fibroblast spheroids
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Author(s):  
Shenyang Li ◽  
Chengcheng Ding ◽  
Yonglong Guo ◽  
Yanan Zhang ◽  
Hao Wang ◽  
...  

Suspended spheroid culture using ultralow attachment plates (ULAPs) is reported to effect corneal fibroblast reprogramming. Polydimethylsiloxane (PDMS), with hydrophobic and soft substrate properties, facilitates adherent spheroid formation that promotes cellular physical reprogramming into stem-like cells without using transcription factors. However, it is still unknown whether the biophysical properties of PDMS have the same effect on adult human corneal keratocyte reprogramming. Here, PDMS and essential 8 (E8) medium were utilized to culture keratocyte spheroids and fibroblast spheroids, and the reprogramming results were compared. We provide insights into the probable mechanisms of the PDMS effect on spheroids. qPCR analysis showed that the expression of some stem cell marker genes (OCT4, NANOG, SOX2, KLF4, CMYC, ABCG2 and PAX6) was significantly greater in keratocyte spheroids than in fibroblast spheroids. The endogenous level of stemness transcription factors (OCT4, NANOG, SOX2, KLF4 and CMYC) was higher in keratocytes than in fibroblasts. Immunofluorescence staining revealed Klf4, Nanog, Sox2, ABCG2 and Pax6 were positively stained in adherent 3D spheroids but weakly or negatively stained in adherent 2D cells. Furthermore, OCT4, NANOG, SOX2, KLF4, HNK1, ABCG2 and PAX6 gene expression was significantly higher in adherent 3D spheroids than in adherent 2D cells. Meanwhile, SOX2, ABCG2 and PAX6 were more upregulated in adherent 3D spheroids than in suspended 3D spheroids. The RNA-seq analysis suggested that regulation of the actin cytoskeleton, TGFβ/BMP and HIF-1 signaling pathways induced changes in mechanotransduction, the mesenchymal-to-epithelial transition and hypoxia, which might be responsible for the effect of PDMS on facilitating reprogramming. In conclusion, compared to corneal fibroblasts, keratocytes were more susceptible to reprogramming due to higher levels of endogenous stemness transcription factors. Spheroid culture of keratocytes using PDMS had a positive impact on promoting the expression of some stem cell markers. PDMS, as a substrate to form spheroids, was better able to promote reprogramming than ULAPs. These results indicated that the physiological cells and culture conditions herein enhance reprogramming. Therefore, adherent spheroid culture of keratocytes using PDMS is a promising strategy to more safely promote reprogramming, suggesting its potential application for developing clinical implants in tissue engineering and regenerative medicine.


Author(s):  
Melanie Krüger ◽  
Bart Spee ◽  
Andreas Walther ◽  
Laura De Laporte ◽  
Linda M. Kock

Abstract Nanofibrillar cellulose as a naturally biocompatible scaffold material is very promising for tissue engineering. It is shear thinning but has the downside of not being degradable in animals, it can only be degraded by cellulase enzymes. In this study, a newly developed bioreactor was used to culture fibroblast spheroids under flow conditions inside nanocellulose hydrogels with and without the presence of cellulase. The aim was to control the tissue size and ideally find a match between degradation and tissue formation within this promising material. Both the concentration of cellulase and the flow rate were varied and their influence on the activity and growth of fibroblast clusters was assessed. Cluster diameters, degradation, metabolic activity, and tissue production increase with higher cellulase concentration, although concentrations above 1 g/l does not have an additional benefit. Flow leads to more viable cells, more proliferation and migration, leading to overall larger tissue constructs compared to static conditions. This is most likely due to the shear thinning effect of flow on cellulose nanofibrils (CNFs) in addition to the increased nutrient supply through perfusion. At a constant cellulase concentration of 1 g/l, a flow of 2 ml/min proved to be optimal for tissue production. Therefore, degradation in combination with flow leads to more effective tissue production in CNF hydrogels, which is a very potent scaffold material for tissue engineering.


ACS Nano ◽  
2019 ◽  
Vol 13 (10) ◽  
pp. 11273-11282 ◽  
Author(s):  
Shiqi Hu ◽  
Zhenhua Li ◽  
Jhon Cores ◽  
Ke Huang ◽  
Teng Su ◽  
...  

2018 ◽  
Vol 29 ◽  
pp. 151-151
Author(s):  
Klara Janjic ◽  
Barbara Cvikl ◽  
Andreas Moritz ◽  
Hermann Agis

2018 ◽  
Vol 13 (2) ◽  
pp. 025006 ◽  
Author(s):  
Gili Kaufman ◽  
Ryan A Whitescarver ◽  
Laiz Nunes ◽  
Xavier-Lewis Palmer ◽  
Drago Skrtic ◽  
...  

2016 ◽  
Vol 345 (1) ◽  
pp. 17-24 ◽  
Author(s):  
Pertteli Salmenperä ◽  
Piia-Riitta Karhemo ◽  
Kati Räsänen ◽  
Pirjo Laakkonen ◽  
Antti Vaheri

2016 ◽  
Vol 202 (5-6) ◽  
pp. 343-354 ◽  
Author(s):  
Gili Kaufman ◽  
Laiz Nunes ◽  
Alex Eftimiades ◽  
Wojtek Tutak

Tissue engineering-based therapies rely on the delivery of monolayered fibroblasts on two-dimensional polystyrene-coated and extracellular matrix (ECM) surfaces to regenerate connective tissues. However, this approach may fail to mimic their three-dimensional (3D) native architecture and function. We hypothesize that ECM fibrous proteins, which direct the migration of cells in vivo, may attach and guide polystyrene- and Matrigel™-ECM (M-ECM)-adherent fibroblasts to rearrangement into large multicellular macrostructures with the ability to proliferate. Gingival monolayered fibroblasts and their derived spheroids were added and adhered to tissue culture polystyrene and M-ECM surfaces. The cells were covered with a layer of collagen1 hydrogel combined with vitronectin, fibronectin or fibrin, or 10% M-ECM. The development of 3D cell constructs was characterized by epifluorescence and confocal scanning microscope image analysis. The ECM turnover and the proliferative capabilities of the fibroblasts were determined via gene expression profiling of collagen1, fibronectin, matrix metalloproteinase/metallopeptidase 2, Nanog, and SRY (sex-determining region Y)-box2 (Sox2). Expression of the Sox2 protein was followed by immunostaining. The collagen1 protein had the strongest effect on monolayered and spheroid cell rearrangements, forming large spherical shapes and fused 3D macroconstructs. The addition of fibrin protein was typically required to achieve a similar effect on M-ECM-adherent monolayered fibroblasts. The spheroid fusion process was followed by an increase in cell density and the formation of tight clusters. The fused spheroids continued to maintain their intracellular ECM turnover and proliferation capacities. Collagen1 is a valuable component in the rearrangement of adherent fibroblast monolayers and spheroids. Fibroblast spheroids should preferably be used as basic building blocks to assemble multicellular connective tissue-like macrostructures.


2011 ◽  
Vol 82 (5) ◽  
pp. 790-797 ◽  
Author(s):  
Zurairah Berahim ◽  
Keyvan Moharamzadeh ◽  
Andrew Rawlinson ◽  
Adrian K. Jowett

2008 ◽  
Vol 314 (19) ◽  
pp. 3444-3452 ◽  
Author(s):  
Pertteli Salmenperä ◽  
Esko Kankuri ◽  
Jozef Bizik ◽  
Vappu Sirén ◽  
Ismo Virtanen ◽  
...  
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