Bu eser Ulusal Sağlık Enstitüleri tarafından (NIMHD-G12MD007581) Jackson Devlet Üniversitesi ve savunma, fikir Ödülü 11-1-0205 ABD Bakanlığı çevre sağlığı için RCMI merkezinden destek verdi.

Not: Bu protokol McArdle vd. anlatılan 11. bir zaman çizelgesi şekil 1' de verilmiştir.
1. kültür plaka hazırlanması
<ol><li>Bir 10 mm cam alt 1 mL 1 M hidroklorik asit (HCl) ile iyi 15dk için cam alt hydrophobicity alt içeren 35-mm kültür bulaşık yıka.</li>
	<li>Kültür plaka fosfat tampon salin (PBS) tüm HCl kurtulmak için 1 mL ile iki kez yıkayın.</li>
	<li>Kültür plaka 1 mL % 70 etanol ile iki kez yıkayın.</li>
	<li>Hücre belirli kültür orta 1 mL ile kültür plaka durulama (burada, kullanım α-MEM % 10 ısı ile desteklenmiş FBS ve % 1 esansiyel olmayan amino asitler inaktive olur). Not: Kültür ortamı içeren tedavi kültür plaka CO2 kuluçka 37 ° C'de bir nemlendirici ile ertesi güne kadar saklanabilir.</li>
	<li>(MSCs ve MCF-7 ortak kültür) hücreleri tedavi cam-alt içinde de 35 mm kültür çanak % 100 confluency için büyümek. 35.000 MSC hücreleri ve 75.000 MCF7 hücre 24 h için ortak kültür.</li>
</ol>2. kollajen jel hazırlanması
<ol><li>Kollajen temas üzerine polimerizasyon önlemek için kollajen jel dondurucuya pipetting için kullanılan micropipette ipuçları saklayın. Not: Bu en önemli adımdır.</li>
	<li>Bir birim belirlemek sıçan kuyruk kollajen ben kullanılmak üzere kollajen hisse senedi toplama üzerinde tabanlı. Kollajen jel 100 µL bu tahlil için hazırlayın. Not: Ben stok sıçan kuyruk kollajen yüksek yoğunlukta daha iyi çalışır. 10 x kollajen buna göre sulandırmak için kullanılmak üzere Dulbecco'nın modifiye kartal orta hacmi belirlemek.</li>
	<li>Birleştirmek buz sıçan kuyruk kollajen üzerinde ben (3.8 µg/mL hisse senedi), 10 x Dulbecco'nın modifiye kartal orta ve 1 x hücre kültür % 2 fetal sığır serum ve kollajen 2.4 mg/ml nihai bir konsantrasyon için uygun chemoattractant ile takıma orta boy.</li>
	<li>Kollajen jel nötralize etmek için sodyum bikarbonat çözüm karışımı %4,5 ekleyin. Not: Sodyum bikarbonat çözüm eklendi hacmi diğer reaktifleri tam pH göre değişebilir. Bu karışımı tarafsız çözüm sağlamak veya bir pH göstergesi kültür ortamında kullanmak için pH şeritler ile test etmek en iyisidir.</li>
	<li>80 µL kollajen karışımı cam-alt de kültür çanak içindeki hücreleri üzerinde yatıyordu.</li>
	<li>Kollajen jel 30 dk 37 ° C'de bir nemlendirici ile CO2 kuluçka için polimerize izin</li>
	<li>Kollajen jel hücre orta düşük serum (% 2) ile 2 mL ile kapak ve CO2 kuluçka bir nemlendirici ile 24, 48 veya 72 h 37 ° C'de hücreler kuluçkaya. Uyarı: Plaka cam alttan ayırma kollajen jel önlemek için itina ile ele alınmalıdır.</li>
</ol>3. hazırlık kollajen örneği jel 3.8 µg /mL bir sıçan kuyruk kollajen stokunun kullanarak
<ol><li>Buz üzerinde birleştirmek 114.5 µL sıçan kuyruk kollajen, 10 DMEM x 16,6 µL ve kültür chemoattractant içeren orta 33.1 µL.</li>
	<li>Kollajen karışımı sodyum bikarbonat 14,0 µL ile etkisiz hale getirin.</li>
	<li>Adım 2,5 olduğu gibi devam edin.</li>
</ol>4. hücreleri görüntüleme
<ol><li>Yavaşça medya çanak çıkarın.</li>
	<li>Yavaşça jel PBS 1 mL ile yıkayın.</li>
	<li>%4 paraformaldehyde 15dk için 1 mL ile hücreleri tamir.</li>
	<li>Hücreleri iki kez PBS 1 mL ile yıkayın.</li>
	<li>DAPI çözüm (100 ng/mL) oda sıcaklığında 20 dk için hücrelerle leke.</li>
	<li>Hücreleri farklı yerlerde confocal bir mikroskop kullanarak ve 400 µm z-yığın görüntüleri her iki yerde de 16 µm adımları alarak görüntü. Kullanılan mikroskop kullanarak bir beyaz ışık, ark uyarma kaynak confocal görüntüleme sağlar birim tarama bir diski var. 0,75 sayısal diyafram ile kullanılan bir 20 X lens kullanabilirsiniz.</li>
	<li>Görüntüleri yeniden oluşturma.<ol>
<li>Görüntüler için verilen jel (yaklaşık 25 resim) açın ve görüntü → yığınları → görüntüler yığmak için tıklatarak görüntü yığını oluşturun. İlk görüntü jel altına denk (z = 0 µm), z yönde ilk adım için ikinci görüntü denk (z = 16 µm), üçüncü resim z yönünde ikinci adımına tekabül (z 32 µm =). Her çekirdek her görüntü derinlikte değerlendirmek ve hangi keskin odak olarak işgal derinliği belirli hücre için çekirdek yapıldı derinliği belirleyebilirsiniz.</li>
	</ol>
</li>
</ol>

<ol>
	<li>Friedl, P., Brocker, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biology+of+cell+locomotion+within+three-dimensional+extracellular+matric.">The biology of cell locomotion within three-dimensional extracellular matric.</a> Cell Mol Life Sci. 57, 41-64 (2000).</li><li>Bissell, M. J., Rizki, A., Mian, I. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+architecture:+the+ultimate+regulator+of+breast+epithelial+function.">Tissue architecture: the ultimate regulator of breast epithelial function.</a> Curr Opin Cell Biol. 15, 753-762 (2003).</li><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, 362-374 (2003).</li><li>Yarrow, J. C., Perlman, 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+comparison+of+image-based+readout+methods.">A high-throughput cell migration assay using scratch wound healing, a comparison of image-based readout methods.</a> BMC Biotechnol. 4, 21 (2004).</li><li>Wolf, K., 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=Compensation+mechanism+in+tumor+cell+migration:+mesenchymal-amoeboid+transition+after+blocking+of+pericellular+proteolysis.">Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis.</a> J Cell Biol. 160, 267-277 (2003).</li><li>Sahai, E., Marshall, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differing+modes+of+tumour+cell+invasion+have+distinct+requirements+for+Rho/ROCK+signalling+and+extracellular+proteolysis.">Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis.</a> Nat Cell Biol. 5, 711-719 (2003).</li><li>Kam, Y., Guess, C., Estrada, L., Weidow, B., Quaranta, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+circular+invasion+assay+mimics+in+vivo+invasive+behavior+of+cancer+cell+lines+and+distinguishes+single-cell+motility+in+vitro.">A novel circular invasion assay mimics in vivo invasive behavior of cancer cell lines and distinguishes single-cell motility in vitro.</a> BMC Cancer. 8, 198 (2008).</li><li>Staton, C. A., Reed, M. W., Brown, N. 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+critical+analysis+of+current+in+vitro+and+in+vivo+angiogenesis+assays.">A critical analysis of current in vitro and in vivo angiogenesis assays.</a> Int J Exp Pathol. 90, 195-221 (2009).</li><li>Poujade, M., 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=Collective+migration+of+an+epithelial+monolayer+in+response+to+a+model+wound.">Collective migration of an epithelial monolayer in response to a model wound.</a> Proc Natl Acad Sci USA. 104, 15988-15993 (2007).</li><li>Noubissi, F. K., Harkness, T., Alexander, C. M., Ogle, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptosis-induced+cancer+cell+fusion:+a+mechanism+of+breast+cancer+metastasis.">Apoptosis-induced cancer cell fusion: a mechanism of breast cancer metastasis.</a> FASEB J. 29, 4036-4045 (2015).</li><li>McArdle, T. J., Ogle, B. M., Noubissi, F. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+In+Vitro+Inverted+Vertical+Invasion+Assay+to+Avoid+Manipulation+of+Rare+or+Sensitive+Cell+Types.">An In Vitro Inverted Vertical Invasion Assay to Avoid Manipulation of Rare or Sensitive Cell Types.</a> J Cancer. 7, 2333-2340 (2016).</li><li>Noubissi, F. K., Ogle, B. 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Yazarlar rakip hiç ilgi var olduğunu ilan etti.

Burada, nadir ve/veya onların microenvironment bağımlı olan hücreleri de dahil olmak üzere hücre türü için uygun olan bir basit ters dikey işgali tahlil açıklayın. Bu 3D işgali tahlil hücreler içinde onların orijinal microenvironment kalır ve bir chemoattractant içeren kollajen jel tarafından karşılanmaktadır. Hücreler göç ve kollajen istila. Bu tahlil, hücreler lekeli ve kolayca jel içinde izlenir. Basitlik ve tekrarlanabilirlik Bu testin bir 3D sisteminde kanser hücre göç ve işgali içi...

Hücre hareketliliği hücreleri bir normal gelişim ve fizyolojik süreçtir. Ancak, değişiklikleri desen ve üretilen hücre göç ve istila için inflamatuar hastalıkları ve kanser metastaz1,2,3gibi patolojik koşullarla ilişkilidir. Metastaz tartışmalı Kanserinde en kötü anlaşılır yönüdür. Metastaz için kanser hücrelerinin ekstra hücresel matris (ECM) aşağılamak, yerel doku ve intravasate istila. Bir kez dolaşımda, kanser hücrelerinin uzak siteye yaymak, extravasate ve ikincil tümörler oluşturur. Bu nedenle, yetenek hücreleri ECM aşağılamak için göç ve istila metastatik potansiyellerini ilgilidir. Çözümlemek ve hücrelerin göç ve işgal yeteneklerini ölçmek için farklı yaklaşımlar geliştirilmiştir. En çok kullanılan yaklaşımlar tahlil, hızlandırılmış mikroskobu ve Boyden odası tahlil şifa yara içerir. Yara iyileşme tahlil4 ve zaman atlamalı mikroskobu hücre göç içine ön fikir verecek basit ve kolay deneyleri var. Ancak, her iki hücre içinde vivovar 3D çevre yansıtmıyor 2D deneyleri vardır. Hücreleri farklı bir 2D bir5,-6karşılaştırıldığında 3D ortamına yanıt çalışmalar göstermiştir. Ayrıca, yara iyileştirici deneyleri çoğaltmak ve nicel sonuçları7,8,9verim için zordur. Tahlil şifa yara 3D bir değişiklik olduğunu, hücre dışlama bölgesi tahlil daha fizyolojik ilgili bir tahlil ama hücre içinde sayı hücre füzyon olaylar10,11 kaynaklanan hibrit hücreler gibi düşük için uygun değil .
Boyden odası tahlil için hücresel çalışmalar ilgili microenvironments sağlar 3 boyutlu bir tahlil olduğunu. Ancak, hücrelerin kendi özgün microenvironment kaldırılacak ve geçirmek ve aşağıya doğru chemoattractant içeren orta istila Boyden tahlil sisteminin üst odaya yatırılması için gerekli. Bu 3D yaklaşım onların microenvironment ve/veya sınırlı sayı10,11,12güvenlik açığı bulunan hücrelerin göç ve işgal yeteneği analiz uygun değildir. Hücre göç ve işgal çözümlemek için yeni bir yaklaşım mikrosıvısal degrade odası tahlil13yaşında. Her ne kadar uygun ve güvenilir geçiş ve işgal çalışmaları, bu tahlil daha pahalı ve teknik olarak tipik bir laboratuvar için zor olabilir. Biz burada hücreleri için onların microenvironment duyarlı da dahil olmak üzere birçok hücre tipleri ile uyumlu ve/veya sınırlı sayıda bir basit ters dikey işgali tahlil tanımlamak. Bu tahlil Hooper ve ark. tarafından önerilen iletişim kuralından adapte oldu 14. bu tahlil, hücreleri kendi özgün microenvironment ve onların geçiş kalır ve işgal chemoattractant11içeren kollajen jel içinde yukarı doğru ilerlerken izlenir. Bu tahlil tekrarlanabilir ve yapılmış en iyi duruma getirilmiş ve 35 mm kültür tabakta doğrulanmış. Farklı hücre kültür boyutları, farklı matrisler veya gücü yapışma ve farklı chemoattractants de dahil olmak üzere farklı tahlil şartlarına adapte. Bu tahlil ilaç bulma işleminin başlangıç tarama için bir vitro platform olarak hizmet verebilir.

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">MatTek P35G-1.5-10C</td>
			<td style="width:269px;">MatTek Corporation, Ashland, MA</td>
			<td style="width:151px;">P35G-1.5-10-C</td>
			<td style="width:232px;">mattek glass bottom dishes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rat tail collagen I</td>
			<td>Corning, Corning, NY, USA</td>
			<td align="right">354236</td>
			<td>Collagen gel</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hydrochloric acid</td>
			<td>Sigma-Aldrich, St. Louis, MO 63178 USA</td>
			<td>H1758</td>
			<td>HCl</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffered saline</td>
			<td>Thermo Fisher Scientific Inc., Whaltham, MA, USA</td>
			<td align="right">10010023</td>
			<td>PBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10X Dulbecco&#39;s Modified Eagle&#39;s Medium</td>
			<td>Sigma-Aldrich, St. Louis, MO 63178 USA</td>
			<td>D2429</td>
			<td>10X DMEM</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium bicarbonate</td>
			<td>Sigma-Aldrich, St. Louis, MO 63178 USA</td>
			<td>S5761</td>
			<td>NaHCO3</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal Fluorescent microscope</td>
			<td>Olympus America, Center Valley, PA, USA</td>
			<td>Olympus IX81</td>
			<td>Confocal&#160; microscope</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Defined Fetal Bovine Serum</td>
			<td>GE Healthcare Life Sciences HyClone Laboratories, Utah, USA</td>
			<td>SH30070.03</td>
			<td>FBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ti:Sapphire multi-photon laser scanning microscope</td>
			<td>Prarie Technologies, Sioux Falls, SD, USA</td>
			<td>40x NA 0.8 objective</td>
			<td>MPLSM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imaris software</td>
			<td>Bitplane Inc. Zurich, CH</td>
			<td>Imaris 7.5.2</td>
			<td>Imaris software</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DAPI</td>
			<td style="width:269px;">Sigma-Aldrich, St. Louis, MO 63178, USA</td>
			<td>D9542</td>
			<td>DAPI</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde</td>
			<td>Thermo Fisher Scientific Inc., Whaltham, MA, USA</td>
			<td align="right">50980487</td>
			<td>PFA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#945;-minimum essential medium</td>
			<td>Sigma-Aldrich, St. Louis, MO 63178, USA</td>
			<td>M0894</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MDA-MB-231</td>
			<td>ATCC; Manassas, VA, USA</td>
			<td>HBT-22</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MSC</td>
			<td>Trivedi and Hematti, 2007</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">20X lens UPLAPO</td>
			<td>Olympus America, Center Valley, PA, USA</td>
			<td>36-064</td>
			<td>Olympus UPLSAPO 20X Objective</td>
		</tr>
	</tbody>
</table>

moving upwards: a simple and flexible in vitro three-dimensional invasion assay protocol

3D işgali deneyleri geliştirilmiştir rağmen onların microenvironment bütünlüğünü etkilemeden hücreleri çalışmaya meydan kalır. Boyden odası gerektiren hücreleri özgün kültür konumdan yerlerinden ve yeni bir ortama taşınmış gibi geleneksel 3D deneyleri. Sadece bu microenvironment için içsel hücresel süreçler bozmak yok, ama bu kez bir hücre kaybolmasına neden. Bu nadir, ya da onların microenvironment için son derece hassas hücreler ile ilgili özellikle zorlu sorunlardır. Burada, her iki endişelerini önler bir 3D işgali tahlil gelişimi açıklayın. Bu tahlil, hücreler de içinde küçük bir kaplama ve bir chemoattractant içeren bir ECM Matris hücreleri koydu. Bu herhangi bir hücre öteleme gerektirir ve hücreleri matrisin yukarı doğru istila sağlar. Bu tahlil, hücre işgali gibi hücre morfolojisi kollajen jel içinde değerlendirilebilir. Bu tahlil kullanarak, nadir ve hassas hücreler invaziv kapasitesini karakterize; füzyon meme kanseri hücreleri MCF7 ve Mezenkimal/multipotent arasında ortaya çıkan melez hücreleri (MSCs) kök/stroma hücreleri.

Biz-si olmak kullanılmış bir 35 mm kültür yemek bir cam alt ve sıçan kuyruk kollajen ile ben geliştirmek ve vitro 3D istila en iyi duruma getirmek için tahlil protokolü. Bu tahlil, bu meme kanseri hücreleri MCF7 ve MSC gösterdik kullanarak hücreleri kollajen jel 0,04 ± 1.8 µm ve 41,3 ± 76.0 µm ortalama bir mesafe içine sırasıyla 48 saat sonra işgal ne zaman IGF1 (18,6 ng/mL) chemoattractant (Şekil 2) kullanılmıştır. Daha da ...

Watch this Scientific Journal Video about Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol at JoVE.com

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Bu iletişim kuralı hücreleri hücre microenvironment koruma sırasında bir üç boyutlu ortamda göç ve işgal yeteneklerini ölçmek için kullanılabilir bir ters dikey işgali tahlil açıklar. Bu tahlil nadir ve/veya hassas hücreler için uygundur.

Yukarı doğru hareket: Bir basit ve esnek Vitro üç boyutlu işgali tahlil iletişim kuralı

Although 3D invasion assays have been developed, the challenge remains to study cells without affecting the integrity of their microenvironment. ...

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Cancer Research

Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

6.0K Görüntüleme.  University of Minnesota - Twin Cities. Bu iletişim kuralı hücreleri hücre microenvironment koruma sırasında bir üç boyutlu ortamda göç ve işgal yeteneklerini ölçmek için kullanılabilir bir ters dikey işgali tahlil açıklar. Bu tahlil nadir ve/veya hassas hücreler için uygundur.

Video: Moving Upwards: A Simple and Flexible In Vitro Three-dimensional Invasion Assay Protocol

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is exposed to in vivo. Solid tumors grow not as a sheet attached to plastic, but instead as a collection of clonal cells in a three-dimensional (3D) space interacting with their neighbors, and with distinct spatial properties such as the disruption of normal cellular polarity. These interactions cause 3D-cultured cells to acquire morphological and cellular characteristics which are more relevant to in vivo tumors. Additionally, a tumor mass is in direct contact with other cell types such as stromal and immune cells, as well as the extracellular matrix from all other cell types. The matrix deposited is comprised of macromolecules such as collagen and fibronectin.
In an attempt to increase the translation of research findings in oncology from bench to bedside, many groups have started to investigate the use of 3D model systems in their drug development strategies. These systems are thought to be more physiologically relevant because they attempt to recapitulate the complex and heterogeneous environment of a tumor. These systems, however, can be quite complex, and, although amenable to growth in 96-well formats, and some now even in 384, they offer few choices for large-scale growth and screening. This observed gap has led to the development of the methods described here in detail to culture tumor spheroids in a high-throughput capacity in 1536-well plates. These methods represent a compromise to the highly complex matrix-based systems, which are difficult to screen, and conventional 2D assays. A variety of cancer cell lines harboring different genetic mutations are successfully screened, examining compound efficacy by using a curated library of compounds targeting the Mitogen-Activated Protein Kinase or MAPK pathway. The spheroid culture responses are then compared to the response of cells grown in 2D, and differential activities are reported. These methods provide a unique protocol for testing compound activity in a high-throughput 3D setting.

Across a wide variety of disease indications, more physiologically relevant models are being developed and implemented into drug discovery programs. The new model system described here demonstrates how three-dimensional tumor spheroids can be cultured and screened in a high-throughput 1536-well plate-based system to search for new oncology drugs.

Yüksek işlem hacmi üç boyutlu tümör pulcuklarının nesil uyuşturucu taraması

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

Kanser Araştırmaları

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Gerçek zamanlı hafiye-in Vitro tümör hücre Apoptosis içinde CD8 tarafından indüklenen+ T hücreleri Myeloid hücrelerin tümör infiltre bağışıklık baskılayıcı işlevlerini çalışmaya

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

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.

Otomatik bir canlı hücre Imager kullanarak basit geçiş/işgali iş akışı

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

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Üç boyutlu kemik hücre dışı matriks modeli osteosarkom için

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Fully Human Tumor-based Matrix in Three-dimensional Spheroid Invasion Assay

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor microenvironment. To obtain more reliable in vitro results, several three-dimensional (3D) cell culture assays have been introduced. These assays allow cancer cells to interact with the extracellular matrix. This interaction affects cell behavior, such as proliferation and invasion, as well as cell morphology. Additionally, this interaction could induce or suppress the expression of several pro- and anti-tumorigenic molecules. Spheroid invasion assay was developed to provide a suitable 3D in vitro method to study cancer cell invasion. Currently, animal-derived matrices, such as mouse sarcoma-derived matrix (MSDM) and rat tail type I collagen, are mainly used in the spheroid invasion assays. Taking into consideration the differences between the human tumor microenvironment and animal-derived matrices, a human myoma-derived matrix (HMDM) was developed from benign uterus leiomyoma tissue. It has been shown that HMDM induces migration and invasion of carcinoma cells better than MSDM. This protocol provided a simple, reproducible, and reliable 3D human tumor-based spheroid invasion assay using the HMDM/fibrin matrix. It also includes detailed instructions on imaging and analysis. The spheroids grow in a U-shaped ultra-low attachment plate within the HMDM/fibrin matrix and invade through it. The invasion is daily imaged, measured, and analyzed using ilastik and Fiji ImageJ software. The assay platform was demonstrated using human laryngeal primary and metastatic squamous cell carcinoma cell lines. However, the protocol is suitable also for other solid cancer cell lines.

Tumor microenvironment is an essential part of cancer growth and invasion. To mimic carcinoma progression, a biologically relevant human matrix is needed. This protocol introduces an improvement for the in vitro three-dimensional spheroid invasion assay by applying a human leiomyoma-based matrix. The protocol also introduces a computer-based cell invasion analysis.

Üç boyutlu Küroid Istilası tahlil tam ınsan tümör tabanlı matris

Two-dimensional cell culture-based assays are commonly used in in vitro cancer research. However, they lack several basic elements that form the tumor ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

In vitro assay tümör çalışma-macrophage etkileşim

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

Kanser Ilişkili fibroblastlar farklılaşma ve Invazyon In vitro etütler için daha fizyolojik bir sistem olarak bir 3D Küroid modeli

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

Hasta Prostat Kanseri Kemik Metastazı Örneklerinden ve Ksenogreftlerinden Elde Edilen Üç Boyutlu (3D) Organoidlerin Oluşturulması ve Analizi

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

Kolon Kanseri Kök Hücrelerini İncelemek İçin Üç Boyutlu Bir Küresel Model

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

Kollajen Bazlı İskeleler Kullanılarak Nöroblastomun Üç Boyutlu İn Vitro Biyomimetik Modeli

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...