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 2-O-α-D-glucopyranosyl-L-ascorbic acid (Vitamin C) by adding 1.5 mL on the top and 1.5 mL on the bottom of each construct.
4. Maintain the cultures in an incubator at 37 °C, 5 % CO₂ for 3 weeks and supply fresh media every other day during the entire study period. No further passage is needed.

2. Cell Differentiation and Co-cultures

1. After 3 weeks, add 8 x 10³ cells/well of SH-SY5Y human neuroblastoma cells on the top of the previously assembled 3D constructs containing 1.5 mL of the EMEM containing 10% FBS, 1% Antibiotic/Antimycotic, and 0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic acid (Vitamin C).
   1. Allow the SH-SY5Y neuroblastoma cells to grow on top of the cornea stromal cells for at least 24 h before initiating cell differentiation.
2. Initiate SH-SY5Y cell differentiation by stimulating the cells with EMEM containing 1% FBS and 1.5 mL of 10 µM Retinoic Acid on top of the construct. Add 1.5 mL of the EMEM 10% FBS 1% Antibiotic/Antimycotic 0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic acid (Vitamin C) to the bottom of the construct. Note that the Retinoic Acid step should be performed in the dark.
   1. Continue to culture the cells in this differentiation media for 5 days.
3. When the cells reach the differentiation phase, switch the cells to 1.5 mL of serum-free media containing 2 nM of Brain-derived neurotrophic factor (BDNF) and 1% Antibiotic/Antimycotic added to the EMEM, by adding to the top of the construct. Add 1.5 mL of the EMEM 10% FBS 1% Antibiotic/Antimycotic 0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic acid (Vitamin C) to the bottom of the construct. Note that the BDNF step should be performed in the dark.
4. Culture the cells for 2 additional days before processing for any further analysis.

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