Bu çalışma, Saint Louis Üniversitesi tarafından Dr. Silviya P Zustiak'a sağlanan başlangıç fonlarının yanı sıra Saint Louis Üniversitesi'ndeki Henry ve Amelia Nasrallah Sinirbilim Merkezi'nden Dr. Silviya P Zustiak'a verilen bir tohum hibesi ile finanse edildi.

Fabrikasyon ve ardından tümör sferoidlerinin PEG hidrojellerinde 3D kapsüllenmesi

<ol>
	<li>Hirschhaeuser, F., 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=Multicellular+tumor+spheroids:+an+underestimated+tool+is+catching+up+again.">Multicellular tumor spheroids: an underestimated tool is catching up again.</a> Journal of Biotechnology. 148 (1), 3-15 (2010).</li><li>Costa, E. C., de Melo-Diogo, D., Moreira, A. F., Carvalho, M. P., Correia, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroids+formation+on+non-adhesive+surfaces+by+liquid+overlay+technique:+Considerations+and+practical+approaches.">Spheroids formation on non-adhesive surfaces by liquid overlay technique: Considerations and practical approaches.</a> Biotechnology Journal. 13 (1), 1700417 (2018).</li><li>Li, Y., Kumacheva, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel+microenvironments+for+cancer+spheroid+growth+and+drug+screening.">Hydrogel microenvironments for cancer spheroid growth and drug screening.</a> Science Advances. 4 (4), eaas8998 (2018).</li><li>Kamatar, A., Gunay, G., Acar, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+and+synthetic+biomaterials+for+engineering+multicellular+tumor+spheroids.">Natural and synthetic biomaterials for engineering multicellular tumor spheroids.</a> Polymers. 12 (11), 2506 (2020).</li><li>Wang, C., Tong, X., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineered+3D+brain+tumor+model+to+elucidate+the+effects+of+matrix+stiffness+on+glioblastoma+cell+behavior+using+PEG-based+hydrogels.">Bioengineered 3D brain tumor model to elucidate the effects of matrix stiffness on glioblastoma cell behavior using PEG-based hydrogels.</a> Molecular Pharmaceutics. 11 (7), 2115-2125 (2014).</li><li>Nakod, P. S., Kim, Y., Rao, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+biomimetic+hyaluronic+acid+hydrogels+to+investigate+glioblastoma+stem+cell+behaviors.">Three-dimensional biomimetic hyaluronic acid hydrogels to investigate glioblastoma stem cell behaviors.</a> Biotechnology and Bioengineering. 117 (2), 511-522 (2020).</li><li>Xiao, W., 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=Bioengineered+scaffolds+for+3D+culture+demonstrate+extracellular+matrix-mediated+mechanisms+of+chemotherapy+resistance+in+glioblastoma.">Bioengineered scaffolds for 3D culture demonstrate extracellular matrix-mediated mechanisms of chemotherapy resistance in glioblastoma.</a> Matrix Biology. 85, 128-146 (2020).</li><li>Pedron, S., 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=Hyaluronic+acid-functionalized+gelatin+hydrogels+reveal+extracellular+matrix+signals+temper+the+efficacy+of+erlotinib+against+patient-derived+glioblastoma+specimens.">Hyaluronic acid-functionalized gelatin hydrogels reveal extracellular matrix signals temper the efficacy of erlotinib against patient-derived glioblastoma specimens.</a> Biomaterials. 219, 119371 (2019).</li><li>Hill, L., Bruns, J., Zustiak, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel+matrix+presence+and+composition+influence+drug+responses+of+encapsulated+glioblastoma+spheroids.">Hydrogel matrix presence and composition influence drug responses of encapsulated glioblastoma spheroids.</a> Acta Biomaterialia. 132, 437-447 (2021).</li><li>Chen, J. -. W. E., 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=Crosstalk+between+microglia+and+patient-derived+glioblastoma+cells+inhibit+invasion+in+a+three-dimensional+gelatin+hydrogel+model.">Crosstalk between microglia and patient-derived glioblastoma cells inhibit invasion in a three-dimensional gelatin hydrogel model.</a> Journal of Neuroinflammation. 17 (1), 346 (2020).</li><li>Thakuri, P. S., Liu, C., Luker, G. D., Tavana, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials-based+approaches+to+tumor+spheroid+and+organoid+modeling.">Biomaterials-based approaches to tumor spheroid and organoid modeling.</a> Advanced Healthcare Materials. 7 (6), 1700980 (2018).</li><li>Shin, S., 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=Alginate-marine+collagen-agarose+composite+hydrogels+as+matrices+for+biomimetic+3D+cell+spheroid+formation.">Alginate-marine collagen-agarose composite hydrogels as matrices for biomimetic 3D cell spheroid formation.</a> RSC Advances. 6 (52), 46952-46965 (2016).</li><li>Pradhan, S., Clary, J. M., Seliktar, D., Lipke, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+three-dimensional+spheroidal+cancer+model+based+on+PEG-fibrinogen+hydrogel+microspheres.">A three-dimensional spheroidal cancer model based on PEG-fibrinogen hydrogel microspheres.</a> Biomaterials. 115, 141-154 (2017).</li><li>Imaninezhad, M., Hill, L., Kolar, G., Vogt, K., Zustiak, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Templated+macroporous+polyethylene+glycol+hydrogels+for+spheroid+and+aggregate+cell+culture.">Templated macroporous polyethylene glycol hydrogels for spheroid and aggregate cell culture.</a> Bioconjugate Chemistry. 30 (1), 34-46 (2018).</li><li>Mirab, F., Kang, Y. J., Majd, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+characterization+of+size-controlled+glioma+spheroids+using+agarose+hydrogel+microwells.">Preparation and characterization of size-controlled glioma spheroids using agarose hydrogel microwells.</a> PLoS One. 14 (1), e0211078 (2019).</li><li>Razian, G., Yu, Y., Ungrin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+large+numbers+of+size-controlled+tumor+spheroids+using+microwell+plates.">Production of large numbers of size-controlled tumor spheroids using microwell plates.</a> Journal of Visualized Experiments: JoVE. 81, e50665 (2013).</li><li>Timmins, N. E., Nielsen, L. K., Hauser, H., Fussenegger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Generation of Multicellular Tumor Spheroids by the Hanging-Drop Method. 140, (2007).</li><li>Zhao, L., 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=A+3D+printed+hanging+drop+dripper+for+tumor+spheroids+analysis+without+recovery.">A 3D printed hanging drop dripper for tumor spheroids analysis without recovery.</a> Scientific Reports. 9 (1), 19717 (2019).</li><li>Amaral, R. L., Miranda, M., Marcato, P. D., Swiech, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+analysis+of+3D+bladder+tumor+spheroids+obtained+by+forced+floating+and+hanging+drop+methods+for+drug+screening.">Comparative analysis of 3D bladder tumor spheroids obtained by forced floating and hanging drop methods for drug screening.</a> Frontiers in Physiology. 8, 605 (2017).</li><li>Gencoglu, M. F., 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=Comparative+study+of+multicellular+tumor+spheroid+formation+methods+and+implications+for+drug+screening.">Comparative study of multicellular tumor spheroid formation methods and implications for drug screening.</a> ACS Biomaterials Science &amp; Engineering. 4 (2), 410-420 (2018).</li><li>Zhang, C., Liu, C., Zhao, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+brain+tissue+based+on+microstructure.">Mechanical properties of brain tissue based on microstructure.</a> Journal of the Mechanical Behavior of Biomedical Materials. 126, 104924 (2021).</li><li>Alifieris, C., Trafalis, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioblastoma+multiforme:+Pathogenesis+and+treatment.">Glioblastoma multiforme: Pathogenesis and treatment.</a> Pharmacology &amp; Therapeutics. 152, 63-82 (2015).</li><li>Zustiak, S. P., Leach, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrolytically+degradable+poly+(ethylene+glycol)+hydrogel+scaffolds+with+tunable+degradation+and+mechanical+properties.">Hydrolytically degradable poly (ethylene glycol) hydrogel scaffolds with tunable degradation and mechanical properties.</a> Biomacromolecules. 11 (5), 1348-1357 (2010).</li><li>Zustiak, S. P., Wei, Y., Leach, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-hydrogel+interactions+in+tissue+engineering:+Mechanisms+and+applications.">Protein-hydrogel interactions in tissue engineering: Mechanisms and applications.</a> Tissue Engineering Part B: Reviews. 19 (2), 160-171 (2013).</li><li>Kroger, S. 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=Design+of+hydrolytically+degradable+polyethylene+glycol+crosslinkers+for+facile+control+of+hydrogel+degradation.">Design of hydrolytically degradable polyethylene glycol crosslinkers for facile control of hydrogel degradation.</a> Macromolecular Bioscience. 20 (10), 2000085 (2020).</li><li>Raeber, G., Lutolf, M., Hubbell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecularly+engineered+PEG+hydrogels:+a+novel+model+system+for+proteolytically+mediated+cell+migration.">Molecularly engineered PEG hydrogels: a novel model system for proteolytically mediated cell migration.</a> Biophysical Journal. 89 (2), 1374-1388 (2005).</li><li>Bruns, J., Egan, T., Mercier, P., Zustiak, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glioblastoma+spheroid+growth+and+chemotherapeutic+responses+in+single+and+dual-stiffness+hydrogels.">Glioblastoma spheroid growth and chemotherapeutic responses in single and dual-stiffness hydrogels.</a> Acta Biomaterialia. 163, 400-414 (2023).</li><li>Kuwajima, T., 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=ClearT:+a+detergent-and+solvent-free+clearing+method+for+neuronal+and+non-neuronal+tissue.">ClearT: a detergent-and solvent-free clearing method for neuronal and non-neuronal tissue.</a> Development. 140 (6), 1364-1368 (2013).</li><li>Holt, S. E., Ward, E. S., Ober, R. J., Alge, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shooting+for+the+moon:+using+tissue-mimetic+hydrogels+to+gain+new+insight+on+cancer+biology+and+screen+therapeutics.">Shooting for the moon: using tissue-mimetic hydrogels to gain new insight on cancer biology and screen therapeutics.</a> MRS Communications. 7 (3), 427-441 (2017).</li><li>Jain, E., Scott, K. M., Zustiak, S. P., Sell, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+polyethylene+glycol-based+hydrogel+microspheres+through+electrospraying.">Fabrication of polyethylene glycol-based hydrogel microspheres through electrospraying.</a> Macromolecular Materials and Engineering. 300 (8), 823-835 (2015).</li><li>Raghavan, S., 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=Comparative+analysis+of+tumor+spheroid+generation+techniques+for+differential+in+vitro+drug+toxicity.">Comparative analysis of tumor spheroid generation techniques for differential in vitro drug toxicity.</a> Oncotarget. 7 (13), 16948 (2016).</li><li>Lee, K. -. H., Kim, T. -. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+multicellular+tumor+spheroid+generation+for+drug+screening.">Recent advances in multicellular tumor spheroid generation for drug screening.</a> Biosensors. 11 (11), 445 (2021).</li></ol>

Yazarların açıklayacak hiçbir şeyi yok.

Hidrojel bazlı çok hücreli tümör sferoid modelleri, kanser terapötik keşiflerini ilerletmek için giderek daha fazla geliştirilmektedir 11,13,29. Faydalıdırlar çünkü tümör mikroçevresinin temel parametrelerini kontrollü bir şekilde taklit ederler ve karmaşıklıklarına rağmen in vivo modellere göre kullanımı daha basit ve daha ucuzdur ve birçoğu yüksek verimli tarama teknolojileriyle uyumludur. Hidroje...

Tümör sferoidleri, kanser etiyolojisi, patolojisi ve ilaç yanıtını incelemede yararlı in vitro araçlar olarak ortaya çıkmıştır1. Geleneksel olarak, sferoidler, hücre-hücre yapışmasının hücre yüzeyi yapışmasına göre tercih edildiği düşük yapışma plakaları veya biyoreaktörler gibi koşullarda kültürlenmiştir2. Bununla birlikte, tümör mikroçevresini daha sadık bir şekilde özetlemek için, in vitro sferoid modellerin hem hücre-hücre hem de hücre-matris etkileşimlerini yakalaması gerektiği artık kabul edilmektedir. Bu, birden fazla grubu, sferoidlerin kapsüllenebileceği hidrojeller gibi iskeleler tasarlamaya sevk etti 3,4. Bu tür hidrojel bazlı sferoid modeller, canlılık, proliferasyon, kök veya tedaviye yanıt verme gibi çeşitli hücre davranışları üzerindeki hücre-hücre ve hücre-matris etkileşimlerinin aydınlatılmasını sağlar3.
Burada, glioblastoma sferoidlerinin polietilen glikol (PEG) hidrojeller içinde kapsüllenmesi için bir protokol açıklıyoruz. Hidrojellerde glioblastoma hücre sferoid kapsüllemesi ile ilgili çok sayıda literatür raporu vardır. Örneğin, hidrojel sertliğinin hücre davranışı üzerindeki etkisini belirlemek için bir RGDS yapışkan ligand ile süslenmiş ve enzimatik olarak bölünebilir bir peptit ile çapraz bağlanmış PEG hidrojellerinde U87 hücrelerinin kapsüllenmesiyle sferoidler oluşturuldu5. U87 hücreleri, kanser kök hücre popülasyonunugenişletmek 6 veya kemoterapi direncinin matris aracılı mekanizmalarını keşfetmek için diğer PEG bazlı veya hyaluronik asit bazlı hidrojellerdede oluşturulmuştur 7,8,9. Glioblastoma sferoidleri, mikroglia ve kanser hücreleri arasındaki karışmayı ve bunun hücre istilası üzerindeki etkisini incelemek için jelatin hidrojeller içinde kapsüllenmiştir10. Genel olarak, bu tür çalışmalar, glioblastoma patolojisini anlamada ve tedavilerin tasarlanmasında hidrojel bazlı in vitro modellerin faydasını göstermiştir.
Ayrıca, tümör sferoid üretimi ve hidrojel kapsülleme için farklı yöntemler vardır11. Örneğin, dağılmış hücreler hidrojeller içinde tohumlanabilir ve zamanla sferoidler oluşturmasına izin verilebilir 5,12. Böyle bir yöntemin bir dezavantajı, diferansiyel hücre tepkilerine yol açabilecek oluşan sferoidlerin polidispersitedir. Tek tip sferoidler üretmek için, hücreler mikrojeller içinde kapsüllenebilir ve jeli13 istila edip yeniden şekillendirene kadar uzun süreler boyunca kültürlenebilir veya hücreler küresel 'deliklere' sahip şablonlu jellerde biriktirilebilir vetoplanmasına izin verilebilir 14. Bu yöntemlerin dezavantajı, göreceli karmaşıklıkları, mikrojeller veya jeldeki 'delikler' oluşturmak için bir damlacık üretecine veya başka araçlara ihtiyaç duyulması ve sferoidlerin büyümesi ve olgunlaşması için geçen süredir. Alternatif olarak, sferoidlermikro kuyucuklarda 9,15,16 veya asılı damla plakalarında17,18 önceden oluşturulabilir ve daha sonra burada açıklanan tekniğe benzer şekilde bir hidrojel içinde kapsüllenebilir. Bu yöntemler daha basittir ve daha yüksek aktarım hızı bir şekilde yapılabilir. İlginç bir şekilde, sferoid oluşum yönteminin, gen ekspresyonu, hücre proliferasyonu veya ilaca yanıt verme gibi sferoid hücre davranışlarını etkileyebileceği gösterilmiştir19,20.
Burada, doğal ortamı yumuşak, nano gözenekli bir hidrojel tarafından taklit edilebilen yumuşak, nano gözenekli beyin matrisi21 olan katı bir tümör olduğu için glioblastoma odaklanıyoruz. Glioblastoma aynı zamanda tedavisi olmayan en ölümcül beyin kanseridir22. Bununla birlikte, burada açıklanan protokol, herhangi bir katı tümörü temsil eden sferoidlerin kapsüllenmesi için kullanılabilir. Michael tipi bir ekleme reaksiyonu23 ile oluşturulan PEG hidrojellerini kullanmayı seçtik. PEG, inert olan ve iskele ve fiziksel hücre desteği görevi gören ancak hücre bağlanmasını desteklemeyen sentetik, bozunmayan ve biyouyumlu bir hidrojeldir23. Hücre yapışkanlığı, tüm proteinlerin veya yapışkan ligandların24 bağlanması yoluyla ayrı olarak eklenebilir ve parçalanabilirlik, PEG polimer zincirinin veya hidrolitik veya enzimatik olarak parçalanabilen çapraz bağlayıcıların25,26 kimyasal modifikasyonları yoluyla eklenebilir. Bu, biyokimyasal özelliklerin, hücre-matris etkileşimlerinin incelenmesinde avantajlı olabilecek mekanik veya fiziksel hidrojel özelliklerinden bağımsız olarak ayarlanmasına izin verir. Michael tipi jelleşme kimyası seçicidir ve fizyolojik koşullarda gerçekleşir; bu nedenle, sferoidleri hidrojel öncü çözeltisi ile karıştırarak sferoid kapsüllemeye izin verir.
Genel olarak, burada sunulan metodolojinin birkaç dikkate değer özelliği vardır. İlk olarak, çok kuyulu bir düzenekte tümör sferoidlerinin üretilmesi verimli, hızlıdır ve gerekli malzemelerin maliyeti düşüktür. İkincisi, sferoidler, düşük polidispersiteye sahip çeşitli boyutlarda büyük partiler halinde üretilir. Son olarak, yalnızca ticari olarak temin edilebilen malzemeler gereklidir. Metodolojinin faydası, substrat özelliklerinin küresel hücre canlılığı, döngüsellik ve hücre saplılığı üzerindeki etkisi araştırılarak gösterilmektedir.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>70% Ethanol</td>
			<td>Fisher Scientific&#160;</td>
			<td>LC22210-4</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Conicals</td>
			<td>FALCON</td>
			<td>352097</td>
			<td></td>
		</tr>
		<tr>
			<td>24-Well Plate Ultra Low Attachment plates</td>
			<td>Fisher Scientific</td>
			<td>07-200-602</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Petri Dish</td>
			<td>Amazon</td>
			<td>706011</td>
			<td></td>
		</tr>
		<tr>
			<td>4-arm poly(ethylene glycol)-acrylate (4-arm PEG-Ac; 10 kDa)</td>
			<td>Laysan Bio</td>
			<td>ACRL-PEG-ACRL-10K-5g</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Conicals</td>
			<td>Fisher Scinetific</td>
			<td>3181345107</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well AggreWell 400&#160;</td>
			<td>StemCell Technologies, Vancouver, Canada</td>
			<td>34421</td>
			<td>Square pyramidal microwells&#160;</td>
		</tr>
		<tr>
			<td>anti-adherence rinsing solution</td>
			<td>StemCell Technologies, Vancouver, Canada</td>
			<td>Cat #: 07010</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspartic Acid-Arginine-Cysteine-Glycine-Valine-Proline-Methionine-Serine-Methionine-Arginine-Glycine-Cysteine-Arginine- Aspartic Acid (DRCG-VPMSMR-GCRD) peptide</td>
			<td>Genic Bio, Shanghai, China</td>
			<td>n/a</td>
			<td>Custom synthesis</td>
		</tr>
		<tr>
			<td>Chemical Fume Hood</td>
			<td>KEWAUNEE</td>
			<td>99151</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Matrigel Basement Membrane Matrix, LDEV Free&#160;</td>
			<td>Corning</td>
			<td>356234</td>
			<td>Basement membrane matrix</td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6-diamidino-2-phenylindole, dihydrochloride)</td>
			<td>Thermo Scientific</td>
			<td>62247</td>
			<td></td>
		</tr>
		<tr>
			<td>Detergent - Triton-X</td>
			<td>Sigma Aldrich</td>
			<td>T8787</td>
			<td>Nonionic surfactant</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Fisher Scientific&#160;</td>
			<td>BP231-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Pipettes (1 mL, 2 mL, 5 mL, 10 mL, 25 mL, 50 mL)</td>
			<td>Fisher Scinetific</td>
			<td>1 mL: 13-678-11B, 2mL: 05214038, 5mL(FALCON): 357529, 10mL: 13-678-11E, 25mL: 13-678-11, 50mL: 13-678-11F</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>HyClone</td>
			<td>SH30073-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde 37% Solution</td>
			<td>Sigma Aldrich</td>
			<td>F1635</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Plates</td>
			<td>Slumpys</td>
			<td>GBS4100SFSL</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Transfer Pipettes</td>
			<td>Fisher Scinetific</td>
			<td>5 3/4&#34;: 1367820A, 9&#34;:136786B</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine-Arginine-Cysteine-Aspartic Acid-Arginine-Glycine-Aspartic Acid-Serine (GRCD-RGDS) peptide</td>
			<td>Genic Bio, Shanghai, China</td>
			<td>n/a</td>
			<td>Custom synthesis</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Bright-Line</td>
			<td>383684</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic solution - Repel Silane&#160;</td>
			<td>GE Healthcare Bio-Sciences</td>
			<td>17-1332-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>NUAIRE</td>
			<td>NU-8500</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted Microscope (Axiovert 25)</td>
			<td>Zeiss</td>
			<td>663526</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitrogen DiOC16(3) (3,3&#39;-Dihexadecyloxacarbocyanine Perchlorate)</td>
			<td>Fisher Scientific&#160;</td>
			<td>D1125</td>
			<td></td>
		</tr>
		<tr>
			<td>Leica Confocal SP8</td>
			<td>Leica Microsystems Inc.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Light and Flourescent Microscope (Axiovert 200M)</td>
			<td>Zeiss</td>
			<td>3820005619</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro centrifuge tubes</td>
			<td>Fisher Scientific</td>
			<td>2 mL: 02681258</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Software</td>
			<td>Zeiss</td>
			<td>AxioVision Rel. 4.8.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Nestin Alexa Fluor 594&#160;</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-23927</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>PARAFILM&#160;</td>
			<td>PM992</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (1x), pH 7.4</td>
			<td>HyClone</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>MP Biomedicals</td>
			<td>1670046</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Aid</td>
			<td>Drummond Scientific Co.</td>
			<td>P-76864</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette Tips (1&#8211;200 &#181;L, 101&#8211;1000 &#181;L)</td>
			<td>Fisher Scinetific</td>
			<td>2707509</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Standard Disposable Transfer Pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-711-9D</td>
			<td></td>
		</tr>
		<tr>
			<td>Plastic Weigh Boats (100 mL)</td>
			<td>Amazon&#160;</td>
			<td>mdo-azoc-1030</td>
			<td></td>
		</tr>
		<tr>
			<td>poly(ethylene glycol)-dithiol (PEG-diSH; 3.4 kDa)</td>
			<td>Laysan Bio</td>
			<td>SH-PEG-SH-3400-5g</td>
			<td></td>
		</tr>
		<tr>
			<td>Polydimehylsiloxane (PDMS) [Slygard 182 Elastomer Kit]</td>
			<td>Elsworth Adhesives</td>
			<td>3097358-1004</td>
			<td>Polydimethylsiloxane</td>
		</tr>
		<tr>
			<td>Powder Free Examination Gloves</td>
			<td>Quest</td>
			<td>92897</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide, 1 mg/mL aqueous soln.&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>AAJ66584AB</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 Medium (1x)</td>
			<td>HyClone</td>
			<td>SH30027-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone spacers - Silicone sheet, 0.5 mm thick/13 cm x 18 cm</td>
			<td>Grace Bio-Labs</td>
			<td>JTR-S-0.5</td>
			<td></td>
		</tr>
		<tr>
			<td>SOX2 Alexa Fluor 488&#160;</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-365823</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Hood</td>
			<td>NUAIRE</td>
			<td>NU-425-600</td>
			<td></td>
		</tr>
		<tr>
			<td>Triethanolamine, &#8805;99.0% (GC)&#160;</td>
			<td>Sigma Aldrich</td>
			<td>90279</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 0.25% (1x)&#160;</td>
			<td>Sigma Aldrich</td>
			<td>SH30042.01</td>
			<td></td>
		</tr>
		<tr>
			<td>U-87 MG human glioblastoma cells</td>
			<td>American Type Culture Collection&#160;</td>
			<td>HTB-14</td>
			<td></td>
		</tr>
	</tbody>
</table>

tumor spheroid fabrication and encapsulation in polyethylene glycol hydrogels for studying spheroid-matrix interactions

Sferoidlerin üç boyutlu (3D) kapsüllenmesi, optimal hücre büyümesi için tümör mikroçevresini yeterince kopyalamak için çok önemlidir. Burada, tümör hücre dışı mikroçevreyi taklit etmek için sferoid kapsülleme için bir in vitro 3D glioblastoma modeli tasarladık. İlk olarak, polidimetilsiloksan kullanarak kare piramidal mikrokuyu kalıpları oluşturduk. Bu mikrokuyu kalıpları daha sonra 50-500 μm arasında sıkı bir şekilde kontrol edilen boyutlara sahip tümör sferoidlerini imal etmek için kullanıldı. Sferoidler oluşturulduktan sonra, hasat edildi ve polietilen glikol (PEG) bazlı hidrojeller içinde kapsüllendi. PEG hidrojelleri, sertlik, bozunabilirlik ve hücre yapışkanlığı gibi hidrojel özellikleri bağımsız olarak ayarlanabildiğinden, küresel kapsülleme için çok yönlü bir platformdur. Burada, glioblastoma sferoidlerini kapsüllemek için temsili bir yumuşak (~8 kPa) hidrojel kullandık. Son olarak, konfokal mikroskopi ile yüksek kaliteli görüntüler elde etmek için sferoidleri boyamak ve görüntülemek için bir yöntem geliştirilmiştir. Yoğun sferoid çekirdek ve nispeten seyrek çevre nedeniyle, görüntüleme zor olabilir, ancak bir temizleme solüsyonu ve konfokal optik kesit kullanmak bu görüntüleme zorluklarını hafifletmeye yardımcı olur. Özetle, tek tip sferoidler üretmek, bunları PEG hidrojellerinde kapsüllemek ve sferoid büyümesini ve çeşitli hücre-matris etkileşimlerini incelemek için kapsüllenmiş sferoidler üzerinde konfokal mikroskopi gerçekleştirmek için bir yöntem gösteriyoruz.

Tumor spheroids have emerged as useful in vitro tools in studying cancer etiology, pathology, and drug responsiveness1. Traditionally, spheroids have been cultured in conditions such as low adhesion plates or bioreactors, where cell-cell adhesion is favored over cell-surface adhesion2. However, it is now recognized that to recapitulate the tumor microenvironment more faithfully, in vitro spheroid models should capture both cell-cell and cell-matrix interactions. This has prompted multiple groups to design scaffolds, such as hydrogels, where spheroids can be encapsulated3,4. Such hydrogel-based spheroid models enable the elucidation of cell-cell and cell-matrix interactions on various cell behaviors, such as viability, proliferation, stemness, or therapy responsiveness3.
Here, we describe a protocol for the encapsulation of glioblastoma spheroids in polyethylene glycol (PEG) hydrogels. There are multiple literature reports of glioblastoma cell spheroid encapsulation in hydrogels. For example, spheroids were formed by encapsulating U87 cells in PEG hydrogels decorated with an RGDS adhesive ligand and crosslinked with an enzymatically cleavable peptide to determine the effect of hydrogel stiffness on cell behavior5. U87 cells have also been formed in other PEG-based or hyaluronic acid-based hydrogels to expand the cancer stem cell population6 or to explore matrix-mediated mechanisms of chemotherapy resistance7,8,9. Glioblastoma spheroids have also been encapsulated in gelatin hydrogels to study the crosstalk between microglia and cancer cells and its effect on cell invasion10. Overall, such studies have demonstrated the utility of hydrogel-based in vitro models in understanding glioblastoma pathology and devising treatments.
Further, there are different methods for tumor spheroid fabrication and hydrogel encapsulation11. For example, dispersed cells could be seeded in hydrogels and allowed to form spheroids over time5,12. One drawback of such a method is the polydispersity of the formed spheroids, which could lead to differential cell responses. To produce uniform spheroids, cells could be encapsulated in microgels and cultured for extended periods until they invade and remodel the gel13, or cells could be deposited in templated gels with spherical &#39;holes&#39; and allowed to aggregate14. The drawback of these methods is their relative complexity, the need for a droplet generator or other means to form microgels or the &#39;holes&#39; in the gel, and the time it takes for spheroids to grow and mature. Alternatively, spheroids could be pre-formed in microwells9,15,16 or in hanging-drop plates17,18 and then encapsulated in a hydrogel, similar to the technique described here. These methods are simpler and can be done in a higher throughput fashion. Interestingly, it has been shown that the method of spheroid formation can affect spheroid cell behaviors, such as gene expression, cell proliferation, or drug responsiveness19,20.
Here, we focus on glioblastoma since it is a solid tumor whose native environment is the soft, nanoporous brain matrix21, which can be mimicked by a soft, nanoporous hydrogel. Glioblastoma is also the deadliest brain cancer for which there is no available cure22. However, the protocol described here can be used for the encapsulation of spheroids representing any solid tumor. We chose to use PEG hydrogels that are formed through a Michael-type addition reaction23. PEG is a synthetic, non-degradable, and biocompatible hydrogel that is inert and serves as scaffolding and physical cell support but does not support cell attachment23. Cell adhesiveness can be added separately via tethering of whole proteins or adhesive ligands24, and degradability can be added via chemical modifications of the PEG polymer chain or hydrolytically or enzymatically degradable crosslinkers25,26. This allows for biochemical properties to be tuned independently of mechanical or physical hydrogel properties, which could be advantageous in studying cell-matrix interactions. The Michael-type gelation chemistry is selective and happens at physiological conditions; hence, it allows for spheroid encapsulation by simply mixing the spheroids with the hydrogel precursor solution.
Overall, the methodology presented here has several notable characteristics. First, fabricating tumor spheroids in a multiwell assembly is efficient, quick, and the cost of the required materials is low. Second, the spheroids are produced in large batches in a variety of sizes with low polydispersity. Finally, only commercially available materials are required. The utility of the methodology is illustrated by exploring the effect of substrate properties on spheroid cell viability, circularity, and cell stemness.

Yazar Spotlight: Glioblastoma Mikroçevre Çalışması için İn Vitro Hidrojel Modeli

Sferoid-Matriks Etkileşimlerini İncelemek için Polietilen Glikol Hidrojellerde Tümör Sferoid Üretimi ve Kapsüllenmesi

Our lab focuses on designing in vitro hydrogel models for glioblastoma steroid encapsulation to mimic the tumor extracellular microenvironment. The effective substrate, mechanical and biochemical properties on steroid cell viability, circularity, stemness, and spreading are investigated with such models. Current experimental challenges in tumor module development revolve around a desire for complexity to mimic all aspects of the tumor microenvironment for physiological relevance, and also a desire for simplicity to make the model accessible, robust, low cost, and easy to use, and compatible with multiplex screening approaches.We present a protocol that enables fast, robust, and low-cost fabrication of tumor steroids and encapsulation in hydrogels made to mimic the tumor microenvironment. The model does not require any specialized equipment. It will be particularly useful for exploring steroid matrix interactions in building in vitro tissue physiology or pathology models.Understanding the interactions between glioblastoma cancer cells and their microenvironment can encourage development of new therapies to better treat this cancer. Furthermore, development of an in vitro model for glioblastoma cancer can enhance development of this model for other cancer diseases.

Kemoterapötik etkileri incelemek için sferoid bazlı ilaç tarama platformları, doğal dokuyu kopyalayan biyomalzemelerde sferoid kapsülleme üzerine tümör mikroçevresinin modüle edilmesine yapılan vurgu nedeniyle giderek daha fazla aranmaktadır. Burada, çok hücreli tümör sferoid hazırlığı ve ardından bir 3D hidrojel içinde kapsülleme ve görüntüleme için bir yöntem geliştirdik. Sferoidler, mikrokuyu kalıplarında hazırlanır (Şekil 3A,B), bu da ...

Watch this Scientific Journal Video about Tumor Spheroid Fabrication and Encapsulation in Polyethylene Glycol Hydrogels for Studying Spheroid-Matrix Interactions at JoVE.com

Burada, tümör sferoidlerinin hızlı, sağlam ve ucuz bir şekilde üretilmesini ve ardından hidrojel kapsüllenmesini sağlayan bir protokol sunuyoruz. Özel ekipman gerektirmediği için yaygın olarak uygulanabilir. Sferoid-matriks etkileşimlerini araştırmak ve in vitro doku fizyolojisi veya patoloji modelleri oluşturmak için özellikle yararlı olacaktır.

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth.  Here, we ...

Laboratuvarımız, tümör hücre dışı mikro çevreyi taklit etmek için glioblastoma steroid kapsüllemesi için in vitro hidrojel modelleri tasarlamaya odaklanmaktadır. Steroid hücre canlılığı, döngüsellik, saplılık ve yayılma üzerindeki etkili substrat, mekanik ve biyokimyasal özellikler bu modellerle araştırılmaktadır. Tümör modülü geliştirmedeki mevcut deneysel zorluklar, fizyolojik alaka düzeyi için tümör mikro çevresinin tüm yönlerini taklit etmek için karmaşıklık arzusu ve ayrıca modeli erişilebilir, sağlam, düşük maliyetli ve kullanımı kolay hale getirmek için basitlik arzusu etrafında dönmektedir.Tümör steroidlerinin hızlı, sağlam ve düşük maliyetli üretimini ve tümör mikroçevresini taklit etmek için yapılan hidrojellerde kapsüllemeyi sağlayan bir protokol sunuyoruz. Model herhangi bir özel ekipman gerektirmez. İn vitro doku fizyolojisi veya patoloji modellerinin oluşturulmasında steroid matris etkileşimlerini araştırmak için özellikle yararlı olacaktır.Glioblastoma kanser hücreleri ve mikro çevreleri arasındaki etkileşimleri anlamak, bu kanseri daha iyi tedavi etmek için yeni tedavilerin geliştirilmesini teşvik edebilir. Ayrıca, glioblastoma kanseri için bir in vitro modelin geliştirilmesi, bu modelin diğer kanser hastalıkları için geliştirilmesini artırabilir.

tumor-spheroid-fabrication-encapsulation-polyethylene-glycol

Research

JoVE Journal

Bioengineering

Tumor Spheroid Fabrication and Encapsulation in Polyethylene Glycol Hydrogels for Studying Spheroid-Matrix Interactions

823 Görüntüleme.  Saint Louis University. Laboratuvarımız, tümör hücre dışı mikro çevreyi taklit etmek için glioblastoma steroid kapsüllemesi için in vitro hidrojel modelleri tasarlamaya odaklanmaktadır. Steroid hücre canlılığı, döngüsellik, saplılık ve yayılma üzerindeki etkili substrat, mekanik ve biyokimyasal özellikler bu modellerle araştırılmaktadır. Tümör modülü geliştirmedeki mevcut deneysel zorluklar, fizyolojik alaka düzeyi için tümör mikro çevresinin tüm yönlerini taklit etmek için ...

Video: Yazar Spotlight: Glioblastoma Mikroçevre Çalışması için İn Vitro Hidrojel Modeli

Three-dimensional (3D) encapsulation of spheroids is crucial to adequately replicate the tumor microenvironment for optimal cell growth. Here, we designed an in vitro 3D glioblastoma model for spheroid encapsulation to mimic the tumor extracellular microenvironment. First, we formed square pyramidal microwell molds using polydimethylsiloxane. These microwell molds were then used to fabricate tumor spheroids with tightly controlled sizes from 50-500 &#956;m. Once spheroids were formed, they were harvested and encapsulated in polyethylene glycol (PEG)-based hydrogels. PEG hydrogels are a versatile platform for spheroid encapsulation, as hydrogel properties such as stiffness, degradability, and cell adhesiveness can be tuned independently. Here, we used a representative soft (~8 kPa) hydrogel to encapsulate glioblastoma spheroids. Finally, a method to stain and image spheroids was developed to obtain high-quality images via confocal microscopy. Due to the dense spheroid core and relatively sparse periphery, imaging can be difficult, but using a clearing solution and confocal optical sectioning helps alleviate these imaging difficulties. In summary, we show a method to fabricate uniform spheroids, encapsulate them in PEG hydrogels and perform confocal microscopy on the encapsulated spheroids to study spheroid growth and various cell-matrix interactions.

Here, we present a protocol that enables fast, robust, and cheap fabrication of tumor spheroids followed by hydrogel encapsulation. It is widely applicable as it does not require specialized equipment. It would be particularly useful for exploring spheroid-matrix interactions and building in vitro tissue physiology or pathology models.

Biyomühendislik

Fabrication of Square Pyramidal Microwells for Tumor Spheroid Fabrication

To begin pour two grams or about one milliliter of negative PDMS precursor solution onto one well of a six well square parametal master mold. After covering the master mold with PDMS, place the plate in a vacuum desiccate to degas the solution. Next, place the plate in an oven at 60 degrees Celsius for 24 hours to cure the PDMS.Once PDMS cures but is still warm, use a spatula to carefully remove the PDMS mold from the master mold. Then with a biopsy punch, cut it into a 35 millimeter diameter slab. Place the properly cut PDMS mold in a Petri dish and cover it with the lid to continue curing it at room temperature for an additional 24 hours.To prepare the positive PDMS mold place a 35 millimeter slab of the negative PDMS mold in a 35 millimeter Petri dish with the textured micro wells facing up. Pour 2.5 grams or about 1.2 milliliters of positive PDMS precursor solution onto the negative mold in the Petri dish. Degas the precursor solution for 30 minutes before placing it into the oven at 60 degrees Celsius for three to four hours.After curing, remove the molds from the Petri dish. Immediately peel the positive mold from the negative mold. Use a 10 millimeter biopsy punch to cut the positive molds into slabs.Next, use tweezers to gently dip the flat side of each 10 millimeter positive mold into the pre-prepared PDMS glue precursor solution. Carefully place one mold per well in a well of a 48 well plate. Gently press each mold to glue it to the well bottom.Incubate the assembled plate at 60 degrees Celsius for four to 24 hours to cure the glue.

Tümör Sferoid Üretimi için Kare Piramidal Mikrokuyuların İmalatı

Multicellular Tumor Spheroid Formation, Harvest, and Encapsulation in Hydrogels

To begin, place the PDMS microwell molds, anti-adherence rinsing solution and necessary lab wear in a tissue culture hood. To wash the PDMS microwell molds, use a 1, 000 microliter pipette to add 300 microliters of anti-adherence rinsing solution to each well. Then centrifuge the plate, 1, 620 G for three minutes.Use a vacuum pump and a Pasteur pipette to aspirate the solution. Next, place 500 microliters of cell suspension at the desired concentration in the microwells and centrifuge the plate at 1, 620 G for three minutes. Place the plate in a humidified incubator to allow spheroid formation.To harvest the spheroids, using a 1, 000 microliter pipette, firmly add 500 microliters of complete medium into each well. Pipette the added medium up and down at each quadrant three to four times to dislodge the speroids. Then use the pipette to gently aspirate the medium containing the spheroids into a microcentrifuge tube.Transfer 50 microliters of the steroid suspension in a microcentrifuge tube. Then sequentially add 4-Arm PEG acrylate and PEG dithiol into it. Mix the resultant solution by pipetting it up and down about 10 times.To yield 100 microliters of a 10%weight by volume PEG hydrogel precursor solution, pipette 20 microliters of the gel precursor solution in between two parafilm-lined glass slides separated with one millimeter silicon spacers and incubate the slides with gel precursor solution to allow for gelation. Once hydrogel gelation is complete, using a spatula, gently peel the gels off the separated glass slides. Place one gel per well into a 24-well plate, ensuring the surface containing the spheroids faces up.Add 500 microliters of complete medium to each well to submerge the hydrogel completely. Place the multi-well plate in a humidified incubator to culture the cells. The described technique using microwell molds resulted in the formation of spheroids with spherical shapes and tightly-controlled polydispersity.For spheroids with 3, 300 cells per microwell, the average spheroid size was about 250 microns and circularity was greater than 0.8, considering a value of 1 as a perfect sphere. Spheroid diameters were dependent on the microwell size and the number of cells per microwell.

Hidrojellerde Çok Hücreli Tümör Sferoid Oluşumu, Hasadı ve Kapsüllenmesi

Immunofluorescence Fixation, Staining, and Clearing of Encapsulated Spheroids for Confocal Microscopy

To begin, aspirate the media from the wells where the hydrogel-encapsulated cells are cultured in the 24-well plate. Rinse the hydrogels by pipetting 500 microliters of PBS directly onto each well containing the hydrogels, and then gently aspirate the PBS. Using a 1, 000 microliter pipette, add 500 microliters of fixative solution per well to fix the spheroids in the 24-well plate.Allow the fixative to soak the gels for 30 minutes at room temperature, before pipetting out the fixative solution and discarding it in a designated waste container. Next, rinse the hydrogels with PBS three times as demonstrated previously. If not used immediately, store the hydrogels in 500 microliters of PBS per well at four degrees Celsius for up to one week.To stain in the hydrogel-encapsulated cells, first, prepare appropriately diluted primary antibodies for nestin and SOX2. After aspirating the PBS from the wells, add 50 microliters of the diluted antibodies to each well. Incubate the cells with the antibodies for 24 hours to stain them completely.Then using a 1, 000 microliter pipette, remove the staining solution and discard the waste appropriately. After rinsing the hydrogels three times, store them in PBS at four degrees Celsius for up to two weeks prior to imaging, or image immediately. To clear the stained spheroid for improving imaging transparency, first aspirate the PBS from each well.Then sequentially treat the spheroids with 500 microliters of 20%40%and 80%formamide per well for 90 minutes each. Finally incubate the hydrogels in 100%formamide for 24 hours prior to imaging. The spheroids were imaged at varying z-stack depths, allowing for the cell viability assessment at each location within the z-stack.A maximum projection utilizing the spheroid stack represented the highest point of light intensity within each location, simplified into one image. Representative images of stained spheroids revealed that the self-renewal transcription factor, SOX2, was co-localized with DAPI in the nucleus. On the contrary, stem cell marker nestin was present throughout the cells.There was no difference between the free floating spheroid without gel and the encapsulated spheroids, possibly due to the inertness of the PEG gel used. The representative images of optical sections at about 90 microns into cleared and uncleared spheroids revealed that when clearing was performed, the spheroid core could be imaged about 30 microns deeper compared to an uncleared spheroid.