Biz derin termosensitif micropatterned kültür plakaları yanı sıra bilimsel danışmanlık sağlamak için Toyo Gosei, Tokyo, Japonya&#39;da Dr Takeshi Ikeya ve teknik personel için teşekkür ederiz. Biz de hayvan deneyleri ile teknik yardım için Sayın Satomi Ogura, Bayan Sae Suzuki, Bayan Asuka Miyoshi ve Bayan Katsue Morii teşekkür ederiz. Bu eser mali JSPS KAKENHI Grant-Aid Bilimsel Araştırma tarafından kısmen desteklenen, İnovasyon (ÇÇ) Programı Merkezi ve Japonya Bilim ve Teknoloji Kurumu (JST) den S yenilik programı ve JSPS core için Çekirdekli Programı, A. İleri Araştırma Ağları.

Tüm hayvan çalışmaları, Tokyo Üniversitesi Hayvan Bakımı ve Kullanımı Komitesi, Tokyo, Japonya onayı ile yürütülmüştür. 1. Hücre Hazırlanması <ol><li> Birincil hepatosit için, bir tadil edilmiş, iki basamaklı bir kollajenaz digestiyonu işlemi 13,14 sıçan hepatosit izolasyonu için protokol takip edin. <ol><li> Izofluran ile inhalasyon anestezi altında Sprague Dawley (SD) sıçanları (erkek, 5 haftalık) anestezisi. Bölmesi için izofluran sağlamak için bir anestezi makinesine bağlı bir odasında bir sıçan yerleştirin. Uykuya dalmakta sonra sıçan dışarı atın ve bir maske kullanarak havalandırma ile ameliyat masasında ayarlayın. Sıçan koşullarını kontrol ederek 0,7 L / dk - izofluran 0,4 yaklaşık akış kontrol edin. 8 g / L sodyum klorid oluşan özel bir çözeltisi (NaCl), 400 mg / L potasyum klorid (KCl), 78 mg ile hepatik portal ven Sprague Dawley (SD) sıçanları (erkek, 5 haftalık) karaciğerler serpmek / Lsodyum dihidrojen fosfat dihidrat (NaH 2 PO 4 · 2H 2 O), 151 mg / L disodyum hidrojen fosfat dodekahidrat (Na 2 HPO 4 · 12H 2 O), 2.38 g / L 2- [4- (2-hidroksietil) -1 piperazinil] etansülfonik asit (HEPES), 190 mg / L etilen glikol tetraasetik asit (EGTA), 350 mg / L sodyum hidrojen karbonat (NaHCO3) ve 900 mg / L glikoz. </li><li> Karaciğerden kolajenaz çözeltisi sirküle. NOT: Çözelti 500 mg / L kolajenaz, 9.8 g / L Hank tamponlu tuz, oluşan 2,38 g / ml HEPES, 556 mg / ml kalsiyum klorür hidrat (CaCl2 · H2O), 350 mg / L NaHCO 3, ve pH değeri ile 50 mg / L tripsin inhibitörü, 7.2 olacak şekilde ayarlanır. </li><li> Dikkatli bir şekilde karaciğer çıkarın ve bir bisturi bıçağını kullanarak bir tabak üzerine yavaşça kıyma, ekleme Dulbecco Modifiye Eagle Ortamı,% 10 fetal sığır serumu (FBS) ile desteklenmiş (DMEM), ve 100 um hücre süspansiyonu filtrenaylon örgü. Ek enkaz kaldırmak için, 1 dakika boyunca 20 × g hücre süspansiyonu santrifüj. Bu adımda, hepatositler süpernatan içinde bulunmaktadır. </li><li> Iki kez santrifüj adımı (üç santrifüj toplam) tekrarlayın. 3 dk pelet formunda onları kurtarmak için Nihayet, 50 × g hepatositler santrifüj. </li><li> % 10 FBS ile takviye edilmiş DMEM içinde oluşan özel bir kültür ortamı içinde 4 x 10 5 hücre / ml&#39;lik bir konsantrasyonda,% 1 Pen-Strep-Glut (PSQ),% 1 dimetil sülfoksit, hepatositlerin yeniden askıya (DMSO), 0.1 umol / L deksametason, / ml insülin, 0.5 ug, 10 mmol / L nikotinamid, 0.2 mmol / L fosforile askorbat (ASC-2P), ve 10 ng / ml insan epidermal büyüme faktörü (hEGF) 15. Bu özel ortam in vitro koşullarda karaciğer fonksiyon korumak için zorunludur. </li></ol></li><li> Sıçan MKH&#39;lerin elde etmek için, isoflur aşın uygulanması ile Sprague Dawley (SD) sıçanları (erkek, 5 haftalık), ötenaziane. Uyluk ve kaval kemiklerinden rezeke ve DMEM, 10 ml% 10 FBS ile takviye edilmiş bunu temizlemek için kemik şaftı bir 22 G iğne sokarak, kemik iliği toplamak. 100 mikron naylon elekle filtrelenmiştir hücreleri toplamak. </li><li> DMEM,% 10 FBS ve% 1 penisilin / streptomisin içeren kullanarak 10 cm&#39;lik kültür tabakları üzerine hücreleri tohumu. Sfero deneyleri için, 5 pasajlar içinde MKH&#39;lerin kullanın. </li></ol> 3D Hücre sferoidlerin 2. Hazırlık <ol><li> Ticari olarak, hücre yapışkan alanlar düzenli PEG matris ile kaplanmış yapışkan olmayan alanlar tarafından çevrelenmiş, iki boyutlu bir şekilde, 100 um çapında dizilmiş edildiği micropatterned kültür plakaları elde. Not: Hücre nakli için, ısıya duyarlı bir polimer PIPAAm bir ilave kaplama plakaları, soğutma hücre çıkarılabilmesi için gerekli olan (adım 5.1). Sıcaklık dalgalanmaları bu polimerin kimyasında değişikliklere neden olabilir8-10. 37 ° C&#39;de, PIPAAm hücreler normal koşullar altında kültürlenebilir sağlayan, hafif hidrofobiktir. Hücrelerin spontan dekolmanı gelen polimerin hızlı hidrasyon 32 ° C sonuçları aşağıda sıcaklığında bir azalma. </li><li> De,% 5 CO2 içeren nemli bir atmosferde 37 ° C &#39;de bunları andincubate / 4 x 10 5 hücre yoğunluğunda 12 oyuklu plakalar üzerinde micropatterned hepatositleri ve mezenkimal tohumu. Hücreler micropatterned yapışma alanlarda birikir ve yavaş yavaş 2 gün içinde yuvarlak parçacıklarının oluşturacaktır. NOT: Bir tek tabakalı kültürü, kontrol hücreleri hazırlamak için, normal bir 12 gözlü levhalar kullanmak, yukarıda tarif edilene benzer bir prosedür takip edilerek, aynı yoğunlukta hücreler tohum. </li></ol> Polyplex Nanomicelles 3. hazırlanması <ol><li> (PEG Mw = 12, bir blok kopolimer, PEG-PASP (DET) (poli [(2-aminoetil) -2-aminoetil] aspartamit - - [N- N &#39;]) sentez0.00, polimerizasyon derecesi PASP (DET) segmentin (DP) = 59) ve daha önce yazarlar 11 tarafından tarif edilen prosedürler takip edilerek, yalnızca katyonik segmenti [PASP (DET)] (DP = 55) oluşan bir homopolimer, 16, 17. </li><li> 33.3 ug polimer konsantrasyonunun ayarlanması ile 10 mM HEPES tamponu (pH 7.3) artık amino grupları arasında eşit bir molar oranda, PEG-PASP (DET) blok kopolimer karışık çözeltisi ve PASP (DET) homopolimer hazırlanması / ml sırasıyla, 19.1 ug / mL olduğunu göstermiştir. Not: Yukarıda tarif edilen polimer konsantrasyonları değişebilir 10. N / P oranı, pDNA (N / P oranı) fosfat grupları iki polimerden toplam amino grupları bir artık mol oranı nanomicelles hazırlamak için temsili değerler (detaylar için, Tartışma) hücre tipine ve amacına bağlı olarak. İki polimerlerin birlikte kullanımı endozomal geliştirmek için etkili PEG kalkan ve PASP (DET) işleyişini hem elde edebilirsinizkaçış 18 (detaylar için, tartışma bakınız). </li><li> PCAG-GS plazmide ilgili genleri eksprese eden bir segment (http://www.cdb.riken.jp/pcs/protocol/vector/map/m36.html klonlanmasıyla pDNA kodlayan Gaussia lusiferaz, GL4 lusiferaz veya eritropoietin hazırlanması ) kullanılarak üreticinin protokolüne göre, ticari bir kit kullanılarak CAG promoter / güçlendirici altında açığa çıkışı elde edildi. Yetkin bir Escherichia coli içinde pDNA yükseltmek ve bir endotoksin içermeyen plazmid DNA arıtma sistemi kullanarak saflaştırılması. 10 mM HEPES tamponu (pH 7.3) ve 150 ug / ml bir solüsyon elde etmek için 260 nm &#39;lik bir emişteki pDNA konsantrasyonunun belirlenmesi. </li><li> (Hacim ile) 1: Polyplex nanomicelles hazırlamak için iyice pDNA çözeltisi (10 mM HEPES tampon içinde, 150 ug / ml) ve 2 oranında iki polimerin önceden karıştırılmış çözeltisi karıştırılır. </li></ol> Sferoidlerin içine 4. Gen Transfeksiyon <ol><li> (Hepatositleri hücreleri inkübeOlgun sferoidlerin oluşumuna izin vermek için micropatterned plakalar üzerine tohumlama sonra 72 saat boyunca ya da MSC&#39;ler). Gen Transfeksiyon için, taze ortam içinde 1 ml kültür ortamı değiştirilmesinden sonra her bir oyuğa (pDNA 10 ug içermektedir) Polyplex nanomicelle çözeltisi 100 ul ilave edin. 24 saat süre ile nanomicelle çözeltisi ile inkübasyon devam edin. </li><li> Bir lipid-bazlı transfeksiyon ayıracı kullanılarak, bir kontrol için, lipit bazlı bir reaktif ve nanomicelle yöntemleri için eşit olması pDNA nihai dozu ayarlama 3 bir ağırlık oranı, reaktif / pDNA ile bir reaktif ile pDNA çözümler karıştırın. </li></ol> Hücre sferoidlerin 5. Kurtarma ve Transplantasyon <ol><li> Soğutulmuş PBS içinde 200 ul kültür ortamı yerine ve buz üzerinde plakaları yerleştirin. Not: Genellikle, sferoidler, yaklaşık 15 dakika içinde ayırmak ve nakli için bir süspansiyon formunda elde edilebilir. </li><li> Yavaşça 200 ul hücreler aspirein vivo enjeksiyonu için 23 G veya 27 G iğne ile bir şırınga kullanılarak süspansiyon. </li></ol> Transjen 6. Değerlendirme <ol><li> Tam olarak 24 saat taze ortam ile değiştirildikten sonra ortam, 100 ul - Kültür ortamında salgılanan Gaussia lusiferaz ekspresyonu in vitro olarak değerlendirilmesi için, 50 toplar. Ticari bir Renilla lusiferaz deney sistemi ve üreticinin protokolüne uygun olarak, bir luminometre kullanılarak lusiferaz ekspresyonunu tahmin. NOT: Gaussia lusiferaz, bir haftadan fazla bir kültür ortamı içinde dengeli olarak kalır. Bu durumda, transgen ekspresyonunun gerçek zamanlı etkinliğinin değerlendirilmesi örnek ortamın toplanması öncesinde taze bir orta yerine. Orta değişimin zamanlaması esnek olabilir. </li><li> Hücre transplantasyonu, aşağıdaki ana hayvanlarda transgen ekspresyonu in vivo olarak değerlendirilmesi için, BALB / c çıplak fareler (dişi anestezi; 7 haftaizofluran ile inhalasyon anestezi altında eski). <ol><li> Bölmesi için izofluran sağlamak için bir anestezi makinesine bağlı bir odasında bir fare yerleştirin. Uykuya dalmakta sonra fareyi dışarı atın ve bir maske kullanarak havalandırma ile ameliyat masasında ayarlayın. Fare şartlarını kontrol ederek 0,5 L / dk - izofluran 0,2 yaklaşık akış kontrol edin. </li><li> Karın bölgesi içinde deri altı dokuya (4.1&#39;de tarif edildiği gibi) GL4 lusiferaz sentezleyen pDNA ile transfekte kürecikler içeren hücre süspansiyonu 200 ul enjekte edilir. </li><li> (150 mg / kg; damar içi yol ile) hemen D-luciferase enjekte sonra, üreticinin protokolüne uygun olarak IVIS görüntüleme sistemi kullanılarak lusiferaz ekspresyonunu ölçer. </li></ol></li><li> Hücre nakli tedavi edici etkilerini değerlendirmek için, içine eritropoetin kodlayan pDNA ile transfekte sferoitleri içeren hücre süspansiyonu 200 ul enjekteKarın bölgesi subkutan doku. <ol><li> Kan 19 yaklaşık 200 ul elde etmek submandibüler kanama kan örnekleri toplamak. Bir kan örneği analiz cihazı kullanılarak hemoglobin ve hematokrit ölçün. Not: nakli için hücre sayısı tabakalarına tohumlanmış ile regüle edilir. Ne yazık ki, sferoitler içindeki sayı ölçülen olamaz, çünkü tam hücre sayısını belirlemek zordur. </li></ol></li><li> Deneylerden sonra, anestezi uyanıyor kadar sıcaklık kontrolörü ile bağlı bir ısıtma yastığı fareler yerleştirin. </li></ol>

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The authors declare that they have no competing financial interests.

Bu protokol, gen girişi ve küremsi iyileşme aşamaları sırasında sferoitlerin 3 boyutlu yapısını muhafaza etmek önemlidir. Hücreler, hücre ölümünü ya da hücre aktivitesi kaybını önlemek için bir uygun mikroçevrelerde korumak için esastır. Geleneksel tek katmanlı kültürdeki hepatositler, hızla 12 tohumlamadan sonra, bir kaç gün kendi salgılama kapasitesini kaybeder Örneğin, albümin salgılama, hepatositlerin temsili bir doğuştan gelen işlevi, iyi, hepatosit sferoidler korun...

Hücre nakli tedavisi çeşitli inatçı hastalıkların tedavisi için yaygın ilgi gördü. nakledilen hücreler tarafından salgılanan aktivite ve biyolojik olarak aktif faktörlerin yarı-ömrü, bir hücre transplantasyonu sisteminin geliştirilmiş bir terapötik etkinliği için önemlidir. Transplantasyon öncesinde hücrelerin genetik modifikasyon düzenleyen ve biyolojik olarak aktif faktörlerin salgılanması da dahil olmak üzere hücresel fonksiyonları, işlemek için faydalı bir tekniktir. Bu hücre ölümünü ya da hücre aktivite kaybını önlemek için bir hücre için uygun bir mikro-ortam sağlamak için de önemlidir. Hücre-hücre etkileşimleri iyi korunmuş olan üç boyutlu (3D) küremsi hücre kültürü, (primer hepatosit albümini sekresyonu iyileştirilmesi ve mezenkimal kök hücrelerinden çok soyundan farklılaşmasını teşvik etmek için, örneğin, bu amaç için MKH&#39;lerin umut verici ) 1-7. Spheroi Bu çalışmada, yeni bir kombinasyon sistemid kültürü ve gen transfeksiyon genetiği değiştirilmiş hücre nakli için bir platform olarak hizmet için kullanılır. Küremsi hücreleri oluşturmak için, micropatterned kültür plakaları üzerinde küremsi kültür sistemi kullanılır. Bu plakalar halinde, 100 um çapında hücre yapışma alanları düzenli olarak iki boyutlu bir şekilde dizilir ve bir PEG matrisi 3 ile kaplanmış yapışkan olmayan bölgelerde ile çevrilidir. Hücrelerin yeterli sayıda tohum olarak, çapı 100 um 3D sferoitlerin dizileri micropatterned kültür yatağı tekabül oluşturulur. sferoidler bir ısıya duyarlı bir polimer, poli (izo-propylacrylamide) ile kaplanmıştır ısıya duyarlı hücre-kültür plakaları kullanılarak, 3B yapısına (PIPAAm) 8-10 bozmadan geri kazanılır. micropatterned mimari termosensitif plakalar üzerinde inşa edilmiştir (özel yapım). Sadece plakaların sıcaklığını düşürerek, sferoidler kültür yatağı ayrılır ve dağıtmak vardırfosfat D salin (PBS) tamponlu. Bu durumda, 100 um muntazam bir boyutu olan sferoitlerin çok sayıda enjekte edilebilir bir süspansiyon formunda elde edilebilir. <img alt="Figür 1" p="" src="/files/ftp_upload/52384/52384fig1.jpg" /> Şekil 1. micropatterned plaka üzerinde küremsi kültür sisteminin şematik temsili. Genetik değişiklik, orijinal viral olmayan gen taşıyıcı, Polyplex nanomicelle kullanılarak gen transfeksiyonu ile elde edilir. Bu plasmid DNA (pDNA) ve polietilen glikol (PEG) -polycation blok kopolimerleri 11 oluşmaktadır. Bunlar karakteristik çekirdek-kabuk yapısını, PEG kabuk ve yoğunlaştırılmış pDNA bir iç çekirdekten oluşan tedavi amaçlı 11 hücre içine güvenli ve etkili gen tanıtım izin var. <a href="https://www.jove.com/files/ftp_upload/52384/52384fig1large.jpg" target="_blank">Thi büyük halini görmek için tıklayınız</a>s rakam. <img alt="Şekil 2," src="/files/ftp_upload/52384/52384fig2.jpg" /> Şekil nükleik asitler ve PEG-blok-polikatyon blok kopolimerlerinin kompleks oluşturduğu Polyplex nanomicelle 2. yapısı. Bu çalışmada, bu tekniğin önemli avantajı, sferoid yapısı nanomicelles gen transfeksiyon sırasında bozulmamasına olmasıdır. Sıçan primer hepatosit küremsi nanomicelle aracılı transfeksiyonlar sonra, uzun süreli bir transgen sentezleme transfekte edilmemiş küremsi 12 ile karşılaştırılabilir bir seviyede hepatosit sürekli albümini sekresyonu fazla bir ay elde edilir. sferoidlerin gelen transgen ifadesi ve albumin salgısı da termosensitif plakalardan iyileştikten sonra sürdürülür. Bu nanomicelles güvenle HEP doğuştan fonksiyonlarını bozmadan gen tanıtım kolaylaştırabilir açıktıratocytes. Böylece, gen tanıtım nanomicelles kullanarak genetiği değiştirilmiş hücre nakli için umut verici bir platformdur ile termosensitif micropatterned plakalar üzerinde kültüre sfero hücrelerin kombinasyonu. <a href="https://www.jove.com/files/ftp_upload/52384/52384fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pen-Strep-Glut</td>
			<td>GIBCO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Wako Pure Chemical Industries</td>
			<td>041-18861</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Wako Pure Chemical Industries</td>
			<td>141-01202</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s buffered salt and L-ascorbic acid 2-phosphate (Asc-2P)</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>Human epidermal growth factor (hEGF)</td>
			<td>Toyobo</td>
			<td>PT10015</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell-able multi-well plates</td>
			<td>Toyo Gosei</td>
			<td>PP-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermosensitive cell culture plates (Upcell)</td>
			<td>CellSeed Inc</td>
			<td></td>
			<td>The micropatterned architecture is constructed on the thermosensitive plates (custom-built by Toyo Gosei)</td>
		</tr>
		<tr>
			<td>Lipid-based transfection reagent (FuGENE HD)</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>Renilla Luciferase Assay System</td>
			<td>Promega</td>
			<td>E2810</td>
			<td></td>
		</tr>
		<tr>
			<td>pGL4 Luciferase Reporter Vector</td>
			<td>Promega</td>
			<td>E6651</td>
			<td></td>
		</tr>
		<tr>
			<td>pDNA expressing Gaussia luciferase</td>
			<td>New England BioLabs</td>
			<td>N8082S</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse erhthropoietin-expressing vector</td>
			<td>Origene</td>
			<td>MC208445</td>
			<td></td>
		</tr>
		<tr>
			<td>pCAG-GS</td>
			<td></td>
			<td></td>
			<td>Kindly provided by Laboratory for Pluripotent Cell Studies, Center for Developmental Biology, RIKEN</td>
		</tr>
		<tr>
			<td>Escherichia coli DH5&#945; competent cells</td>
			<td>Takara</td>
			<td>9057</td>
			<td></td>
		</tr>
		<tr>
			<td>Endotoxin-free plasmid DNA purification system</td>
			<td>Nippon Genetics</td>
			<td>NucleoBond Xtra EF</td>
			<td></td>
		</tr>
		<tr>
			<td>collagenase</td>
			<td>Wako Pure Chemical Industries</td>
			<td>639-00951</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin inhibitor</td>
			<td>GIBCO</td>
			<td>R-007-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Promega</td>
			<td>GloMax&#8482; 96 Microplate Luminometer</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Imaging System</td>
			<td>Xenogen Corp.</td>
			<td>Xenogen IVIS Spectrum in vivo imaging system</td>
			<td></td>
		</tr>
		<tr>
			<td>blood sample analyzer</td>
			<td>Sysmex</td>
			<td>pocH-100i Automated Hematology Analyzer</td>
			<td></td>
		</tr>
	</tbody>
</table>

gene transfection toward spheroid cells on micropatterned culture plates for genetically-modified cell transplantation

Hücre nakli terapötik etkinliğini artırmak için, genetik olarak tadil edilmiş, enjekte küremsi bir transplantasyon sistemi geliştirilmiştir. Hücre sferoidler bir ısıya duyarlı bir polimer ile kaplanmış micropatterned plakalar üzerinde bir kültür sistemi içinde hazırlanır. Sferoidlerin bir dizi, bir polietilen glikol (PEG) matris ile kaplanır yapıştırıcı olmayan bölgeler tarafından sarılmış düzenli olarak iki boyutlu bir şekilde dizilmiş olan 100 um çaplı hücre yapışma alanları, tekabül eden plakalar üzerinde oluşturulur. sferoidler kolay plakaların sıcaklığın düşürülmesiyle, bir sıvı süspansiyon olarak elde edilebilir, ve bunların yapısı iyi (27 g fazla) yeteri kadar büyük bir kalibreye sahip enjeksiyon iğneleri içinden geçirilerek muhafaza edilmektedir. Genetik modifikasyonlar küremsi yapıyı bozmadan hücrelerine gen dahil etme yeteneğine sahip olan, orijinal viral olmayan gen taşıyıcı, Polyplex nanomicelle, kullanılarak gen transfeksiyonu ile elde edilir. Prim içinalbümin ekspresyonu ile gösterildiği gibi, bir lusiferaz sentezleyen bir gen ile transfekte ary hepatosit sferoidler, lusiferaz sürdürülebilir, korunmuş hepatosit işlevi ile birlikte, nakledilen hayvanlarda elde edilir. Bu sistem, mezenkimal kök hücreleri dahil olmak üzere hücre tiplerinin çeşitli uygulanabilir.

Gaussia lusiferaz sentezleyen pDNA gene Polyplex nanomicelles veya kontrol lipit bazlı transfeksiyon reaktifi 12&#39;yi kullanarak hepatositlere ya da MSC tarafından oluşturulan sferoidler uygulandı. kontrol reaktif anlamlı transfeksiyon (Şekil 3) bir gün sonra bir yapı kesintiye ise nanomicelles micropatterned tabakalarına transfekte-olmayan sferoidler kıyasla küremsi yapısı hemen hemen herhangi bir değişiklik neden olmuştur. Nanomicelles tutarlı albümini sekresyonu ve tran...

Watch this Scientific Journal Video about Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation at JoVE.com

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

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

Genetik olarak tadil edilmiş hücre transplantasyonu için Micropatterned kültür plakalarında Sferoid hücrelere karşı gene

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

gene-transfection-toward-spheroid-cells-on-micropatterned-culture

Research

JoVE Journal

Bioengineering

8.7K Görüntüleme.  The University of Tokyo. This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

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

Micropatterned Surfaces to Study Hyaluronic Acid Interactions with Cancer Cells

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix (ECM) components within the tumor microenvironment. Hyaluronic acid (HA) is one stromal ECM component that is known to facilitate tumor progression by enhancing invasion, growth, and angiogenesis1. Interaction of HA with its cell surface receptor CD44 induces signaling events that promote tumor cell growth, survival, and migration, thereby increasing metastatic spread2-3. HA is an anionic, nonsulfated glycosaminoglycan composed of repeating units of D-glucuronic acid and D-N-acetylglucosamine. Due to the presence of carboxyl and hydroxyl groups on repeating disaccharide units, native HA is largely hydrophilic and amenable to chemical modifications that introduce sulfate groups for photoreative immobilization 4-5. Previous studies involving the immobilizations of HA onto surfaces utilize the bioresistant behavior of HA and its sulfated derivative to control cell adhesion onto surfaces6-7. In these studies cell adhesion preferentially occurs on non-HA patterned regions.
To analyze cellular interactions with exogenous HA, we have developed patterned functionalized surfaces that enable a controllable study and high-resolution visualization of cancer cell interactions with HA. We utilized microcontact printing (uCP) to define discrete patterned regions of HA on glass surfaces. A "tethering" approach that applies carbodiimide linking chemistry to immobilize HA was used 8. Glass surfaces were microcontact printed with an aminosilane and reacted with a HA solution of optimized ratios of EDC and NHS to enable HA immobilization in patterned arrays. Incorporating carbodiimide chemistry with mCP enabled the immobilization of HA to defined regions, creating surfaces suitable for in vitro applications. Both colon cancer cells and breast cancer cells implicitly interacted with the HA micropatterned surfaces. Cancer cell adhesion occurred within 24 hours with proliferation by 48 hours. Using HA micropatterned surfaces, we demonstrated that cancer cell adhesion occurs through the HA receptor CD44. Furthermore, HA patterned surfaces were compatible with scanning electron microscopy (SEM) and allowed high resolution imaging of cancer cell adhesive protrusions and spreading on HA patterns to analyze cancer cell motility on exogenous HA.

A novel approach that allows the high-resolution analysis of cancer cell interactions with exogenous hyaluronic acid (HA) is described. Patterned surfaces are fabricated by combining carbodiimide chemistry and microcontact printing.

Micropatterned Yüzeyler Kanser Hücreleri ile Hyaluronik Asit Etkileşimleri Eğitim

Cancer invasion and progression involves a motile cell phenotype, which is under complex regulation by growth factors/cytokines and extracellular matrix ...

Biyomühendislik

Improved Visualization and Quantitative Analysis of Drug Effects Using Micropatterned Cells

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck 1-2.
To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

Adhesive micropatterns that normalize cellular architecture can be used to increase sensitivity in the detection of drug effects, improve reproducibility and simplify automated image acquisition and analysis. Such technology will benefit drug/siRNA screening assays, performed on conventional cell culture supports and consequently suffering from excessive cell-to-cell variability.

İlaç Etkisi Micropatterned Hücreler Geliştirilmiş Görselleştirme ve Kantitatif Analiz

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated ...

Fabrication of Micropatterned Hydrogels for Neural Culture Systems using Dynamic Mask Projection Photolithography

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the need for 3D environments to represent in vitro experiments which better mimic in vivo qualities 1-3. Studies in fields such as cancer research 4, neural engineering 5, cardiac physiology 6, and cell-matrix interaction7,8have shown cell behavior differs substantially between traditional monolayer cultures and 3D constructs.
Hydrogels are used as 3D environments because of their variety, versatility and ability to tailor molecular composition through functionalization 9-12. Numerous techniques exist for creation of constructs as cell-supportive matrices, including electrospinning13, elastomer stamps14, inkjet printing15, additive photopatterning16, static photomask projection-lithography17, and dynamic mask microstereolithography18. Unfortunately, these methods involve multiple production steps and/or equipment not readily adaptable to conventional cell and tissue culture methods. The technique employed in this protocol adapts the latter two methods, using a digital micromirror device (DMD) to create dynamic photomasks for crosslinking geometrically specific poly-(ethylene glycol) (PEG) hydrogels, induced through UV initiated free radical polymerization. The resulting "2.5D" structures provide a constrained 3D environment for neural growth. We employ a dual-hydrogel approach, where PEG serves as a cell-restrictive region supplying structure to an otherwise shapeless but cell-permissive self-assembling gel made from either Puramatrix or agarose. The process is a quick simple one step fabrication which is highly reproducible and easily adapted for use with conventional cell culture methods and substrates.
Whole tissue explants, such as embryonic dorsal root ganglia (DRG), can be incorporated into the dual hydrogel constructs for experimental assays such as neurite outgrowth. Additionally, dissociated cells can be encapsulated in the photocrosslinkable or self polymerizing hydrogel, or selectively adhered to the permeable support membrane using cell-restrictive photopatterning. Using the DMD, we created hydrogel constructs up to ~1mm thick, but thin film (&lt;200 &mu;m) PEG structures were limited by oxygen quenching of the free radical polymerization reaction. We subsequently developed a technique utilizing a layer of oil above the polymerization liquid which allowed thin PEG structure polymerization.
In this protocol, we describe the expeditious creation of 3D hydrogel systems for production of microfabricated neural cell and tissue cultures. The dual hydrogel constructs demonstrated herein represent versatile in vitro models that may prove useful for studies in neuroscience involving cell survival, migration, and/or neurite growth and guidance. Moreover, as the protocol can work for many types of hydrogels and cells, the potential applications are both varied and vast.

Simple techniques are described for the rapid production of microfabricated neural culture systems using a digital micromirror device for dynamic mask projection lithography on regular cell culture substrates. These culture systems may be more representative of natural biological architecture, and the techniques described could be adapted for numerous applications.

Dinamik Maske Projeksiyon Fotolitografi kullanarak Sinir Kültür Sistemleri Micropatterned Hidrojeller Fabrikasyon

Increasingly, patterned cell culture environments are becoming a relevant technique to study cellular characteristics, and many researchers believe in the ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

The field of human trophoblast research aids in understanding the complex environment established during placentation. Due to the nature of these studies, human in vivo experimentation is impossible. A combination of primary cultures, explant cultures and trophoblast cell lines1 support our understanding of invasion of the uterine wall2 and remodeling of uterine spiral arteries3,4 by extravillous trophoblast cells (EVTs), which is required for successful establishment of pregnancy. Despite the wealth of knowledge gleaned from such models, it is accepted that in vitro cell culture models using EVT-like cell lines display altered cellular properties when compared to their in vivo counterparts5,6. Cells cultured in the rotating cell culture system (RCCS) display morphological, phenotypic, and functional properties of EVT-like cell lines that more closely mimic differentiating in utero EVTs, with increased expression of genes mediating invasion (e.g. matrix metalloproteinases (MMPs)) and trophoblast differentiation7,8,9. The Saint Georges Hospital Placental cell Line-4 (SGHPL-4) (kindly donated by Dr. Guy Whitley and Dr. Judith Cartwright) is an EVT-like cell line that was used for testing in the RCCS.
The design of the RCCS culture vessel is based on the principle that organs and tissues function in a three-dimensional (3-D) environment. Due to the dynamic culture conditions in the vessel, including conditions of physiologically relevant shear, cells grown in three dimensions form aggregates based on natural cellular affinities and differentiate into organotypic tissue-like assemblies10,11,12 . The maintenance of a fluid orbit provides a low-shear, low-turbulence environment similar to conditions found in vivo. Sedimentation of the cultured cells is countered by adjusting the rotation speed of the RCCS to ensure a constant free-fall of cells. Gas exchange occurs through a permeable hydrophobic membrane located on the back of the bioreactor. Like their parental tissue in vivo, RCCS-grown cells are able to respond to chemical and molecular gradients in three dimensions (i.e. at their apical, basal, and lateral surfaces) because they are cultured on the surface of porous microcarrier beads. When grown as two-dimensional monolayers on impermeable surfaces like plastic, cells are deprived of this important communication at their basal surface. Consequently, the spatial constraints imposed by the environment profoundly affect how cells sense and decode signals from the surrounding microenvironment, thus implying an important role for the 3-D milieu13.
We have used the RCCS to engineer biologically meaningful 3-D models of various human epithelial tissues7,14,15,16. Indeed, many previous reports have demonstrated that cells cultured in the RCCS can assume physiologically relevant phenotypes that have not been possible with other models10,17-21. In summary, culture in the RCCS represents an easy, reproducible, high-throughput platform that provides large numbers of differentiated cells that are amenable to a variety of experimental manipulations. 
In the following protocol, using EVTs as an example, we clearly describe the steps required to three-dimensionally culture adherent cells in the RCCS.

Traditional, two dimensional cell culture techniques often result in altered characteristics with respect to differentiation markers, cytokines and growth factors. Three-dimensional cell culture in the rotating cell culture system (RCCS) reestablishes expression of many of these factors as shown here with an extravillous trophoblast cell line.

İnsan Hücre Kültürü Döner Hücre Kültür Sistemleri: Bir Model Olarak İnsan trofoblast Hücreleri

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Mass Cytometry: Protocol for Daily Tuning and Running Cell Samples on a CyTOF Mass Cytometer

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the issue of spectral overlap of fluorophore emissions has limited the number of simultaneous probes. In contrast, the new CyTOF mass cytometer by DVS Sciences couples a liquid single-cell introduction system to an ICP-MS.1 Rather than fluorophores, chelating polymers containing highly-enriched metal isotopes are coupled to antibodies or other specific probes.2-5 Because of the metal purity and mass resolution of the mass cytometer, there is no "spectral overlap" from neighboring isotopes, and therefore no need for compensation matrices. Additionally, due to the use of lanthanide metals, there is no biological background and therefore no equivalent of autofluorescence. With a mass window spanning atomic mass 103-203, theoretically up to 100 labels could be distinguished simultaneously. Currently, more than 35 channels are available using the chelating reagents available from DVS Sciences, allowing unprecedented dissection of the immunological profile of samples.6-7
Disadvantages to mass cytometry include the strict requirement for a separate metal isotope per probe (no equivalent of forward or side scatter), and the fact that it is a destructive technique (no possibility of sorting recovery). The current configuration of the mass cytometer also has a cell transmission rate of only ~25%, thus requiring a higher input number of cells.
Optimal daily performance of the mass cytometer requires several steps. The basic goal of the optimization is to maximize the measured signal intensity of the desired metal isotopes (M) while minimizing the formation of oxides (M+16) that will decrease the M signal intensity and interfere with any desired signal at M+16. The first step is to warm up the machine so a hot, stable ICP plasma has been established. Second, the settings for current and make-up gas flow rate must be optimized on a daily basis. During sample collection, the maximum cell event rate is limited by detector efficiency and processing speed to 1000 cells/sec. However, depending on the sample quality, a slower cell event rate (300-500 cells/sec) is usually desirable to allow better resolution between cells events and thus maximize intact singlets over doublets and debris. Finally, adequate cleaning of the machine at the end of the day helps minimize background signal due to free metal.

The steps necessary for daily tuning and optimization of the performance of a CyTOF mass cytometer are described. Comments on optimal sample preparation and flow rate are discussed

Kütle Sitometri: Bir CyTOF Kütle Sitometre Daily Tuning ve Koşu Hücre Örnekleri için protokol

In recent years, the rapid analysis of single cells has commonly been performed using flow cytometry and fluorescently-labeled antibodies. However, the ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Kas Yenilenmesinin Fare Modellerinde İndüklenmiş Pluripotent Kök Hücre türevi Mezoangioblast benzeri Miyojenik Progenitörlerin Nakli

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

PDMS Zarında Micropatterned Hücreler germe

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues.
Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

We present a method for microfluidic deposition of patterned genipin and fibronectin on PDMS substrates, allowing extended viability of vascular smooth muscle cell-dense tissues. This tissue fabrication method is combined with previous vascular muscular thin film technology to measure vascular contractility over disease-relevant time courses.

Micropatterned Vasküler Musküler İnce Filmlerin Genişletilmiş Kültür mikroakışkan Genipin Biriktirme Tekniği

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Segmenting Growth of Endothelial Cells in 6-Well Plates on an Orbital Shaker for Mechanobiological Studies

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and multidirectional shear stresses have all been postulated to stimulate a pro-atherosclerotic phenotype in endothelial cells, whereas high magnitude and unidirectional or uniaxial shear are thought to promote endothelial homeostasis. These hypotheses require further investigation, but traditional in vitro techniques have limitations, and are particularly poor at imposing multidirectional shear stresses on cells. 
One method that is gaining increasing use is to culture endothelial cells in standard multi-well plates on the platform of an orbital shaker; in this simple, low-cost, high-throughput and chronic method, the swirling medium produces different patterns and magnitudes of shear, including multidirectional shear, in different parts of the well. However, it has a significant limitation: cells in one region, exposed to one type of flow, may release mediators into the medium that affect cells in other parts of the well, exposed to different flows, hence distorting the apparent relation between flow and phenotype. 
Here we present an easy and affordable modification of the method that allows cells to be exposed only to specific shear stress characteristics. Cell seeding is restricted to a defined region of the well by coating the region of interest with fibronectin, followed by passivation using passivating solution. Subsequently, the plates can be swirled on the shaker, resulting in exposure of cells to well-defined shear profiles such as low magnitude multidirectional shear or high magnitude uniaxial shear, depending on their location. As before, the use of standard cell-culture plasticware allows straightforward further analysis of the cells. The modification has already allowed the demonstration of soluble mediators, released from endothelium under defined shear stress characteristics, that affect cells located elsewhere in the well.

This protocol describes a coating method to restrict endothelial cell growth to a specific region of a 6-well plate for shear stress application using the orbital shaker model.

Mechanobiyolojik Çalışmalar için Orbital Çalkalayıcıda Endotel Hücrelerinin 6 Kuyu Plakalarında Parçalanması

Shear stress imposed on the arterial wall by the flow of blood affects endothelial cell morphology and function. Low magnitude, oscillatory and ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Sessiz İnsan Hematopoetik Kök Hücrelerini Korumak İçin Bir Kültür Yöntemi

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...