Isolation of HCECs
1. The corneoscleral tissues were rinsed three times with primary cell culture system containing 50 mg/mL gentamicin and 1.25 mg/mL amphotericin B.
2. The central cornea was removed by a trephine of 8-mm diameter.
3. Descemet’s membrane and corneal endothelial cells were stripped from the posterior surface of the peripheral corneoscleral tissue under a dissecting microscope.
4. Tissues were digested at 37℃ for 1.5 to 16 hours with 2 mg/mL collagenase A in supplemented hormonal epithelial medium (SHEM)
5. Primary cell culture system
Primary cell culture medium
5% FBS
0.5% dimethyl sulfoxide
2 ng/mL mouse EGF
5 μg/mL insulin
5 μg/mL transferring
5 ng/mL selenium
0.5 μg/mL hydrocortisone
1 nM cholera toxin
50 μg/mL gentamicin
1.25 μg/mL amphotericin B
6. After digestion, HCECs formed aggregates, which were collected by centrifugation at 2000 rpm for 3 minutes to remove the digestion solution.
7. As a control, Descemet’s membrane strips were also digested in 10 mg/mL Dispase II in SHEM and trypsin/EDTA for up to 3 hours.

Preservation of Isolated HCEC Aggregates
1. The resultant aggregates of HCECs were preserved in keratinocyte serum-free medium (KSFM) with complete supplement (storage medium 1), DMEM/F12 with KSFM supplements (storage medium 2), or DMEM/F12 with supplemented hormonal epithelial medium (SHEM) supplements without FBS (storage medium 3).
2. All these media are serum free, one of the major differences among them is the calcium concentration, which was 0.09 mM in storage medium 1, but was 1.05 mM in storage media 2 and 3.
3. HCEC aggregates were stored in a tissue culture incubator at 37℃ for up to 3 weeks.
4. Cell viability was determined and also evaluated by subculturing them in SHEM.

Expansion of Isolated HCEC Aggregates
1. The resultant HCEC aggregates, either immediately after digestion or after a period of preservation in a storage medium, were then cultured in SHEM with or without additional growth factors on a plastic dish under 37℃and 5% CO^₂.
40 ng/mL bFGF
0.1 mg/mL bovine pituitary extract
0.2 20 ng/mL nerve growth factor
2. The media were changed every 2 to 3 days.
3. Some HCEC aggregates were pretreated with trypsin/EDTA at 37℃ for 10 minutes to dissociate endothelial cells before the aforementioned cultivation.

Reference
1. Li W, Sabater AL, Chen YT, Hayashida Y, Chen SY, He H, Tseng SC. A novel method of isolation, preservation, and expansion of human corneal endothelial cells. Invest Ophthalmol Vis Sci. 2007; 48: 614-620.

2. Ying-Ting Zhu, Yasutaka Hayashida, Ahmad Kheirkhah, Hua He, Szu-Yu Chen and Scheffer C. G. Tseng. Characterization and comparison of intercellular adherent junctions expressed by human corneal endothelial cells in vivo and in vitro. IOVS. 2008; 49: 3879-3886.

3. Masahiro Yamaguchi, Nobuyuki Ebihara, Nobuyuki Shima, Miwa Kimoto, Toshinari Funaki, Seiichi Yokoo, Akira Murakami, Satoru Yamagami. Adhesion, Migration, and proliferation of cultured human corneal endothelial cells by laminin-5. IOVS. 2011; 52: 679-684.