Yazar Dr. Dongxuan Jia teknik yardım için teşekkür etmek istiyorum. Bu makalede ortaya çıkan görüntülerin bazıları Dr Malcolm S. Steinberg, Moleküler Biyoloji Anabilim Dalı, Princeton Üniversitesi ile işbirliği içinde. Yazar ayrıca, Savunma Prostat Kanseri Araştırma Programı (hibe 030.482 PC ve PC-991.552) Bakanlığı ve NCI / NIH (hibe R01CA118755) cömert desteği için teşekkür etmek istiyorum.

1. Tek Bir Hücre Süspansiyon hazırlanması <ol><li> Bunun üzerine mono tabakaları yapışmış hücre kültürleri,% 90 izdiham büyüdü olmalıdır PBS ile iki kez durulanır olmalıdır. De boşalttıktan sonra, 2 ml% 0.05 tripsin-1 mM EDTA (100 mm plaka için) ekleyin ve 37 inkübe ° C hücreleri ayırmak kadar. 2 ml tam orta ekleme ve hücreleri süspansiyon kadar karışımı hafifçe çiğnemek için 5 ml pipet kullanın trypsinization Dur. 15 ml konik tüp hücreleri aktarın. </li><li> RT az 5 dakika için 10 mg / ml DNAz stok ve inkübe 40 ul ekleyin. 5 dakika boyunca 200 XG Vortex kısa ve santrifüj. </li><li> Süpernatant atın ve 1 ml tam doku kültür ortamı ile yıkayın pelet. Tekrarlama, daha sonra tekrar süspansiyon hücrelerin doku kültürü orta 2 ml. </li><li> Hemasitometre, ya da otomatik hücre sayıcı kullanarak hücre sayımı ve 2.5 x 10 6 hücre / ml konsantrasyon ayarlayabilirsiniz. Bu gösteri için bir BioRad TC10 otomatik hücre sayıcı kullanılmıştır. </li></ol> 2. Asma Damlalar oluşumu. <ol><li> 60 mm doku kültürü çanak ve çanak alt PBS 5 ml kapağını kaldırın. Bu hidrasyon odası olarak hareket edecektir. </li><li> Kapağını ters çevirin ve alt kapağın üzerine 10 ul damla yatırmak için 20 ul pipet kullanın. Dokunmatik olarak damla yeteri kadar ayrı yerleştirildiğinde olduğunu emin olun. Bulaşık başına en az 20 damla yerleştirmek mümkündür. </li><li> 37 ° PBS-dolu alt odası ve inkübe ° C /% 5 CO 2 /% 95 nem üzerine kapağı, invert günlük damla izlemek ve hücre yaprak veya agrega ya oluşturmuşlardır kadar inkübe. Stereo mikroskop toplam oluşumu değerlendirmek için kullanılabilir. </li><li> Yaprak formunda yuvarlak alt cam çalkalayıcı transfer olabilir tam orta 3 ml içeren ve 37 sallayarak bir su banyosunda inkübe şişeler ° C ve% 5 CO 2 parçacıklarının formu kadar. </li></ol> 3. Notlar <ol><li> Hücre boyutu bağlı olarak, konsantrasyon yukarı veya aşağı ayarlanması gerekebilir. </li><li> Hücreler, membran asılı damla oluşumu önce floresan boyalar intercalating ile boyanmış olabilir. </li><li> İki farklı hücre tipleri farklı asılı damla oluşumu öncesinde bir 1:1 (veya diğer) oranı boyanmış ve karışık olabilir. Bu kanser ve stromal hücreler, embriyonik dokuların veya hücrelerin genetik özel yapıştırıcı imzaları için tasarlanmış olabilir. </li><li> Hücreler, doku kültürü yemekleri kaderin fonksiyonunu korumak için% 0.05 tripsin / 2 mM kalsiyum kullanarak ayrılabilir. </li><li> Deney bağlı olarak, levha boyutları da ölçülen olabilir, ya da büyüklükler mekanik test veya biyokimyasal veya moleküler analiz için kullanılabilir. </li></ol> 4. Temsilcisi Sonuçlar: Yaprak veya sfero oluşumu genellikle 24 saat içinde ortaya çıkar, ancak hücre tipine bağlı olarak uzun sürebilir. Şekil 1, tedavi edilmemiş prostat kanserinin MAT-LyLu (MLL) açılan kültür asılı hücreleri (Şekil 1A) ya da 25 mikron MEK inhibitörü PD98059 ile tedavi edilen hücreler inkübasyon 18 saat sonra çekilen görüntülerin temsil eder. Not hücre sac belirgin bir sıkışma olduğunu MEKi tedavi sonuçları. Sıkıştırma 10-20 boyutunu (veya daha fazla) damla asılı ve tedavi grupları arasında ortalama büyüklüğü karşılaştırılarak ölçerek belirlenebilir. Toplam piksel görüntü alanını ortaya çıkarmak için parçacık analizi uyguladıktan bunun üzerine Biz genellikle, İkili Mod görüntüleri daha sonra her bir görüntü eşikleme dönüştürme ImageJ yazılımı kullanarak görüntüleri analiz. Bu kare mikron sonra kolayca dönüştürülebilir. Şekil 2, tedavi edilmeyen ve tedavi edilen MEKi MLL hücreler için bir boyut karşılaştırma temsil eder. Student t-testi (p &lt;0.0001) ile karşılaştırıldı olarak belirtildiği gibi, MEKi tedavi, yaprak boyutu önemli ölçüde azaltır. Bu karşılaştırma için, tedavi edilmeyen ve tedavi edilen hücrelerde her biri için 10 agrega ölçüldü. Şekil 3, 24 saat ve 2 gün çalkalayÛcÛ banyosu sonra açılan kültür asılı oluşan bir civciv embriyonik karaciğer sfero temsil eder. Asılı damla tahlil de mekansal konumlandırma benimsedi ortaya çıkarmak için birden fazla hücre tipi içerecek şekilde modifiye edilebilir. Örneğin, bir membran intercalating floresan boya ile boyanmış ve farklı bir floresan işaretleyici ile boyandı civciv embriyonik kalp hücreleri ile karışık civciv embriyonik karaciğer hücreleri izole edilebilir. Yerine kalan daha karışmış Şekil 4A gösterir, karaciğer ve kalp hücrelerinin biri diğerine &quot;sort-out&quot; eğilimi ve benimseyen bir küre içinde bir küre kalp hücreleri, karaciğer hücreleri tarafından zarflı olduğu yapılandırma. Bu yapılandırma, karaciğer ve kalp hücreleri arasındaki interselüler adezyon farklılıklarla açıklanabilir. Bu kavram, pankreas adacık hücre organizasyonu 5 ve hücre aynı iki hücre popülasyonları arasındaki sıralama için, amfibi 3 ve teleost 4 embriyolar için embriyonik germ tabakası doku arasındaki davranış sıralama hücre açıklamak için kullanılır olmuştur Diferansiyel Yapışma Hipotez kodlanmış .n kaderin aynı tip (6 ve Şekil 4B) ekspresyon düzeyleri dışında her açıdan. Bu sonuç, yapışma, hücre popülasyonlarının arasında basit bir fark mühendislik nasıl göstermek için özellikle önemlidir, örneğin, organ yapısı nesil neden olabilir, pankreas adacık 5. <img src="/files/ftp_upload/2720/2720fig1.jpg" alt="Şekil 1" /> Şekil 1 damla kültür, ya da (A) yokluğu ya da varlığı 18 saat asılı inkübe hücreler Görüntüler (B) MEK inhibitörü PD98059 25 mikron . Görüntüler Nikon Eclipse TE 300 Epifloresans mikroskop ve bir Photometrics CoolSnap ES dijital fotoğraf makinesi ele geçirildi. <img src="/files/ftp_upload/2720/2720fig2.jpg" alt="Şekil 2" /> Şekil 2 MEKi indüklenen sıçan prostat kanseri MLL hücreleri sıkıştırma Niceleme. Tedavi edilmemiş ve tedavi edilen MEKi MLL hücrelerinin asılı damla kültürleri yukarıda açıklandığı gibi toplam büyüklüğü tespit edildi bunun üzerine 18 saat süreyle inkübe edildi. Toplam büyüklüğü istatistiksel analizi Student t-testi ile oldu. Yıldız işareti, önemli ölçüde daha küçük ve daha kompakt agrega toplam boyutu tedavi edilmeyen ve tedavi edilen MEKi hücreleri ve önerir MEKi tedavi sonuçları arasında istatistiksel olarak anlamlı fark (p &lt;0.0001) temsil eder. <img src="/files/ftp_upload/2720/2720fig3.jpg" alt="Şekil 3" /> Şekil 3, 18 saat sonra bir kuluçka bırak kültür asılı / 48 saat boyunca banyo sallayarak civciv embriyonik karaciğer hücreleri kuluçka oluşan sfero . Bu görüntü Nikon SMZ800 stereo mikroskop ve bir Sony Siyah CCD kamera kullanılarak yakalandı. <img src="/files/ftp_upload/2720/2720fig4.jpg" alt="Şekil 4" /> Şekil 4 damla kültürü farklı hücre türleri arasındaki davranış Hanging sıralama ortaya koyuyor. Panelinde, civciv embriyonik karaciğer hücreleri PKH-2 membran intercalating boya ile boyandı ve PKH-26 lekeli civciv embriyonik kalp hücrelerinin eşit oranda karıştırılır. Panel B, 2.4:1 oranı yüzeyler N-cad ifade iki N-kaderin-transfekte L hücre klonlarının (LN4 ve LN2a), floresan membran intercalating boyalar PKH-2 ve PKH-26 ile boyandı (Sigma-Aldrich) eşit oranda karıştırılır ve damla 7 asılı olarak kültür. 18 saat sonra kültür, hücre sayfaları sallayarak şişeler transfer edildi ve 48 saat inkübe edilebilir. Büyüklüklerdeki% 2 paraformaldehid sabit tuzlu fosfat tamponlu ve Nikon Optiphot-2 mikroskop bağlı bir BioRad MRC600 lazer tarama konfokal mikroskop sistemi ile görüntülenebilir. (A) yeşil ve kırmızı her iki kanal Optik bölümleri toplanmış ve Vital Resimler ile donatılmış bir Silikon Graphix Crimson VGX iş istasyonu Voksel Ultra görüntüleme yazılımı anatomik yapılandırmaları ifşa sahte renk, her iki kanal atamak ve görüntüleri birleştirmek için kullanılmıştır. Pseudocolor mavi karaciğer hücreleri (8), kalp hücrelerinin bir toplamı gösteren zarflama yoluyla Konfokal optik bölümü (burada sözde-renk sarı) . (B) N-kaderin (kırmızı) (6) düşük seviyelerde ifade ile yüksek N-kaderin ifade hücreleri (yeşil) bir toplamı gösteren zarflama ile Konfokal optik bölümü.

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Çalışmalar, üç boyutlu bir bağlamda (3D) hücreleri kültür farklı hücresel morfoloji ve katı bir iki boyutlu (2D) kültür sistemi, 9 kıyasla sinyalizasyon ürettiğini göstermiştir . Örneğin, fibroblast nüfuslu kollajen jeller 3D fibroblast morfolojisi 2D 10,11 gözlenen oldukça farklı olduğunu göstermektedir. Benzer şekilde, 3D kültürü meme epitel hücrelerinin doku-spesifik farklılaşma neden olabilir. 3D kültür sistemleri, normal ve malign hücrelerin ayırt etmek i?...

<ul><li> otomatik hücre sayıcı (BioRad TC10) </li><li> CO 2 beşik örtüsü Model 3540 (Lab-line) ile su banyosunda sallayarak </li></ul>

a simple hanging drop cell culture protocol for generation of 3d spheroids

Katı yüzeye yapışık tek tabaka kültürler, hücre-hücre uyum ve hücre alt tabaka yapışma çalışmalar tarihsel yapılmamıştır. Ancak, Hücreler, doku içinde hücrelerin çok yakın komşuları ile ve ekstrasellüler matriks bileşenleri ile yakın bağlantıları kurmak yakından paketlenmiş bir doku kitlesi içinde genellikle kaplı bulunmaktadır. Buna göre, kimyasal ortam ve 3D doku hücreleri içinde yaşadığı fiziksel kuvvetlerin tek tabaka kültüründe yetişen hücreleri tarafından deneyimli dışında temelde farklı. Bu hücresel morfoloji ve sinyalizasyon belirgin etkisi gösterilmiştir. Kollajen jeller 1 veya biyomalzeme iskelelerde 2 hücre de dahil olmak üzere kapsülleme 3D hücre kültürleri oluşturmak için çeşitli yöntemler geliştirilmiştir. Bu tür yöntemler, yararlı olmakla beraber, normal dokularda bulunan samimi doğrudan hücre-hücre adezyon mimarisi özetlemek değil. Aksine, onlar daha yakından yaklaşık kültür sistemleri tek hücre, gevşek bir 3D ECM ürünleri ağ örgüsü içinde dağılmış bulunmaktadır. Burada, basit bir yöntem hücreleri açılan kültür asılı olarak yerleştirilir ve hücrelerin birbirleri ile ve ekstrasellüler matriks bileşenleri ile doğrudan temas halinde olan gerçek 3D parçacıklarının oluşana kadar fizyolojik şartlar altında inkübe olduğu açıklanmaktadır. Bu yöntem hiçbir özel ekipman gerektirir ve hücre-hücre veya hücre-ECM etkileşimi üzerindeki etkileri nedenlerine açıklık ilgi olabilir çok küçük miktarlarda herhangi bir biyolojik ajan eklenmesi için adapte edilebilir. Yöntemi de ko-kültür iki (veya daha fazla) farklı hücre popülasyonlarının hücreleri arasındaki mekansal ilişkileri belirterek, hücre-hücre veya hücre-ECM etkileşimleri rolünü aydınlatmak için kullanılabilir. , Hücre-hücre uyum ve hücre-ECM yapışma malign invazyon, yara iyileşmesi, doku mühendisliği uygulamaları için embriyonik gelişim, stromal tümör hücre etkileşimi çalışmaları temel taşlarından. Bu basit bir yöntem gibi doku biyomekanik özellikleri ölçümü veya hücresel, fizyolojik ilgili bir model moleküler ve biyokimyasal analiz için agrega üreten bir araç sağlayacaktır.

Watch this Scientific Journal Video about A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids at JoVE.com

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Biz 3D doku parçacıklarının ve potansiyel uygulama üreten hücre-hücre etkileşimleri farklılıkları ölçmek için basit ve hızlı bir yöntem açıklanmaktadır.

3D parçacıklarının Üretimi için Basit Asma Bırak Hücre Kültürü Protokolü

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

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56.6K Görüntüleme.  UMDNJ-Robert Wood Johnson Medical School. Biz 3D doku parçacıklarının ve potansiyel uygulama üreten hücre-hücre etkileşimleri farklılıkları ölçmek için basit ve hızlı bir yöntem açıklanmaktadır.

Video: A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

3D Fibrin hazırlanması Kök Hücre Kültürü Uygulamaları için iskeleleri

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Biyomühendislik

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &amp;#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

Adım büyüme PEG-peptid Hidrojeller kaynaktan β-hücre sferoidlerin Üretimi ve Kurtarma

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

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 ...

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 ...

A Protocol for Real-time 3D Single Particle Tracking

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although various RT-3D-SPT methods have been put forward in recent years, tracking high speed 3D diffusing particles at low photon count rates remains a challenge. Moreover, RT-3D-SPT setups are generally complex and difficult to implement, limiting their widespread application to biological problems. This protocol presents a RT-3D-SPT system named 3D Dynamic Photon Localization Tracking (3D-DyPLoT), which can track particles with high diffusive speed (up to 20 &#181;m2/s) at low photon count rates (down to 10 kHz). 3D-DyPLoT employs a 2D electro-optic deflector (2D-EOD) and a tunable acoustic gradient (TAG) lens to drive a single focused laser spot dynamically in 3D. Combined with an optimized position estimation algorithm, 3D-DyPLoT can lock onto single particles with high tracking speed and high localization precision. Owing to the single excitation and single detection path layout, 3D-DyPLoT is robust and easy to set up. This protocol discusses how to build 3D-DyPLoT step by step. First, the optical layout is described. Next, the system is calibrated and optimized by raster scanning a 190 nm fluorescent bead with the piezoelectric nanopositioner. Finally, to demonstrate real-time 3D tracking ability, 110 nm fluorescent beads are tracked in water.

This protocol details the construction and operation of a real-time 3D single particle tracking microscope capable of tracking nanoscale fluorescent probes at high diffusive speeds and low photon count rates.

Gerçek zamanlı 3D tek parçacık izleme için bir iletişim kuralı

Real-time three-dimensional single particle tracking (RT-3D-SPT) has the potential to shed light on fast, 3D processes in cellular systems. Although ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

3D dinamik kültür sistemi ile bir çok katmanlı mezenkimal kök hücre levha inşaatı

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

Bir İnsan 3D Ekstrasellüler Matrix-Adiposit Kültür Modeli Matrix-Hücre Metabolik Crosstalk Çalışma için

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

3D Biyobaskı için Adipoz Türevi Kök Hücre Sferoidlerinin Büyük Ölçekli, Otomatik Üretimi

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.

Üç Boyutlu Hücre Kültürü Substratlarının UV-Fotodesenlenmesi ile Multicue Hücresel Mikro Ortamların Oluşturulması

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand ...

Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We recently published a cost-effective strategy for the massive expansion of hiPSC-CMs in two dimensions (2D). Two major limitations are cell immaturity and a lack of three-dimensional (3D) arrangement and scalability in high-throughput screening (HTS) platforms. To overcome these limitations, the expanded cardiomyocytes form an ideal cell source for the generation of 3D cardiac cell culture and tissue engineering techniques. The latter holds great potential in the cardiovascular field, providing more advanced and physiologically relevant HTS. Here, we describe an HTS-compatible workflow with easy scalability for the generation, maintenance, and optical analysis of cardiac spheroids (CSs) in a 96-well-format. These small CSs are essential to fill the gap present in current in vitro disease models and/or generation for 3D tissue engineering platforms. The CSs present a highly structured morphology, size, and cellular composition. Furthermore, hiPSC-CMs cultured as CSs display increased maturation and several functional features of the human heart, such as spontaneous calcium handling and contractile activity. By automatization of the complete workflow, from the generation of CSs to functional analysis, we increase intra- and inter-batch reproducibility as demonstrated by high-throughput (HT) imaging and calcium handling analysis. The described protocol allows modeling of cardiac diseases and assessing drug/therapeutic effects at the single-cell level within a complex 3D cell environment in a fully automated HTS workflow. In addition, the study describes a straightforward procedure for long-term preservation and biobanking of whole-spheroids, thereby providing researchers the opportunity to create next-generation functional tissue storage. HTS combined with long-term storage will substantially contribute to translational research in a wide range of areas, including drug discovery and testing, regenerative medicine, and the development of personalized therapies.

Presented here is a set of protocols for the generation and cryopreservation of cardiac spheroids (CSs) from human-induced pluripotent stem cell-derived cardiomyocytes cultured in a high-throughput, multidimensional format. This three-dimensional model functions as a robust platform for disease modeling, high-throughput screenings, and maintains its functionality after cryopreservation.

İnsan Kaynaklı Pluripotent Kök Hücre Kaynaklı Kardiyak Sferoidlerin Üretimi, Yüksek Verimli Taraması ve Biyobankacılığı

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are of paramount importance for human cardiac disease modeling and therapeutics. We ...