Name: preparation of chitosan-based injectable hydrogels and its application in 3d cell culture
Uploaded: 2017-09-29T14:00:00.000+00:00
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Bu araştırma Ulusal Bilim Vakfı Çin tarafından (21474057 ve 21604076) destek verdi.

dikkat: ilgili tüm malzeme güvenlik bilgi formları (MSDS) kullanmadan önce lütfen danışın. Lütfen uygun güvenlik uygulamaları kimya deneyleri, bir duman hood ve kişisel koruyucu ekipman (koruyucu gözlük, koruyucu eldiven, önlük, vb) kullanımı da dahil olmak üzere yerine getirirken kullanın. Standart hücre işleme teknikleri (sterilizasyon, hücre kurtarma, cep passaging, hücre dondurma, hücre boyama, vb) protokolü gerektirir.
 1. hazırlık Hydrogels <ol><li><ol><li>öncesi kuruma, PEG benzaldehyde sentezi di functionalized polietilen glikol (DF PEG) sona polimer <ol><li>tartmak 4.00 g PEG (MW 4.000, 1.00 mmol), bir yuvarlak alt şişesi (250 mL) aktarmak ve toluen (100 mL) ekleyin. 
			Not: Farklı molekül ağırlıkları ile diğer mandal hidrojel oluşumu için çalışabilir ancak farklı sertlik götürecek.</li>
			<li>Isı tabancası (ya da sıcak bir tabak yaklaşık 40 ° C) hafif polimer çözülür yardımcı olmak için çözüm ısı. Tüm polimerler geçiyoruz sonra bir Buharlaştırıcı tüm çözücüler kaldırmak için kullanın. Bu erime ve kuru süreç kurutulmuş PEG polimerler yapmak için iki kez yineleyin. 
			Dikkat: Bu bir duman başlıklı aşırı özenle yapılmalıdır. Toluen yanıcı.</li>
		</ol></li> <li>Benzaldehyde fesih tepki PEG <ol><li>şişesi için manyetik karıştırıcı ve tetrahydrofuran (THF, 100 mL) ekleyip PEG bir karıştırıcı kullanarak geçiyoruz. 4-carboxybenzaldehyde (0,90 g, 6.0 mmol) ve 4-dimethylaminopyridine (0,07 g, 0.6 mmol) sırayla tüm katı tamamen erimesi için çözümler ekleyin.</li>
			<li>Ekle N, N &#39;-dicyclohexylcarbodiimide (DCC, 1,25 g, 6.0 mmol) çözümü ve susuz CaCl 2 şişeye doldurulur bir kurutma tüp ekleyin. Reaksiyon karıştırma altında oda sıcaklığında tutmak (~ 20 ° C) için yaklaşık 12 h.</li>
		</ol></li> <li>Sonrası tepki süreci <ol><li>reaksiyon tamamlandıktan sonra vakum tarafından çözümde oluşturulan beyaz katı filtre. Çözüm beyaz katı çökelti için karıştırma altında soğuk Dietil eter (500 mL) dökün. Beyaz katı göre filtrele toplamak ve katı bir duman başlıklı kuru.</li>
			<li>Beyaz katı THF yeniden dağıtılması, çözünmez herhangi bir beyaz katılar filtre, çözüm taze soğuk Dietil beyaz katı çökelti eter dökün ve kuru. 2-3 kez bu adımları tekrarlayın ve sonra beyaz katı bir vakum kurutma fırın (20 ° C, 0.1 mbar) onları tamamen kurumasını yerleştirin. Nihai ürün olarak beyaz toz toplamak: benzaldehyde sona DF PEG.</li>
		</ol></li></ol></li> <li>Hydrogels hazırlanması <ol><li>tartmak DF PEG (0,11 g, 0,028 mmol 0,22 g, 0,055 mmol; 0,44 g, 0.110 mmol) farklı miktarda bir tüp (10 mL), deiyonize su (5.0 mL) ekleyin ve bir girdap veya manyetik karıştırıcı yardımcı olması için kullanın Polimer çözülür.</li>
		<li>Glikol chitosan (0.495 g, x 10 -3 mmol 6,18) deiyonize su (15,0 mL) bir tüp (50 mL) dağıtılması ve kitosan çözüm (3 wt %) homojenize için birkaç dakika için bir girdap kullanın.</li>
		<li>Glikol kitosan çözüm (0.2 mL, 3 wt %) ve DF PEG Çözümleri (0.2 mL) sırayla bir tüp (2.0 mL) ekleyin. Bir girdap formu hydrogels homojen çözümlerinize birkaç dakika içinde karıştırmak için kullanın. Hydrogels farklı mekanik güçlü yapmak için Tablo 1 ' deki oranları izleyin. 
		Not: hidrojel zaten oluşmuş olup olmadığını belirlemek için tüp ters çevirme yöntemini kullanın.</li>
	</ol></li> <li>Reolojisi analizleri <ol><li>Reolojisi analizleri dönüş rheometer paralel bir çelik plaka üzerinde yürütmek (çapı: 20 mm). Jelleşme test için Glikol kitosan çözüm (0.2 mL, 3 wt %) daha düşük bir tabağa yayıldı. O zaman, DF PEG sulu çözümler (0.2 mL, 2 wt %) eşit olarak dropwise kitosan çözüm yüzeye ve hızlı bir şekilde bir pipet ile karıştırın. Düşük aşağı üst plaka ve test için başlar</li>
		<li>İçin hidrojel &#39; s sertlik testi, kesilmiş bir hidrojel bir çember içine (çapı: 20 mm) ve depolama modülü ölçmek (G &#39;) değerleri frekans analizleri % 1 zorlanma, karşı. Tipik G &#39;: 6,3 rad s değerleri &#8722; 1 Tablo 1 ' de listelenen. 
		Not: 0,5 h hidrojel Reolojisi analizleri hidrojel dinamik bağ istikrar sağlamak için hidrojel oluşumu sonra taşımak.</li>
		<li>İçin hidrojel &#39; s kendi kendine healable özelliği testi, bir hidrojel paramparça ve duyulursa ayrılmaz bir parçası için parçaları bir araya toplamak. Bir daire yapmak hidrojel parça kesme (çapı: 20 mm) ve Reolojisi analizlerini incelemek rheometer üzerinde koydu.</li>
	</ol></li></ol> 2. Hydrogels 3D cep ekimi <ol><li>hidrojel jelleşme çözümleri hazırlanması <ol><li>tartmak DF PEG (0,44 g, 0,11 mmol) steril tüp (4.0 mL), hücre kültürü medya (RPMI-1640, 2.0 eklemek mL) ve bir girdap veya karıştırıcı polimer çözüm (20 wt %) elde etmek için polimer çözülür yardımcı olması için kullanın. Çözüm bir şırınga (10.0 mL) içinde yük ve sonra mikrop bakteri kuvvetli filtreden (0,22 µm) geçirerek sterilize.</li>
		<li>Glikol chitosan (0,165 g, 2,06 x 10 -3 mmol) bir steril tüp (15,0 mL) tartmak, hücre kültürü medya (RPMI-1640, 4.0 mL) ve girdap glikol kitosan çözüm (4.0 wt %) elde etmek için polimer çözülür yardımcı olmak için ekleyin. Bir şırınga (10.0 mL) çözümde yük ve 0,22 µm bakteri kuvvetli filtre ile filtre.</li>
	</ol></li> <li>Hydrogels hücre ekimi dikkat: gerçekleştirmek tüm doku kültürü başlıklı ilgili yordamları cep. Steril tekniği konusunda temel bilgi bekleniyor.
	<ol><li>Hücre süspansiyon <ol><li>RPMI-1640 hücrelerde kültür L929 hazırlanması orta % 10 ile desteklenmiş FBS, %5 penisilin (10 mL) bir Petri çanak (çapı 10 cm) ve 37 ° C, % 5 CO 2 kuluçkaya. Orta kullanmadan önce her gün değiştirin.</li>
			<li>Hasat L929 hücreleri tripsin (0.025 w/v %) ve EDTA içeren PBS ile (% 0,01 w/v), (70 x g, 5 min) santrifüj kapasitesi ve RPMI-1640 orta (1.0 mL) hücreleri yeniden askıya alma. Kan sayım kurulu standart operasyon kullanarak hücre sayımı gerçekleştirmek. Hücre toplama ayarlamak için hücreleri yeniden askıya ~ 3.75 × 10 6 hücre/mL.</li>
		</ol></li> <li>Hydrogels hücre katma <ol><li>L929 hücre süspansiyonlar (0.4 mL, 3,75 x 10 6 hücre mL -1) glikol kitosan çözüm (0.4 mL) girdap tarafından bir tüp (4.0 mL) ile karıştırın. L929/glikol kitosan çözüm (0.8 mL) confocal Petri kabına ortasına pipette (çapı 2,0 cm). DF PEG Çözümleri (0.2 mL) aynı çanak içine pipette ve yavaşça çözüm mix ve hidrojel oluşumu teşvik pipet. 
			Not: hidrojel oluşumu petri kabına eğerek değerlendirmek.</li>
			<li>Doğrudan hücre kültürü için ek RPMI-1640 kültür medya (1.0 mL) hidrojel üstüne miktarda ekleyebilirsiniz. Gömülü hücre hydrogels (1.0 mL, 1,5 wt % glikol kitosan, 4.0 wt % DF PEG, 1.5 × 10 6 hücre mL -1) (37 ° C, % 5 CO 2) bir kuluçka makinesine koy ve orta her gün değiştirin. Gün 1, 3, 5 ve 7'den sonra hücre kapsülleme hücre görüntüleme için hazır olun.</li>
		</ol></li> <li>3D cep sonrası kültür için hydrogels enjeksiyon sonra yüklü hücre hidrojel hazırlamak (1.0 mL, bkz: adım 2.2.2) bir şırınga (10.0 mL, 48 G iğne) içinde. Sonra hidrojel formları hidrojel yavaş yavaş bir petri confocal görüntüleme için içine enjekte. Üstünde tepe-in hidrojel (1.0 mL) kültür ortamının ek bir miktar ekleyin ve her gün değiştirin. Petri kabına (37 ° C, % 5 CO 2) bir kuluçka makinesine koy ve daha sonra görüntüleme için hazır olun. 
		Dikkat: Lütfen kontrol edin ve bir şırınga Emanet işlem protokolü izleyin.</li>
	</ol></li> <li>Hücre canlılığı analizi <ol><li>Confocal gözlem <ol>
			<li>PBS (1.0 mL) ile hydrogels için iki kez yıkayın. Floresein diacetate (FDA, 0.5 mL, 0,05 mg/mL) ve propidium iyodür (PI, 0.5 mL, 0,08 mg/mL) çözümleri için 15 dk ile hydrogels leke. Boyama sonra tüm maddeleri kaldırın.</li>
			<li>Gözlemlemek confocal mikroskop altında uyarma dalgaboyu 488, kullanarak hydrogels nm ve 543 canlı görselleştirmek için nm ve ölü hücreleri, anılan sıraya göre. Z-yığınlar hücreler eşit dağılımı doğrulamak için hydrogels her 2 µm derinliğe üzerinden alın. 
			Not: PI ölü hücreleri lekeleri ise FDA canlı hücreleri lekeleri.</li>
		</ol></li> <li>Hidrojel (1.0 mL) Asetik asit (HAc, 3 v %, 1.0 mL) 5 min ve pipet ile bir tüpe (4.0 mL) bozulmasına yol açar. Hücreleri tarafından santrifüj (70 x g, 5 min) toplamak ve RPMI-1640 hücre kültür orta (1.0 mL) hücreleri yeniden askıya alma. Bir kan sayım kurulu kullanarak hücre sayımı gerçekleştirmek.</li>
	</ol>
	</li>
</ol>

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Bu Protokolü (şekil 1) sunulan hidrojel iki ana bileşenden oluşur: doğal polimer glikol chitosan ve sentetik bir benzaldehyde sona polimer gelator DF PEG, hangi her iki Biyouyumlu malzemelerdir. DF PEG sentezi bir tek adımlı değişiklik tepki kullanılarak sunulur. Moleküler ağırlık 4.000 PEG bu protokol çözünürlük, değişiklik verimliliği gibi hidrojel sertlik endişelerini için seçildi. Hydrogels ile farklı mekanik güçlü bir dizi hazırlanan kullanarak farklı kat?...

Hydrogels çapraz polimer malzemeler büyük miktarda su ve yumuşak mekanik özellikleri ile ve birçok bio-tıbbi uygulamalar1,2' kullanılmıştır. Hydrogels hücreleri içinde vivoiçin fizyolojik çevre çok benzer bir yumuşak ve ıslak ortam sunmaktadır. Bu nedenle, hydrogels 3D hücre kültür3,4için en popüler iskele biri haline gelmiştir. 2D Petri kabına hücre kültürü için karşılaştırıldığında, 3D hücre kültürü, microenvironment başvurun ve yayılmasını önleme ve ayırt etme amacıyla5için bir araya hücreler için bir hücre dışı Matriks (ECM) taklit sunmak için hızla ilerlemiştir. Ayrıca, doğal polimerler içeren hydrogels biyo uyumlu ve teşvik ortamları çoğalırlar ve3ayırt etmek için hücreleri için önerebiliriz. Sentetik polimerler elde Hydrogels hayvan kökenli proteinler veya virüs gibi karmaşık etkileri dışlamak onların basit ve net bileşenleri için tercih edilir. Tüm hidrojel adaylar arasında 3D hücre kültürü için kolayca hazırlanır ve tutarlı bir özellik olan hydrogels her zaman tercih edilir. Farklı araştırma gereksinimleri uyacak hidrojel'ın özelliklerini ayarlamak için tesisin iyi6önemlidir.
Burada bir çok yönlü hidrojel platform 3D hücre kültür7için olur dinamik imin kimya kullanarak bir glikol kitosan tabanlı hidrojel facile hazırlanması tanıtmak. Bu yöntemde, iyi bilinen biyo uyumlu glikol kitosan çerçeveler hidrojel'ın ağlar kurmak için kullanılır. Onun amino grupları ile sona benzaldehyde polietilen glikol dinamik imin Tahvil hydrogels8crosslinkages oluşturmak için polimer gelator olarak tepki. Dinamik imin Tahvil formu ve çevresi, hydrogels mekanik olarak ayarlanabilir çapraz ağlar9,10,11ile endowing için geri döndürülebilir ve responsively ayrıştırmak. Yüksek su içeriği, biyo uyumlu malzemeler ve ayarlanabilir mekanik güçlü nedeniyle hidrojel başarıyla 3D hücre kültürü12,13L929 hücreler için bir iskele olarak uygulanır. İletişim kuralı burada polimer gelator sentezi, hidrojel hazırlama, hücre katıştırma ve 3D hücre kültürü çalışmalarının da dahil olmak üzere yordamlar, ayrıntıları.
Hidrojel aynı zamanda birçok diğer özellikleri nedeniyle çok duyarlı çeşitli biyo-uyarıcılara (asit/pH, B6 vitamini türevi piridoksal, protein papain, vb) dahil olmak üzere, hidrojel olabileceğini gösteren onun dinamik imin crosslinkages gösterir fizyolojik şartlarda8altında çürümeye bağlı. Hidrojel hidrojel minimal invaziv enjeksiyon yöntemi ile idare ve uyuşturucu ve hücre teslimatlar14,15dakika sonra bir avantaj Yani kendi kendine healable ve enjekte edilebilir, da. Hidrojel işlevsel katkı maddeleri veya belirli önceden tasarlanmış polimer gelators ekleyerek, manyetik, sıcaklık, pH duyarlı, hangi yerine vb16,17, gibi belirli özellikleri kazanıyor için uyumlu bir araştırma gereksinimleri çok çeşitli. Bu özellikleri bir enjekte edilebilir olmak potansiyel kapasite hidrojel'ın uyuşturucu ve hücreler içinde in vitro ve in vivo bio-Medikal araştırma ve uygulamalar için birden fazla taşıyıcıları ortaya koyuyor.

<table><tbody><tr><td>Glycol chitosan</td><td>Wako Pure Chemical Industries</td><td>39280-86-9</td><td>90% degree of deacetylation</td></tr><tr><td>4-Carboxybenzaldehyde</td><td>Shanghai Aladdin&amp;nbsp;Bio-Chem Technology Co.,LTD</td><td>619-66-9</td><td>99%</td></tr><tr><td>N, N&#039;-dicyclohexylcarbodiimide</td><td>Shanghai Aladdin&amp;nbsp;Bio-Chem Technology Co.,LTD</td><td>538-75-0</td><td>99%</td></tr><tr><td>Calcium chloride anhydrous</td><td>Shanghai Aladdin&amp;nbsp;Bio-Chem Technology Co.,LTD</td><td>10043-52-4</td><td>96%</td></tr><tr><td>4-dimethylamiopryidine</td><td>Shanghai Aladdin&amp;nbsp;Bio-Chem Technology Co.,LTD</td><td>1122-58</td><td>99%</td></tr><tr><td>Polyethyleneglycol</td><td>Sino-pharm Chemical Reagent</td><td>5254-43-7</td><td>99%</td></tr><tr><td>Tetrahydrofuran</td><td>Sino-pharm Chemical Reagent</td><td>109-99-9</td><td>99%</td></tr><tr><td>Toluene</td><td>Sino-pharm Chemical Reagent</td><td>108-88-3</td><td>99%</td></tr><tr><td>Ethyl ether</td><td>Sino-pharm Chemical Reagent</td><td>60-29-7</td><td>99%</td></tr><tr><td>Acetic acid</td><td>Sino-pharm Chemical Reagent</td><td>64-19-7</td><td>99%</td></tr><tr><td>Anhydrous CaCl2</td><td>Sino-pharm Chemical Reagent</td><td>10043-52-4</td><td>99%</td></tr><tr><td>Fluorescein diacetate</td><td>Sigma</td><td>596-09-8</td><td>99%</td></tr><tr><td>Propidium iodide&amp;nbsp;</td><td>Sigma</td><td>25535-16-4</td><td>94%</td></tr><tr><td>RPMI-1640 culture media</td><td>Gibco</td><td></td><td></td></tr><tr><td>Fetal bovine serum</td><td>Gibco</td><td></td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Gibco</td><td></td><td>0.25%</td></tr><tr><td>PBS</td><td>Solarbio</td><td></td><td>0.01 M</td></tr><tr><td>Penicillin streptomycin solution</td><td>Hyclone</td><td></td><td>10,000 U/mL</td></tr><tr><td>Rheometer</td><td>TA Instrument</td><td></td><td>AR-G2</td></tr><tr><td>Confocal microscope</td><td>Zeiss</td><td></td><td>710-3channel</td></tr><tr><td>L929 Cells</td><td>ATCC</td><td></td><td>NCTC clone 929; L cell, L929, derivative of Strain L</td></tr><tr><td>Evaporator</td><td>EYELA</td><td></td><td>N-1100</td></tr><tr><td>48 guage needle</td><td>ShanghaiZhiyu Medical Material Co., LTD</td><td></td><td>48-guage</td></tr><tr><td>Microscope</td><td>Leica</td><td></td><td>DM3000 B</td></tr><tr><td>Microscope software</td><td>Imaris</td><td></td><td></td></tr><tr><td>Heat gun</td><td>Confu</td><td></td><td>KF-5843&amp;nbsp;</td></tr><tr><td>Petri dish</td><td>NEST</td><td></td><td></td></tr></tbody></table>

preparation of chitosan-based injectable hydrogels and its application in 3d cell culture

Protokol kitosan tabanlı hydrogels dinamik imin kimya kullanarak hazırlamak için facile, verimli ve çok yönlü bir yöntem sunuyor. Hidrojel çözümleri glikol kitosan, sentezlenmiş benzaldehyde sonlandırıldı polimer gelator ile karıştırılarak hazırlanır ve hydrogels verimli bir şekilde oda sıcaklığında birkaç dakika içinde elde edilir. Glikol kitosan, polimer gelator ve su içeriği arasında değişen oranlarda tarafından farklı jelleşme kez ve sertliği ile çok yönlü hydrogels elde edilir. Zarar görmüş, hidrojel onun Görünüşe ve modülü, dinamik imin Tahvil reversibility crosslinkages olarak nedeniyle kurtarabilirsiniz. Kendi kendine healable bu özellik hidrojel enjeksiyon işleminden sonra sıkılmış parçaları bir ayrılmaz toplu hidrojel kendi kendine iyileşmiş olabilir bu yana enjekte edilebilir olmasını sağlar. Hidrojel da çok farklı denge durumları dinamik imin Tahvil nedeniyle birçok biyo-aktif uyaranlara cevap veriyor. Bu hidrojel biyo uyumlu doğrulandı ve L929 fare fibroblast hücreleri katıştırılmış aşağıdaki Standart prosedürler ve Hücre proliferasyonu kolayca 3D cep ekimi işlemi tarafından değerlendirildi. Hidrojel farklı araştırma nerede bir 3D çevre hücreler için fizyolojik bir taklit kazançlı için ayarlanabilir bir platform sunar. Çok duyarlı, kendi kendine healable ve enjekte edilebilir özellikleri ile birlikte, hydrogels potansiyel olarak uyuşturucu ve gelecekteki bio-tıbbi uygulamalar hücrelerde birden çok taşıyıcıları olarak uygulanabilir.

Bu protokol hidrojel hazırlık ve 3D hücre kültürü olarak kullanılmasını şematik bir sunumunu şekil 1' de sunulmaktadır. Bilgi hidrojel'ın içindekiler ve farklı mekanik güçlü ile hazırlanan oranları Tablo 1' de özetlenmiştir. Hidrojel kendi kendine healable ve depolama modülü Frekans testi Şekil 2' deki karşı tarafından hidrojel'ın sertlik Reolojisi özelliği sunar. Hücre confocal görüntüleri ve gün ile hücre s...

Watch this Scientific Journal Video about Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture at JoVE.com

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

Burada facile hazırlanması enjekte edilebilir hydrogels kitosan tabanlı dinamik imin kimya kullanarak açıklar. Hidrojel'ın mekanik mukavemeti ve 3D hücre kültürü kendi uygulamasında ayarlamak için yöntemleri sunulmaktadır.

Kitosan-esaslı enjekte edilebilir Hydrogels ve kendi uygulamasında 3D hücre kültürü hazırlanması

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

preparation-chitosan-based-injectable-hydrogels-its-application-3d

Universidad San Sebastian

Research

JoVE Journal

Chemistry

19.0K Görüntüleme.  Tsinghua University. Burada facile hazırlanması enjekte edilebilir hydrogels kitosan tabanlı dinamik imin kimya kullanarak açıklar. Hidrojel'ın mekanik mukavemeti ve 3D hücre kültürü kendi uygulamasında ayarlamak için yöntemleri sunulmaktadır.

Video: Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

Easy Manipulation of Architectures in Protein-based Hydrogels for Cell Culture Applications

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field of 3D templates is rising due to the potential resemblance of those materials to the natural extracellular matrix. Protein-based hydrogels are particularly promising because they can easily be functionalized and can achieve defined structures with adjustable physicochemical properties. However, the production of macroporous 3D templates for cell culture applications using natural materials is often limited by their weaker mechanical properties compared to those of synthetic materials. Here, different methods were evaluated to produce macroporous bovine serum albumin (BSA)-based hydrogel systems, with adjustable pore sizes in the range of 10 to 70 &#181;m in radius. Furthermore, a method to generate channels in this protein-based material that are several hundred microns long was established. The different methods to produce pores, as well as the influence of pore size on material properties such as swelling ratio, pH, temperature stability, and enzymatic degradation behavior, were analyzed. Pore sizes were investigated in the native, swollen state of the hydrogels using confocal laser scanning microscopy. The feasibility for cell culture applications was evaluated using a cell-adhesive RGD peptide modification of the protein system and two model cell lines: human breast cancer cells (A549) and adenocarcinomic human alveolar basal epithelial cells (MCF7).

Different methods to manipulate three-dimensional architecture in protein-based hydrogels are evaluated here with respect to material properties. The macroporous networks are functionalized with a cell-adhesive peptide, and their feasibility in cell culture is evaluated using two different model cell lines.

Protein bazlı Hydrogels mimarilerde kolayca idare hücre kültür uygulamaları için

Hydrogels are recognized as promising materials for cell culture applications due to their ability to provide highly hydrated cell environments. The field ...

Biyokimya

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

Kök Hücre yüklü Chitosan Mikrokürelerin teslimi için bir Kollajen Hidrojel oluşturma

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Biyomühendislik

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

Eklem Kondrosit Kültür ve Kıkırdak Üretimi için 3D hidrojel iskeleleri

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) substrates such as tissue culture polystyrene (TCPS). However, characteristic changes of PDL cells have been observed during in vitro culture. This phenomenon is probably because the 2D TCPS differs from the in vivo three-dimensional (3D) microenvironment. Compared to cells cultured on 2D substrates, cells grown in a 3D microenvironment exhibit more similarities to in vivo cells. Therefore, 3D cell culture models provide a promising alternative for conventional 2D monolayer cell culture. To improve conventional PDL cell culture models, we have recently developed a 3D cell culture method, which is based on spheroid formation of PDL cells on chitosan films. Here, we present detailed cell spheroid culture protocols based on chitosan films. The 3D culture system of PDL cellular spheroids overcome some of the limitations related to conventional 2D monolayer cell culture, and thus may be suitable for producing PDL cells with an enhanced therapeutic efficacy for future periodontal tissue regeneration.

Here, we present protocols of culturing human periodontal ligament (PDL) cell spheroids by chitosan films. The culture of three-dimensional (3D) cellular spheroids provides an alternative to conventional tissue culture polystyrene (TCPS) culture system.

Kitosan filmlerde ınsan periodontal ligament hücre kürleri oluşumu

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) ...

Gelişim Biyolojisi

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

3D Hücre Kültürü için Pulmoner Ekstrasellüler Matrix Ayarlanabilir Hidrojellerinin

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

Enjekte edilebilir rejeneratif terapi ve yüksek-den geçerek uyuşturucu tarama için 3D Microtissues

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

Elastin benzeri Protein Hydrogels üretimi için saklama ve Immunostaining hücre 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

Bioprintable aljinat/jelatin hidrojel 3D Vitro Model sistemleri hücre küresel oluşumu teşvik

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Jelatin Metacryloyl Hidrojel Bazlı Bioinks 3D Biyobaskı Protokolleri

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...

Fabrication of Size-Controlled and Emulsion-Free Chitosan-Genipin Microgels for Tissue Engineering Applications

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, chitosan microgels are commonly fabricated using emulsion methods that require organic solvent rinses, which are toxic and harmful to the environment. The present protocol presents a rapid, non-cytotoxic, non-emulsion-based method for fabricating chitosan-genipin microgels without the need for organic solvent rinses. The microgels described herein can be fabricated with precise size control. They exhibit sustained release of biomolecules, making them highly relevant for tissue engineering, biomaterials, and regenerative medicine. Chitosan is crosslinked with genipin to form a hydrogel network, then passed through a syringe filter to produce the microgels. The microgels can be filtered to create a range of sizes, and they show pH-dependent swelling and degrade over time enzymatically. These microgels have been employed in a rat growth plate injury model and were demonstrated to promote increased cartilage tissue repair and to show complete degradation at 28 days in vivo. Due to their low cost, high convenience, and ease of fabrication with cytocompatible materials, these chitosan microgels present an exciting and unique technology in tissue engineering.

The present protocol describes a non-emulsion-based method for the fabrication of chitosan-genipin microgels. The size of these microgels can be precisely controlled, and they can display pH-dependent swelling, degrade in vivo,&#160;and be loaded with therapeutic molecules that release over time in a sustained manner, making them highly relevant for tissue engineering applications.

Doku Mühendisliği Uygulamaları için Boyut Kontrollü ve Emülsiyonsuz Kitosan-Genipin Mikrojellerinin İmalatı

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, ...