Generating a 3D Co-Culture Model of Human Corneal Fibroblasts and Neurons

Abstract

Source: Sharif, R., et al. Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea. J. Vis. Exp. (2018)

This video demonstrates a technique to generate a three-dimensional co-culture model system of primary human corneal fibroblasts and differentiated neuronal cells. This co-culture system can be used to study the stromal-nerve interactions.

Protocol

1. Primary HCFs (human corneal fibroblasts) Culture and 3D Constructs Assembly

1. To assemble 3D constructs from explants previously grown in EMEM 10% FBS 1% Antibiotic/Antimycotic, passage and count HCFs.
2. Seed approximately 1 x 10⁶ cells/well of primary HCFs on polycarbonate membrane inserts with 0.4-µm pores from the 3D constructs, along with 1.5 mL of fresh EMEM 10% FBS 1% Antibiotic/Antimycotic to the top of the construct. Add another 1.5 mL of the same media to the bottom of each construct.
3. After 24 h of cell seeding, culture the cells with EMEM 10% FBS 1% Antibiotic/Antimycotic, stimulated with 0.5 mM....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Dulbecco's Phosphate Buffered Solution (1X)	Gibco by Life Technologies	14190-144
Sterile forceps	Fischer Scientific	13-812-42	Fisherbrand Dissecting Extra-Fine-Pointed Splinter Forceps
Single edge razor blades	Personna	270100
Sterile surgical scalpel blades No.10	Feather Surgical Blade	2976#10
Eagle's Minimum Essential Medium	ATCC	30-2003
Fetal Bovine Serum	Atlanta Biologicals	S11550	10% FBS is required for media preparation
Antibiotic-Antimycotic (100X)	Gibco by Life Technologies	15240-062	1% Antibiotic-Antimycotic is required for media preparation
0.05% Trypsin EDTA(1X)	Gibco by Life Technologies	25300062
Polycarbonate membrane inserts with 0.4-μm pores	Corning Costar	3412
2-O-α-Dglucopyranosyl-L-ascorbic acid (Vitamin C)	Sigma-Aldrich	SMB00390-14	A concentration of 0.5 mM should be used for the study
SH-SY5Y Neuroblastoma cells	ATCC	SHSY5YATCC CRL-2266
Retinoic Acid	Sigma-Aldrich	SRP3014-10UG	Final concentration of 10uM needs to be used
BDNF	Sigma-Aldrich	R2625-100MG	Final concentration of 2nM needs to be used
Dimethyl Sulfoxide(DMSO)	VWR-Alfa Aesar	67-68-5	Ultra Pure Grade-Sterile DMSO to be used
Thermo Scientific Nunc Cell Culture Treated EasYFlasks (T25)	Fisher Scientific	12-565-351
Thermo Scientific Nunc Cell Culture Treated EasYFlasks (T75)	Fisher Scientific	12-565-349