Corneal bioprinting utilizing collagen‐based bioinks and primary human keratocytes

2019 ◽  
Vol 107 (9) ◽  
pp. 1945-1953 ◽  
Author(s):  
Daniela F. Duarte Campos ◽  
Malena Rohde ◽  
Mitchell Ross ◽  
Parham Anvari ◽  
Andreas Blaeser ◽  
...  
Keyword(s):  
Human Keratocytes

The effect of dichlorotriazinyl aminofluorescein on human keratocytes in vitro

1996 ◽  
Vol 10 (2) ◽  
pp. 63 ◽  
Author(s):  
Won Ryang Wee ◽  
Roya Rofougaran ◽  
Alireza Pakkar ◽  
Seiji Hayashi ◽  
Peter J. McDonnell
Keyword(s):  
Human Keratocytes

scholarly journals Expression Profiles of Neuropeptides, Neurotransmitters, and Their Receptors in Human Keratocytes In Vitro and In Situ

PLoS ONE ◽  
2015 ◽  
Vol 10 (7) ◽  
pp. e0134157 ◽  
Author(s):  
Marta Słoniecka ◽  
Sandrine Le Roux ◽  
Peter Boman ◽  
Berit Byström ◽  
Qingjun Zhou ◽  
...  
Keyword(s):  
Expression Profiles ◽  
Human Keratocytes

scholarly journals Single-cell analysis reveals IGF-1 potentiation of inhibition of the TGF-β/Smad pathway of fibrosis in human keratocytes in vitro

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Tomislav Sarenac ◽  
Martin Trapecar ◽  
Lidija Gradisnik ◽  
Marjan Slak Rupnik ◽  
Dusica Pahor
Keyword(s):  
Single Cell ◽  
Single Cell Analysis ◽  
Cell Analysis ◽  
Smad Pathway ◽  
Human Keratocytes

scholarly journals Cell signaling pathways in human mutant PAX6 corneal cells: an in vitro model for aniridia-related keratopathy

2021 ◽  
Author(s):  
Marta Słoniecka ◽  
André Vicente ◽  
Berit Byström ◽  
Fátima Pedrosa Domellöf
Keyword(s):  
Protein Expression ◽  
Signaling Pathways ◽  
Cell Fate ◽  
Expression Patterns ◽  
Pax6 Gene ◽  
Cas9 Nuclease ◽  
Extracellular Matrix Components ◽  
Gene And Protein Expression ◽  
Human Keratocytes

ABSTRACTPURPOSETo establish an in vitro model of aniridia-related keratopathy (ARK) using CRISPR/Cas9 engineered human keratocytes with mutations in the PAX6 gene, and to study the Notch Homolog 1, Translocation-Associated (Notch1), sonic hedgehog (SHH), mammalian target of rapamycin (mTOR), and Wnt/β-catenin signaling pathways in the PAX6 mutant keratocytes.METHODSPrimary human keratocytes were isolated from healthy corneas. Keratocytes were transduced with Cas9 lentiviral particles in order to create cells stably expressing Cas9 nuclease. Lentiviral particles carrying PAX6 sgRNA were transduced into the Cas9 keratocytes creating mutants. Analysis of signaling pathways was assessed by RT-qPCR for gene expression and western blot for protein expression.RESULTSHuman keratocytes stably expressing Cas9 nuclease were created. Keratocytes carrying PAX6 gene mutation were successfully generated. PAX6 mutant keratocytes showed modified expression patterns of extracellular matrix components such as collagens and fibrotic markers. Analysis of the Notch1, SHH, mTOR, and Wnt/β-catenin signaling pathways in the PAX6 mutant keratocytes revealed altered gene and protein expression of the key players involved in these pathways.CONCLUSIONSA properly functioning PAX6 gene in keratocytes is crucial for the regulation of signaling pathways important for cell fate determination, proliferation, and inflammation. Pax6 mutation in the in vitro settings leads to changes in these pathways which resemble those found in corneas of patients with ARK.

scholarly journals A cellular and proteomic approach to assess proteins extracted from cryopreserved human amnion in the cultivation of corneal stromal keratocytes for stromal cell therapy

2019 ◽  
Vol 6 (1) ◽  
Author(s):  
Beau J. Fenner ◽  
Nur Zahirah B. M. Yusoff ◽  
Matthias Fuest ◽  
Lei Zhou ◽  
Francisco Bandeira ◽  
...  
Keyword(s):  
Epithelial Mesenchymal Transition ◽  
Protein Extraction ◽  
Culture Media ◽  
Cell Metabolism ◽  
Absolute Quantitation ◽  
Human Amnion ◽  
Mesenchymal Transition ◽  
Proteomic Approach ◽  
Isobaric Tagging ◽  
Human Keratocytes

Abstract Background Human corneal stromal keratocytes propagated in culture media supplemented with human amnion extract (AME) can correct early corneal haze in an animal model. Clinical application of cultivated keratocytes is limited by infectious disease screening before amnion products can be used in humans. It remains unclear if AME from cryopreserved versus fresh human amnion can support human keratocyte propagation, and which components of the extract promote keratocyte growth. Methods Three placentas were collected for the preparation of fresh and cryopreserved amnion tissues followed by homogenization and protein extraction. AME protein profiles were studied using isobaric tagging for relative and absolute quantitation (iTRAQ) proteomics. Enriched gene ontology (GO) terms and functional classes were identified. Primary human keratocytes from 4 donor corneas were cultured in media supplemented with fresh AME (F-AME) or cryopreserved AME (C-AME). Cell viability, proliferation and keratocyte marker expression were examined by confocal immunofluorescence and flow cytometry. Results AME proteomics revealed 1385 proteins with similar expression levels (between 0.5- and 2-fold) between F- and C-AME, while 286 proteins were reduced (less than 0.5-fold) in C-AME. Enriched GO term and biological pathway analysis showed that those proteins with comparable expression between F-AME and C-AME were involved in cell metabolism, epithelial-mesenchymal transition, focal adhesion, cell-extracellular matrix interaction, cell stress regulation and complement cascades. Human corneal stromal keratocytes cultured with F-AME or C-AME showed similar morphology and viability, while cell proliferation was mildly suppressed with C-AME (P > 0.05). Expression of aldehyde dehydrogenase 3A1 (ALDH3A1) and CD34 was similar in both cultures. Conclusion AME from cryopreserved amnion had limited influence on keratocyte culture. It is feasible to use protein extract from cryopreserved amnion to propagate human keratocytes for potential translational applications.

Stability of freeze-dried plasma rich in growth factors eye drops stored for 3 months at different temperature conditions

2020 ◽  
pp. 112067212091303 ◽  
Author(s):  
Eduardo Anitua ◽  
María de la Fuente ◽  
Francisco Muruzábal ◽  
Jesús Merayo-Lloves
Keyword(s):  
Growth Factors ◽  
Room Temperature ◽  
Storage Condition ◽  
Eye Drops ◽  
Temperature Conditions ◽  
Healthy Donors ◽  
Freeze Dried ◽  
And Storage ◽  
Blood Centrifugation ◽  
Human Keratocytes

Purpose: The purpose of this study was to analyze the biological content and activity of freeze-dried plasma rich in growth factors eye drops after their storage at 4°C and at room temperature for 3 months with respect to fresh samples (time 0). Methods: Plasma rich in growth factors was obtained after blood centrifugation from three healthy donors. After platelet activation, the obtained plasma rich in growth factors eye drops were lyophilized alone or in combination with lyoprotectant (trehalose), then they were stored for 3 months at room temperature or at 4°C. Several growth factors were analyzed at each storage time and condition. Furthermore, the proliferative and migratory potential of freeze-dried plasma rich in growth factors eye drops kept for 3 months at different temperature conditions was evaluated on primary human keratocytes. Results: The different growth factors analyzed maintained their levels at each time and storage condition. Freeze-dried plasma rich in growth factors eye drops stored at room temperature or 4°C for 3 months showed no significant differences on the proliferative activity of keratocytes in comparison with fresh samples. However, the number of migratory human keratocytes increased significantly after treatment with lyophilized plasma rich in growth factors eye drops kept for 3 months compared to those obtained at time 0. No significant differences were observed between the freeze-dried plasma rich in growth factors eye drops whether mixed or not with lyoprotectant. Conclusion: Freeze-dried plasma rich in growth factors eye drops preserve the main growth factors and their biological activity after storage at room temperature or 4°C for up to 3 months. Lyophilized plasma rich in growth factors eye drops conserve their biological features even without the use of lyoprotectants for at least 3 months.

KGF, FGFb, VEGF, HGF and TGFβ1 secretion of human keratocytes following photodynamic inactivation (PDI) in vitro

2013 ◽  
Vol 251 (8) ◽  
pp. 1987-1993 ◽  
Author(s):  
Tanja Stachon ◽  
Jiong Wang ◽  
Timo Eppig ◽  
Achim Langenbucher ◽  
Markus Bischoff ◽  
...  
Keyword(s):  
Photodynamic Inactivation ◽  
Human Keratocytes

Myofibroblast Differentiation of Normal Human Keratocytes and hTERT, Extended-Life Human Corneal Fibroblasts

2003 ◽  
Vol 44 (5) ◽  
pp. 1850 ◽  
Author(s):  
James V. Jester ◽  
Jiying Huang ◽  
Stephen Fisher ◽  
Jennifer Spiekerman ◽  
Jin Ho Chang ◽  
...  
Keyword(s):  
Myofibroblast Differentiation ◽  
Corneal Fibroblasts ◽  
Normal Human ◽  
Extended Life ◽  
Human Keratocytes

Responses of human keratocytes to micro- and nanostructured substrates

2004 ◽  
Vol 71A (3) ◽  
pp. 369-376 ◽  
Author(s):  
Ana I. Teixeira ◽  
Paul F. Nealey ◽  
Christopher J. Murphy
Keyword(s):  
Nanostructured Substrates ◽  
Human Keratocytes
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