TY - JOUR
T1 - Methods for Investigating Corneal Cell Interactions and Extracellular Vesicles In Vitro
AU - McKay, Tina B.
AU - Guo, Xiaoqing
AU - Hutcheon, Audrey E.K.
AU - Karamichos, Dimitrios
AU - Ciolino, Joseph B.
N1 - Funding Information:
We would like to dedicate this manuscript to Dr. James D. Zieske, who was an amazing mentor and a leader in corneal biology. Dr. Zieske's work allowed for significant contributions to establishing the methods to isolate and study the major corneal cell types in vitro. This research was funded by the National Institutes of Health (NIH) 5T32EY007145‐20, R01EY005665 and the National Eye Institute (NEI) Core grant P30EY003790. Human corneal tissues were obtained from the National Disease Research Interchange (NDRI) with support from NIH 2U42 OD011158.
Publisher Copyright:
© 2020 Wiley Periodicals LLC
PY - 2020/12
Y1 - 2020/12
N2 - Science and medicine have become increasingly “human-centric” over the years. A growing shift away from the use of animals in basic research has led to the development of sophisticated in vitro models of various tissues utilizing human-derived cells to study physiology and disease. The human cornea has likewise been modeled in vitro using primary cells derived from corneas obtained from cadavers or post-transplantation. By utilizing a cell's intrinsic ability to maintain its tissue phenotype in a pre-designed microenvironment containing the required growth factors, physiological temperature, and humidity, tissue-engineered corneas can be grown and maintained in culture for relatively long periods of time on the scale of weeks to months. Due to its transparency and avascularity, the cornea is an optimal tissue for studies of extracellular matrix and cell-cell interactions, toxicology and permeability of drugs, and underlying mechanisms of scarring and tissue regeneration. This paper describes methods for the cultivation of corneal keratocytes, fibroblasts, epithelial, and endothelial cells for in vitro applications. We also provide detailed, step-by-step protocols for assembling and culturing 3D constructs of the corneal stroma, epithelial- and endothelial-stromal co-cultures and isolation of extracellular vesicles.
AB - Science and medicine have become increasingly “human-centric” over the years. A growing shift away from the use of animals in basic research has led to the development of sophisticated in vitro models of various tissues utilizing human-derived cells to study physiology and disease. The human cornea has likewise been modeled in vitro using primary cells derived from corneas obtained from cadavers or post-transplantation. By utilizing a cell's intrinsic ability to maintain its tissue phenotype in a pre-designed microenvironment containing the required growth factors, physiological temperature, and humidity, tissue-engineered corneas can be grown and maintained in culture for relatively long periods of time on the scale of weeks to months. Due to its transparency and avascularity, the cornea is an optimal tissue for studies of extracellular matrix and cell-cell interactions, toxicology and permeability of drugs, and underlying mechanisms of scarring and tissue regeneration. This paper describes methods for the cultivation of corneal keratocytes, fibroblasts, epithelial, and endothelial cells for in vitro applications. We also provide detailed, step-by-step protocols for assembling and culturing 3D constructs of the corneal stroma, epithelial- and endothelial-stromal co-cultures and isolation of extracellular vesicles.
KW - co-culture
KW - corneal endothelial cells
KW - corneal epithelial cells
KW - extracellular vesicles
KW - keratocytes
UR - http://www.scopus.com/inward/record.url?scp=85092119816&partnerID=8YFLogxK
U2 - 10.1002/cpcb.114
DO - 10.1002/cpcb.114
M3 - Article
C2 - 32986311
AN - SCOPUS:85092119816
SN - 1934-2616
VL - 89
JO - Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]
JF - Current protocols in cell biology / editorial board, Juan S. Bonifacino ... [et al.]
IS - 1
M1 - e114
ER -