The authors would like to acknowledge Stanford Department of Orthopaedic Surgery and Stanford Coulter Translational Seed Grant for funding. J.H.L. would like to thank National Science Foundation Graduate Fellowship and DARE Doctoral Fellowship for support.

Tüm deneyler Stanford Üniversitesi İnsan deneklerin esaslara uygun olarak yapılır ve Kurumsal Değerlendirme Kurulu protokolü onaylandı. 1. Eklem Kondrosit İzolasyonu <ol><li> Total diz protezi sırasında atılan doku kıkırdak edinin ve daha önce 8 açıklandığı gibi teşrih. </li><li> Görme pürüzsüz yüzeyler için kıkırdak örneklerinin medial veya lateral femoral kondilleri seçin ve subkondral kemik etkilemeden 4-5 mm kıkırdak biyopsi kaldırmak için bir neşter ile kıkırdak yüzeyinde kesi yapmak. </li><li> Arıtılmış kollajenaz ile O / N tedavi ile doku enzimatik ayrışma ile hücre dışı matris kondrositler izole edin. 37 ° C &#39;de, kondrosit büyüme ortamı kollajenaz II (125 U / ml) ve kollajenaz IV (160 U / ml) 1: 1&#39;lik kullanın. </li><li> Ertesi gün, 70 ile ayrışmış hücreleri ihtiva eden sindirilir doku filtremm boyut Naylon örgü gözenek. 5 dakika boyunca 600 x g&#39;de DMEM / F12 ve santrifüj 20 ml ile iki kez yıkayın. </li><li> % 10 Fetal Sığır Serumu (FBS) ile desteklenmiş DMEM / F12 içinde yeniden süspansiye, 25 ug / ml askorbat, 2 mM L-glutamin, antimikrobik maddeler, aşağıdaki 1 x 10 6 hücre / 60 mm kültür çanağı yoğunluğunda izole kondrositler Plate (100 U / ml penisilin, 100 ug / ml streptomisin ve 0.25 ug / ml fungizon). Önceki biomimetic hidrojeller kapsülleme 37 ° C&#39;de plakaları tutun ve yüksek yoğunluklu bir tek tabaka olarak birincil kondrosit kültürleri korumak. </li></ol> 2. Biyomimetik Hidrojel Fabrikasyon Not: Bu yöntem sağlar poli (etilen glikol diakrilat) (PEGDA, MW 5.000 g / mol), DPBS kondroitin sülfat-metakrilat (CS-MA) ihtiva eden bir biomimetik hidrojel sentezi. Foto-başlatıcı eklenmesi fotoaktif çapraz bağlanmasını sağlar. Nihai hidrojel bileşimi% 7 tartmak içeriyort / hacim (a / h) PEGDA bölgesinin% 3 CS-MS w / v, ve ışık başlatan a / h% 0.05. <ol><li> Metakrilat grupları ile CS polimerin işlevselleştirilmesiyle CS-MA sentezlemek. <ol><li> MES 1,952 g deiyonize su, 200 ml NaCl 5,84 g (dihidroksi 2 O) eritilmesi ile 50 mM 2-morpholinoethanesulfonic asit (MES) ve 0.5 M sodyum klorür (NaCl) tamponlu çözeltisi hazırlayın. Tamponlu çözelti içinde kondroitin sülfat, sodyum tuzu, 5 g çözündürülür. </li><li> (: EDC = 1: 2 NHS mol oranı) 0,532 g (4,62 mmol) N-hidroksisüksinimid (NHS) ve 1,771 g (9,24 mmol) 1-etil-3- (3-dimetilaminopropil) -karbodiimid hidroklorür (EDC) ilave 5 dakika boyunca çözeltisi ve bir karıştırma. </li><li> Çözeltisine ve 24 saat boyunca oda sıcaklığında reaksiyon sıcaklığı (: 2: 1: EDC: AEMA = 1 NHS molar oranı) 0,765 g (4,62 mmol) 2-aminoetil metakrilat (AEMA) ekleyin. </li><li> Diyaliz tüpüne (12-14 kDa&#39;lık bir MWCO) kullanılarak, 4 gün boyunca iyonu giderilmiş suya karşı diyaliz (diH 2 O) karışımın saflaştınlır. </li><li> P Filtre0.22 um&#39;lik bir filtreden urified çözeltisi ve kurutucu ve vakum O / N çözeltisi içeren açık tüpleri. Tüm tüpleri ışıktan korunmasını sağlamak. Nem ve ışıktan korunan -20 ° C &#39;de erimiş polimeri saklayın. </li></ol></li></ol> 3. Hücre Encapsulation <ol><li> (Hücre yüklü 3D hidrojeller yumrukluyor için) PCR film ve silindirik çubuklar Otoklav ve UV ışığı altında doku kültürü kaputu 0,2 um filtrelenmiş% 70 etanol dalma ısmarlama silindirik jel kalıp sterilize edin. </li><li> Hava kabarcıkları veya sızıntıları önlemek için boşluklar olmadan otoklavlanmış PCR film ile jel kalıbın dibine kapatılmalıdır. Steril 150 mm plaka muhafaza ediniz. </li><li> Önceki biomimetic hidrojel hücre kapsülleme günlük, kollajenaz II O / N tedavi ve kollajenaz IV çözümü ile yüksek yoğunluklu kondrosit tek tabaka ayrıştırmaları. Kollajenaz VI, her kondrosit büyüme medya için kollajenaz II için 125 U / ml ve 160 U / ml kullanın37 ° C&#39;de ilave edildi. </li><li> 50 ml&#39;lik bir tüp almak ve ilave,% 5 ağırlık / hacim (ağırlık / hacim), poli (etilen glikol diakrilat) (PEGDA, molekül ağırlığı = 5000 g / mol),% 3 w / v kondroitin sülfat-metakrilat (CS-MA) ve DPBS h ışık başlatan /% 0.05 w. Çözüm karıştırmak ve bir kenara tutmak için tüp Vortex. Işıktan tüp koruyun. </li><li> , 50 ml&#39;lik bir Falcon tüpü içine ayrışmış hücreleri toplamak 5 dakika boyunca 460 x g&#39;de santrifüj sayısı ve. Aspire tüm hücreler tüp ortam 15 x 10 6 hücre / ml&#39;lik bir yoğunlukta hücreler yeniden askıya almak için, yukarıda karışık jel maddesi ekleyin. Hava kaçınarak oluşumunu kabarcıklar ise 30 kez iyice karıştırın. </li><li> Pipet 72 özel yapılmış silindirik jel kalıbına hücre hidrojel süspansiyonunun ya da tek başına hidrojel ul ve 5 dakika için 3 mW / m 2 UV ışığı (365 nm dalga boyu) maruz kalma ile jelleşme neden. Hiçbir hücre içeriği ile bazı hidrojel çözüm hazırlayın. Gelecekteki biyokimyasal ve mekanik test için negatif kontrol olarak bu yapıları kullanın. MeasuUV yoğunluğu metre ile UV ışığın yoğunluğuna yeniden. Yoğunluğunu ayarlamak için, UV ışık kaynağı ve jelleşme sırasında hidrojel iskele arasındaki mesafeyi değiştirin. NOT: hidrojel CS içerdiğinden, aselüler katkısı sadece hücresel GAG temsil hücre yüklü hidrojellerin biyokimyasal GAG içeriğinden çıkarılır. </li><li> Kapalı kolay kabuğu için filmi kesmek için bir neşter kullanın ve dikkatlice çıkarın. Silindirik çubuklar yardımıyla, polimerleşmemiş jel ve gevşek hücreler yıkayın steril DPBS 5 ml ile bir 6 yuvalı plaka içine jel üzerinden itin. </li><li> Kültür, her oyuğa kondrosit büyüme ortamı 1,5 ml içeren 24 gözlü levhalar içine, hücre yüklü hidrojeller. Kuyuya yıkanmış hidrojeller aktarmak için tek kullanımlık spatula kullanın. </li><li> Bir canlılık / Sitotoksisite kiti kullanılarak, canlı cansız boyama 24 saat sonrası kapsülleme hücre canlılığını değerlendirmek. Kültür taze kondrosit Gro değişen 3-6 hafta süreyle hücre yüklü hidrojeller ve boş hidrojeller2 günde analizler için hasat öncesi medya wth. </li></ol> 4. RNA Ekstraksiyon ve Gen İfadesi Analizleri <ol><li> Dikkatle medya aspire ve hücre-Ladin yanı sıra boş kontrolü hidrojeller steril PBS 1X koydu. </li><li> Steril bir spatula yardımı ile, temiz bir 1.5 mi Eppendorf tüpüne her hidrojel transferi ve her tüpe Tri-reaktif madde 250 ul ilave edin. RNA izole etmek için üreticinin talimatlarına uyun. </li><li> RNA izolasyonu ve miktar kullanımı her örnekten bunun 1 mcg ve sonra High Capacity cDNA Ters Transkripsiyon Kit kullanılarak cDNA içine ters transkripsiyonu gerçekleştirin. </li><li> Nicel PCR ilgi genlerin ifadesinin incelenmesi için TaqMan Gen İfade Diziler kullanın. GAPDH dahili gen ifadesi düzeylerini normale. </li><li> PCR, aşağıdaki koşullar için bir 10 ardından urasil DNA glikosilaz önceki amplikonları inaktive etmek için 50 ° C&#39;de 2 dakika inkübasyon yer alır60, 95 ° C&#39;de inkübasyon dakika Taq polimerazı aktive etmek için. Daha sonra 95 ° C de 15 sn meydana gelen PCR&#39;nin devre döngüsü gerçekleştirmek ve 60 ° C&#39;de 1 dak. </li></ol> 5. Biyokimyasal analizler <ol><li> Her hücre hidrojel yapıları için DNA ve aşağıdaki gibi sülfatlı glikozaminoglikan (sGAG) üretimini ölçmek. </li><li> Dikkatle medya aspire ve hücre-Ladin yanı sıra boş kontrolü hidrojeller steril PBS 1X koydu. Boş bir tüp tartılır ve kağıt mendil üzerinde kurutma sonra içine hidrojel koymak ve hidrojellerin ağırlığını veya ıslak ağırlığı kaydedin. </li><li> Enzimatik sindirimi için ayrı bir 1.5 mi Eppendorf tüp -20 ° C &#39;de, her bir hidrojel dondurun ve daha sonra (GAG) (DNA) Picogreen deneyi ve Dimetil-Metilen mavisi analizi çalıştırmak için sindirilmiş ürünün bir kısım kullanın. </li><li> Hidrojeller enzimatik sindirimi için papainase çözeltisi hazırlayın. <ol><li> İlk Sodyum fosfata 7.1 g dışarı tartarak PBE tamponu hazırlamake dibazik (Na 2 HPO 4) ve Etilendiamintetraasetik asit disodyum tuzu (EDTA-Na2) 1.6 g. , DH 2 O 500 ml çözülür pH 6,5&#39;e ayarlanır ve 0.22 mm&#39;lik bir filtreden filtre saflaştırılmış çözeltisi. </li><li> PBE tamponu, 20 ml L-sistein 0,035 g çözündürülür. Çözüm ve steril papain enzimi 100 ul Filtre. </li></ol></li><li> Hazır deneyleri gerçekleştirmek için zaman, -20 ° C ila tüpleri alıp dondurulmuş hidrojellere hazır papain çözüm 300 ul ekleyin. Bir havanda jel Crush ve motor tokmak ile iyice karıştırın. </li><li> Ek papain solüsyonu ile 500 ul hacim kazandırın. Kontrol jeller için aynı prosedürü uygulayın. 16 st 3,9, 60 ° C&#39;de inkübe edin. Sindirilmiş hidrojel ihtiva eden çözelti, dolayısıyla DNA ve GAG ​​ölçümü için kullanılabilir. Üreticinin pr aşağıdaki standart olarak Lambda faj DNA ile Picogreen analizi kullanılarak DNA içeriği ölçünotocol. </li><li> 1,9-dimetilmetilen mavisi (DMMB) standart 10 olarak köpekbalığı kondroitin sülfat ile boya bağlama testi kullanılarak sülfatlı-GAG içeriği ölçmek. İlgili ıslak ağırlık GAG miktarını bölerek GAG içeriği belirler. </li></ol> 6. Mekanik Test <ol><li> 10 N yük hücresi ile donatılmış bir Instron 5944 test sistemi kullanılarak serbest basınç testleri gerçekleştirin. <ol><li> In vitro kültür istenen gün sonra, hücre-hidrojel iskele ve aselüler kontrol hidrojeller sıkıştırma testi gerçekleştirin. </li><li> Oda sıcaklığında PBS banyosu içinde örnekleri daldırın ve yükleme koşulları altında kıkırdak dokunun yaşadığı fizyolojik suşu itibaren 15,% 11,12 maksimum suşuna 1% streyn / sn bir hızda sıkıştırılması% 10-20 olduğu rapor edilmiştir 13,14. </li></ol></li><li> Üçüncü mertebeden polinom denklem kullanılarak gerilme eğrileri ve eğri uyum vs stres oluşturun. Compr belirleme% 15 suş değerlerinde eğri fit denklemden essive teğet modülü. </li></ol>

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The authors have nothing to disclose.

Bu protokolde bildirildiği gibi, 3D hidrojeller genellikle tek tabakalı kültürler 15 ile karşılaştı fibrocartilage hücrelerine hücre dediferansiyasyon süreci kaçınarak, kültürde kondrosit fenotipi muhafaza edebiliyoruz. Ayrıca, hidrojel yapısının chondrocytes- uzun süreli kültürleri yaş ve hastalık ile ilişkili içsel hücre özelliklerini korumaktadır elverişli bir ortam ortaya koymuştur. 3B biomimetik hidrojel kullanılması birçok avantaj vardır. İlk olarak, kondroitin sülfat dahil (CS), eklem kıkırdağının içinde bulunan başlıca bir komponent, kondroitinaz salgılayarak hidrojel matrisi bozan ve ekstra hücresel matris 5, 16 yeni sentezlenmiş kıkırdak bırakmaya hücreleri sağlar. Buna ek olarak, CS gösterilmiştir artritik eklem anti-inflamatuar özelliklere sahip. Biomimetik hidrojel kıkırdak tamir hücre dağıtımı için bir iskele malzemesi olarak kullanılabilir, ve kimyasal olarak modifiye edilebilirDaha iyi doku biyomalzeme entegrasyon 17,18 kolaylaştırmak için. PEG-CS hidrojellerin kullanımı, uzun süreli kondrositlerin kültürleri ve biyokimyasal ve mekanik özelliklerinin değerlendirilmesine olanak verir. İşte biz bu platformu kıkırdak mühendisliği için en uygun hücre tipini tanımlamak amacıyla farklılaştırılmış kondrosit çeşitli kaynaklardan karşılaştırmalı analizler için yararlı olabilir nasıl gösterir. İlginçtir, hidrojeller kapsüllü kondrositlere kendi içsel yeteneklerine göre canlı kalır ve çoğalır. Hidrojel bileşimi destekleri, aslında, sağlıklı genç ve erişkin kondrositlerin büyümesi Şekil 2&#39;de gösterildiği gibi. Açıklanan hidrojel bileşimi ve yapısı da kıkırdak doku formasyonunu teşvik glikozaminoglikan ile değerlendirilen fonksiyonel hücre dışı matris birikimi (GAG ile gösterildiği gibi ) ölçümü. Buluşun bir başka avantajı, kondrosit hidrojel yapıları olanYeni oluşan kıkırdak dokusunun mekanik özellikleri için değerlendirilebilir. Serbest basınç testi karşılaştırma için aselüler hidrojel yapılmalıdır unutmayın. Hidrojeller, aslında, bağlı CS parçalarının sertliği için temel bir sertliğe sahiptir. (Bir gerilme oranı / s% 1 at)% 5-20 serbest basınç gerilme yükleme koşulları altında kıkırdak dokusu yaşadığı fizyolojik zorlanma beri kıkırdak dokusunun 11,12 mekanik testler için uygulanabilir 10-20 olduğu rapor edilmiştir % 13,14. Mekanik test hücre yüklü ve hücresel olmayan iki hidrojellerin tepkisi, kültür son nokta değerlendirilmiştir. Tarif edilen örnekte, OA kıkırdak ihtiva eden konstruktların düşük katılıkta aksine, yetişkin ve juvenil kondrositlerin ihtiva eden konstruktların benzer bir sertliğe gözlenen, yukarıda. Hücre-hidrojel yapısının bu tür mekanik özellikleri fonksiyonel özelliklerinin değerlendirilmesine olanakHücre olgunlaşma yeteneği derinlemesine analizini veren oluşturulan doku. Sonuç olarak, kıkırdak doku üretmek için farklı bir kondrosit nüfusun potansiyel çalışma 3D biomimetik hidrojellerin kullanımı yaygın olarak uygulanabilir. Burada tarif edilen in vitro çalışmaların yanısıra hücre-yüklü yapıları in vivo nakli fizyolojik bağlamda hücre olgunlaşmasını ve rejeneratif potansiyel çalışma tasavvur edilebilir. Ek biomimetik faktörlerle hidrojel platformun bundan başka değişiklikleri de, kondrosit çoğalmasını ve olgunlaşmasını optimize etmek için tasavvur edilebilir.

With its limited self-repair potential, human articular cartilage undergoes frequent irreversible damages. Extensive efforts are currently focused on the development of efficient cell-based approaches for treatment of articular cartilage injuries. The success of these cell-based therapies is highly dependent on the selection of an optimal cell source and the maintenance of its regenerative potential. Chondrocytes are a common cell source for cartilage repair, but they are limited in supply and can de-differentiate during in vitro expansion in 2D monolayer culture thereby limiting their generation of hyaline cartilage 1. 
The aim of this protocol is to establish a 3-dimensional hydrogel platform for an in vitro comparative study of human chondrocytes from different ages and disease state. Unlike conventional two-dimensional (2D) culture, three-dimensional (3D) hydrogels allow chondrocytes to maintain their morphology and phenotype and provides a physiologically relevant environment enabling chondrocytes to produce cartilage tissue 2,3. In addition to providing a 3D physical structure for chondrocyte culture, hydrogels mimic the function of native cartilage extracellular matrix (ECM). Specifically, the inclusion of chondroitin sulfate methacrylate provides a potential reservoir for secreted paracrine factors 4 and enables cell-mediated degradation and matrix turnover 5. Although many 3D hydrogel culture systems have been utilized widely in various studies including agarose and alginate gels, we have used a biomimetic 3D culture system that has some distinct advantages for chondrocyte culture. Chondroitin sulfate (CS) is an abundant component in articular cartilage and the PEG-CS hydrogels have been shown to maintain and even enhance chondrogenic phenotype and facilitate cell-mediated matrix degradation and turnover 2,5. In addition, the mechanical properties of the hydrogel scaffold can be easily modulated by changing concentration of PEG and hence can be utilized to further enhance the regeneration potential of chondrocytes or a related cell type 6,7. PEG/CSMA is also biocompatible and hence has the potential for a direct clinical application in cartilage defects for example. The limitation for this system is its complexity and the use of photopolymerization that can potentially affect cell viability as compared to simpler systems like agarose, however the advantages for the chondrocyte culture outweigh the potential limitations.
The 3D hydrogel culture is compatible with conventional assay for evaluation of cell phenotype (gene expression, protein immunostaining) and functional outcome (quantification of cartilage matrix production, mechanical testing). This favorable 3D environment was tested to compare the tissue regeneration potential of human chondrocytes from three different aged populations in long-term 3D cultures. 
The outcomes were evaluated via both phenotypic and functional assays. Juvenile, adult and OA chondrocytes showed differential responses in the 3D biomimetic hydrogel culture. After 3 and 6 weeks, chondrogenic gene expression was upregulated in juvenile and adult chondrocytes but was downregulated in OA chondrocytes. Deposition of cartilage tissue components including aggrecan, type II collagen, and glycosaminoglycan (GAG) was high for juvenile and adult chondrocytes but not for OA chondrocytes. The compressive moduli of the resulting cartilage constructs also exhibited similar trends. In conclusion, both juvenile and adult chondrocytes exhibited chondrogenic and cartilage matrix disposition up to 6 weeks of 3D culture in hydrogels. In contrast, osteoarthritic chondrocytes revealed a loss of cartilage phenotype and minimal ability to generate robust cartilage.

<table><tbody><tr><td>juvenile chondrocytes (Clonetics Normal Human Chondrocyte Cell System )</td><td>Lonza</td><td>CC-2550</td><td></td></tr><tr><td>adult chondrocytes (Clonetics Normal Human Chondrocyte Cell System)</td><td>Lonza</td><td>CC-2550</td><td></td></tr><tr><td>poly(ethylene glycol diacrylate)</td><td>Laysan Bio</td><td>ACRL-PEG-ACRL-1000-1g</td><td></td></tr><tr><td>2-morpholinoethanesulfonic acid</td><td>Sigma</td><td>M5287</td><td></td></tr><tr><td>photoinitiator</td><td>Irgacure&amp;nbsp;</td><td>2959</td><td></td></tr><tr><td>sodium chloride&amp;nbsp;</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>chondroitin sulfate sodium salt&amp;nbsp;</td><td>Sigma</td><td>C9819</td><td></td></tr><tr><td>N-hydroxysuccinimide&amp;nbsp;</td><td>Sigma</td><td>130672</td><td></td></tr><tr><td>1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride</td><td>Sigma</td><td>E1769</td><td></td></tr><tr><td>2-aminoethyl methacrylate</td><td>Sigma</td><td>516155</td><td></td></tr><tr><td>dialysis tubing</td><td>Spectrum Laboratories&amp;nbsp;</td><td>132700</td><td></td></tr><tr><td>Collagenase 2</td><td>Worthington Biochemical&amp;nbsp;</td><td>LS004177</td><td></td></tr><tr><td>Collagenase 4</td><td>Worthington Biochemical&amp;nbsp;</td><td>LS004189</td><td></td></tr><tr><td>DMEM/F12 media</td><td>HyClone, Thermo Scientific&amp;nbsp;</td><td>SH3002301&amp;nbsp;</td><td></td></tr><tr><td>live/dead assay</td><td>Life Technologies</td><td>&lt;a href=&quot;https://www.lifetechnologies.com/order/catalog/product/L3224&quot;&gt;L3224&lt;/a&gt;</td><td></td></tr><tr><td>Tri reagent</td><td>Life Technologies</td><td>AM9738</td><td></td></tr><tr><td>Quant-iT PicoGreen dsDNA Assay Kit</td><td>Invitrogen</td><td>&lt;a href=&quot;https://www.lifetechnologies.com/order/catalog/product/P11496&quot;&gt;P11496&lt;/a&gt;</td><td></td></tr><tr><td>Sodium phosphate dibasic&amp;nbsp;</td><td>Sigma</td><td>S3264</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt&amp;nbsp;</td><td>Sigma</td><td>E5134</td><td></td></tr><tr><td>L-Cysteine</td><td>Sigma</td><td>C1276</td><td></td></tr><tr><td>1,9-dimethylmethylene blue&amp;nbsp;</td><td>Sigma</td><td>341088</td><td></td></tr><tr><td>Instruments</td><td></td><td></td><td></td></tr><tr><td>UV light equipment - XX-15LW Bench Lamp, 365 nm</td><td>UVP</td><td>95-0042-07</td><td></td></tr><tr><td>Instron 5944 testing system&amp;nbsp;</td><td>Instron Corporation</td><td>E5940</td><td></td></tr></tbody></table>

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.

PEG ve CS kısımları (Şekil 1) içeren biyoaktif hidrojeller insan mafsal kondrositleri 2,3,5,7 kültür ve olgunlaşması için ideal bir platform oluşturmaktadır. Farklı yaşlarda ve hastalık durumlarından Kondrositler tarif edilen yöntem ile kültürlendi ve fenotipi, gen ekspresyonu ve oluşturulan kıkırdak dokusunun biyokimyasal ve mekanik özellikleri için analiz edilebilir. Üç kondrosit örnekleri juvenile- 6 ay, bireylerde erişkin 34 yaş ve osteoarthritic- 74 yaşında, 3D biomimetik hidrojeller kültür 3 hafta sonra hasat edilmiştir. Gen ekspresyonu, normal kondrosit, çocuk ve yetişkin hem analizleri kondrosit genleri Col2a1 ve Col6a1 ekspresyonunda bir artış göstermiştir. Col6a1 (Şekil 2) ekspresyonunu sürdürmek uygun bir biomimetik ortamda kültürlenen olmasına rağmen kondrojenik fenotipinin kaybı gösterirken Aksine, hastalıklı kondrositler Col2a1 dramatik bir azalma olduğunu gösterdi. CS-PEG hidrojeller de kondrositler çoğalmaya için dinamik bir ortam olarak hizmet PEG ve CS önceden var olan matris sindirimi ve hücre etrafındaki ve hücre dışı matris olgun kıkırdak dokusunun başlıca bileşenleri bırakır. In vitro kültür 3 hafta sonra, kondrosit genişlemesi, bu özellikler göz önüne alındığında Picogreen boyası ile DNA miktarının ölçülmesi ile tahmin edilebilir. Hücreler üç grup karşılaştırmalı analizi OA kondrositler kültür 1 gün ile karşılaştırıldığında dramatik bir düşüş sergiledi ise çocuk ve yetişkin nüfusun hücre yoğunluğu değişmeden olduğunu göstermektedir. DNA içeriğinin kaybı aynı zamanda uzun dönemli kültür (Şekil 3) sırasında mümkün hücre ölümünü destek veren kondrositler OA için değil gözlenmiştir. Salgılanmış matris kültürlerin 3 hafta sonra bitiş noktası aşamasında DMMB boya bağlama deneyi ile sülfatlanmış-GAG içeriği olarak belirlendi. GAG içeriği recor olarak hidrojel yaş ağırlık normalize edilmektedirded enzimatik sindirim ve aselüler katkı önce çıkarılır. Şekil 4&#39;te gösterildiği gibi, kondrositler 3D hidrojeller kültür 3 hafta boyunca GAG benzer bir önemli miktarda biriken. Fiziksel iskele varlığı serbest basınç deneyleri 2,3 ile numunelerin biyomekanik özelliklerinin değerlendirilmesine olanak verir. Hücresel olmayan hidrojeller basınç modüllerden bir azalma olur ise, hücreye yüklü yapılar Kültürde 3 hafta (Şekil 4) daha sonra sıkıştırma modülünü muhafaza. Fenotipik, burada açıklanan biyokimyasal ve biyomekanik analizler değerlendirilmesi ve mühendislik kıkırdak için farklı kondrosit popülasyonlarının potansiyelini anlamak için, bu nedenle yararlıdır. <img alt="figür 1" src="/files/ftp_upload/53085/53085fig1.jpg" /> 3D kondrosit cul 1. PEG-CS biomimetic hidrojeller ŞekilTure. Kondrositler poli (etilen glikol diakrilat) (PEGDA) ve kondroitin sülfat-metakrilat (CS-MA) ve ısmarlama silindirik jel kalıbına dökülmüş ihtiva eden bir karışım içinde tekrar süspanse edilir. UV ışınlarına maruz sonra, jeller kalıplardan toplanır ve hücre canlılığı, canlı cansız boyanma 24 saat sonrası kapsülleme değerlendirilir katılaştı. <a href="https://www.jove.com/files/ftp_upload/53085/53085fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 2," src="/files/ftp_upload/53085/53085fig2.jpg" /> Şekil 3D biomimetik hidrojeller kültürlenmiş insan kondrositlerde kondrojenik genlerin 2. ifadesi. 3B biomimetik hidrojeller kültür 3 hafta sonra genç, yetişkin ve OA kondrositler tarafından kıkırdak belirteçleri Col2a1 ve Col6a1 nicel gen ekspresyonu. Değerler gün 1. Hata b gen ifade düzeyine normalize edilmişars ortalama ± SD temsil etmektedir. p *, &lt;iki kuyruklu Student t testi ile tespit edildiği üzere 0.05. Smeriglio ark modifiye. 2 <a href="https://www.jove.com/files/ftp_upload/53085/53085fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 3," src="/files/ftp_upload/53085/53085fig3.jpg" /> 3D biomimetic Hidrojellerde kültürlü insan kondrositlerde Şekil 3. DNA içeriği. 3D biomimetic hidrojellerin kültür 3 hafta sonra çocuk, yetişkin ve OA kondrositlerinde Picogreen testi ile DNA miktar tayini. Değerler gün 1&#39;de DNA düzeyinde normalize <a href="https://www.jove.com/files/ftp_upload/53085/53085fig3large.jpg" target="_blank">edilir, bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 4," src="/files/ftp_upload/53085/53085fig4.jpg" /> &lt;br /&gt; Şekil 4. GAG içerik ölçümü ve 1. günde ve 3D biomimetic hidrojeller kültür 3 hafta sonra insan kondrosit serbest basınç testi. 1. günde kondrositlerde ve kültür 3 hafta sonra DMMB deneyi ile (A) GAG miktar 3D biomimetic hidrojeller. Değerler ağırlığı (ww) ıslak normalize ve ug / g olarak ifade edilmiştir. 1. günde ve 3D biomimetic hidrojeller kültür 3 hafta sonra asellüler ve hücre yüklü hidrojellerin (B) Basınç modülü (kPa). <a href="https://www.jove.com/files/ftp_upload/53085/53085fig4large.jpg" target="_blank">Görüntülemek için tıklayınız Bu rakamın büyük bir versiyonu.</a>

Watch this Scientific Journal Video about 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation at JoVE.com

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

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 ...

3d-hydrogel-scaffolds-for-articular-chondrocyte-culture-cartilage

Research

JoVE Journal

Bioengineering

20.0K Görüntüleme.  Stanford University. 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. 

Video: 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

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

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is prepared by mixing solutions of glycol chitosan with a synthesized benzaldehyde terminated polymer gelator, and hydrogels are efficiently obtained in several minutes at room temperature. By varying ratios between glycol chitosan, polymer gelator, and water contents, versatile hydrogels with different gelation times and stiffness are obtained. When damaged, the hydrogel can recover its appearances and modulus, due to the reversibility of the dynamic imine bonds as crosslinkages. This self-healable property enables the hydrogel to be injectable since it can be self-healed from squeezed pieces to an integral bulk hydrogel after the injection process. The hydrogel is also multi-responsive to many bio-active stimuli due to different equilibration statuses of the dynamic imine bonds. This hydrogel was confirmed as bio-compatible, and L929 mouse fibroblast cells were embedded following standard procedures and the cell proliferation was easily assessed by a 3D cell cultivation process. The hydrogel can offer an adjustable platform for different research where a physiological mimic of a 3D environment for cells is profited. Along with its multi-responsive, self-healable, and injectable properties, the hydrogels can potentially be applied as multiple carriers for drugs and cells in future bio-medical applications.

Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel&#8217;s mechanical strength and its application in 3D cell culture are presented.

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 ...

Kimya

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Üç boyutlu Mürekkep Püskürtmeli Baskı Teknolojisi Kullanımı İnsan Kıkırdak Doku Fabrikasyon

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Biyomühendislik

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Katmanlı Aljinat Yapıları: Heterojen hücre popülasyonlarının Co-kültür için bir platform

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

Kıkırdak Yenileme için 3D Manyetik Kök Hücre Toplama ve Biyoreaktör Olgunlaşma

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

Üç boyutlu peptid iskele memeli hücrelerinde kültür

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

Sinovya Sıvısı Kültürleme İnsan Eklem kondrosit 3D Sistemi

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

Biyoloji

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

Kondrosit-agaroz Hidrojeller mekanik stimülasyonu

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target joint disorders are mostly palliative. The unmet need for effective disease-modifying OA drugs (DMOADs) has been primarily caused by the absence of appropriate models for studying the disease mechanisms and testing potential DMOADs. Herein, we describe the establishment of a miniature synovial joint-mimicking microphysiological system (miniJoint) comprising adipose, fibrous, and osteochondral tissue components derived from human mesenchymal stem cells (MSCs). To obtain the three-dimensional (3D) microtissues, MSCs were encapsulated in photocrosslinkable methacrylated gelatin before or following differentiation. The cell-laden tissue constructs were then integrated into a 3D-printed bioreactor, forming the miniJoint. Separate flows of osteogenic, fibrogenic, and adipogenic media were introduced to maintain the respective tissue phenotypes. A commonly shared stream was perfused through the cartilage, synovial, and adipose tissues to enable tissue crosstalk. This flow pattern allows the induction of perturbations in one or more of the tissue components for mechanistic studies. Furthermore, potential DMOADs can be tested via either &#34;systemic administration&#34; through all the medium streams or &#34;intraarticular administration&#34; by adding the drugs to only the shared &#34;synovial fluid&#34;-simulating flow. Thus, the miniJoint can serve as a versatile in vitro platform for efficiently studying disease mechanisms and testing drugs in personalized medicine.

We provide detailed methods for generating four types of tissues from human mesenchymal stem cells, which are used to recapitulate the cartilage, bone, fat pad, and synovium in the human knee joint. These four tissues are integrated into a customized bioreactor and connected through microfluidics, thus generating a knee joint-on-a-chip.

Eklem hastalıklarını modellemek ve ilaçları test etmek için çip üzerinde diz eklemi oluşturulması

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target ...

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their biocompatibility and ability to mimic natural tissue properties. This protocol aims to produce DC-ECM hydrogels that closely mimic the native ECM of cartilage tissue. The protocol involves a combination of physical and chemical disruption and enzymatic digestion to remove the cellular material while preserving the structure and composition of the ECM. The DC-ECM is cross-linked using a chemical agent to form a stable and biologically active hydrogel. The DC-ECM hydrogel has excellent biological activity, spatial structure, and biological induction function, as well as low immunogenicity. These characteristics are beneficial in promoting cell adhesion, proliferation, differentiation, and migration and for creating a superior microenvironment for cell growth. This protocol provides a valuable resource for researchers and clinicians in the field of tissue engineering. Biomimetic hydrogels can potentially enhance the development of effective tissue engineering strategies for cartilage repair and regeneration.

This paper introduces a new method for the synthesis of decellularized cartilage extracellular matrix (DC-ECM) hydrogels. DC-ECM hydrogels have excellent biocompatibility and provide a superior microenvironment for cell growth. Therefore, they can be ideal cell scaffolds and biological delivery systems.

Decellularize Kıkırdak Hücre Dışı Matriks Hidrojellerinin Sentezi

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their ...

Generating Spheroids from Various Chondrocytes using Low-Adhesive Conditions under Gravity and Homemade Mini-Bioreactors

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a step-by-step method for differentiating induced pluripotent stem cells (iPSCs) into chondrocyte-based spheroids, supporting tissue engineering and cell therapy applications. The differentiation process is carefully structured to promote chondrogenic lineage commitment, beginning with iPSCs cultured in specific media that sequentially guide cells through critical stages of differentiation. Initially, iPSCs are expanded to reach optimal confluency before induction toward chondrogenic lineage using a series of defined media changes. By day 10, cells are transitioned to a chondrogenesis-promoting medium that enhances the formation of chondrocyte-like cells expressing key markers of mature chondrocytes. Further aggregation in 96-well agarose-coated plates leads to the formation of three-dimensional spheroids, which are then cultured in custom mini-bioreactors designed to simulate a microenvironment that encourages extracellular matrix (ECM) deposition. By enabling scalable production of chondrocyte spheroids that mimic native cartilage characteristics, this approach offers a promising, reproducible solution for developing cell-based treatments for cartilage defects, providing broad utility for clinical and research applications in musculoskeletal regenerative medicine.

This study describes a method for producing chondrocytic spheroids by aggregating cells into spheroids under low-adhesion conditions using gravity, followed by culturing the resulting spheroids in mini-bioreactors.

Yerçekimi ve Ev Yapımı Mini Biyoreaktörler Altında Düşük Yapışkan Koşullar Kullanılarak Çeşitli Kondrositlerden Sferoidlerin Üretilmesi

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a ...