션 분석 및 등갈 피부 접기 챔버 에서 생체 외 및 상처 치유의 생체 내 분석

Impaired cutaneous wound healing is a major concern for patients suffering from diabetes and for elderly people, and there is a need for an effective ...

Quantifying Three-Dimensional Cell Migration Within and Into Granular Hydrogel Biomaterials

Granular hydrogel scaffolds hold significant potential in regenerative medicine, functioning either as carriers for cell delivery or as interfaces for tissue integration. This article introduces two novel approaches for quantifying cell migration within and into granular hydrogels, highlighting the distinct applications of these scaffolds. First, a cell monolayer interface assay that simulates tissue growth into granular hydrogels for integration purposes is presented. Second, a spheroid-based assay is described, designed to track cell movement within the hydrogel matrix, specifically suited for applications involving cell delivery. Both methods enable precise and controlled measurements of cell migration, providing a comprehensive toolkit for researchers utilizing granular hydrogel scaffolds. The motivation for these methods stems from the need for tailored control over cell migration within the scaffold to align with specific applications. By optimizing and standardizing these quantification techniques, researchers can iteratively refine granular hydrogel properties, ensuring their effectiveness in diverse regenerative medicine contexts. This robust set of quantitative tools offers new opportunities to enhance granular hydrogel scaffolds, advancing their use in both cell delivery and tissue integration applications.

A protocol for the quantitative evaluation of 3D cell migration within and into the interface of granular hydrogels is presented here.

Quantifying three-dimensional cell migration within and into granulular hydrogel biomaterials(과립 하이드로겔 생체 물질로의 3차원 세포 이동 정량화)

Granular hydrogel scaffolds hold significant potential in regenerative medicine, functioning either as carriers for cell delivery or as interfaces for ...

An Innovative 3D-Printed Insert Designed to Enable Straightforward 2D and 3D Cell Cultures

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of conditioned media remains time-consuming and far from physiological and pathological conditions. Although a few models of co-culture are commercially available, they remain restricted to specific assays and are mostly for two types of cells. 
Here, 3D-printed inserts are used that are compatible with numerous functional assays. The insert allows the separation of one well of a 6-well plate into four compartments. A wide range of combinations can be set. Moreover, windows are designed in each wall of the compartments so that potential intercellular communication between every compartment is possible in the culture medium in a volume-dependent manner. For example, paracrine intercellular communication can be studied between four cell types in monolayer, in 3D (spheroids), or by combining both. In addition, a mix of different cell types can be seeded in the same compartment in 2D or 3D (organoids) format. The absence of a bottom in the 3D-printed inserts allows the usual culture conditions on the plate, possible coating on the plate containing the insert, and direct visualization by optical microscopy. The multiple compartments provide the possibility to collect different cell types independently or to use, in each compartment, different reagents for RNA or protein extraction. In this study, a detailed methodology is provided to use the new 3D-printed insert as a co-culture system. To demonstrate several capacities of this flexible and simple model, previously published functional assays of cell communication were performed in the new 3D-printed inserts and were demonstrated to be reproducible. The 3D-printed inserts and the conventional cell culture using conditioned media led to similar results. In conclusion, the 3D-printed insert is a simple device that can be adapted to numerous models of co-cultures with adherent cell types.

In this paper, a newly designed 3D-printed insert is presented as a model of co-culture and validated through the study of the paracrine intercellular communication between endothelial cells and keratinocytes.

간단한 2D 및 3D 세포 배양이 가능하도록 설계된 혁신적인 3D 프린팅 인서트

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of ...