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Methods for Investigating Corneal Cell Interactions and Extracellular Vesicles In Vitro
Tina B McKay1, Xiaoqing Guo1, Audrey E K Hutcheon1
1Schepens Eye Research Institute of Massachusetts Eye and Ear and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts.
Current Protocols in Cell Biology
|September 28, 2020
Summary
This study details methods for creating advanced in vitro human cornea models using cell culture techniques. These tissue-engineered corneas enable research into drug permeability, scarring, and regeneration.
Area of Science:
- Biomedical Engineering
- Ophthalmology Research
- Tissue Engineering
Background:
- Increasing shift towards human-centric research models in science and medicine.
- Development of sophisticated in vitro tissue models using human-derived cells.
- Need for advanced in vitro models of the human cornea for physiological and disease studies.
Purpose of the Study:
- To describe methods for cultivating human corneal cells for in vitro applications.
- To provide protocols for assembling and culturing 3D corneal constructs.
- To detail the isolation of extracellular vesicles from corneal cell conditioned medium.
Main Methods:
- Cultivation of human corneal keratocytes, fibroblasts, epithelial, and endothelial cells.
- Assembly and culturing of 3D corneal stromal constructs.
- Co-culture of epithelial- and endothelial-stromal components.
- Isolation of extracellular vesicles from conditioned medium.
Main Results:
- Established protocols for isolating and culturing various human corneal cell types.
- Successful assembly and maintenance of 3D corneal stromal and co-culture constructs.
- Methods for cryopreservation of corneal cells developed.
- Protocols for extracellular vesicle isolation from corneal cell cultures provided.
Conclusions:
- Tissue-engineered human corneas can be reliably grown and maintained in vitro for extended periods.
- These models are suitable for studying extracellular matrix interactions, drug toxicology, and corneal regeneration.
- The described methods facilitate advanced research in corneal physiology and disease using human-derived in vitro models.
