Successful Consecutive Expansion of Limbal Explants Using a Biosafe Culture Medium under Feeder Layer-Free Conditions

ABSTRACT

Purpose: Transplantation of in vitro cultured limbal epithelial stem cells (LESCs) is a treatment widely used for LESC deficiency. However, the number of limbal tissue donors is limited, and protocols for LESC cultivation often include compounds and/or feeder layers that can induce side effects and/or increase the cost of the culture procedure. We investigated the feasibility of obtaining more than one limbal primary culture (LPC) from the same biopsy using a culture medium in which several potentially harmful compounds were replaced at the same time by biosafe supplements, allowing the LESC cultivation without feeder layers.

Materials and methods: We established feeder layer-free LPCs with three culture media: (1) a modified supplemental hormonal epithelial medium, containing potential harmful components (cholera toxin, dimethylsulfoxide, and fetal bovine serum [FBS]), (2) IOBA-FBS, a medium with FBS but with no other harmful supplements, and (3) IOBA-HS, similar to IOBA-FBS but with human serum instead of FBS. Additionally, the same limbal explant was consecutively cultured with IOBA-HS producing three cultures. LPCs were characterized by real-time reverse transcription polymerase chain reaction and/or immunofluorescence.

Results: LPCs cultured with the three media under feeder layer-free conditions showed cuboidal cells and no significant differences in the percentage of positive cells for limbal (ABCG2, p63, and K14) and corneal (K3, K12) proteins. Except for ABCG2, the relative mRNA expression of the LESC markers was significantly higher when IOBA-FBS or IOBA-HS was used. LPC1 showed characteristics similar to LPC0, while LPC2 cell morphology became elongated and the expression of some LESC markers was diminished.

Conclusion: IOBA-HS enables the culturing of up to two biosafe homologous LPCs from one limbal tissue under feeder layer-free conditions. The routine use of this culture medium could improve both the biosafety and the number of available LPCs for potential clinical transplantation, as well as decrease the expense of the culture procedure.

KEYWORDS:

• cell culture
• culture media
• limbal cells
• ocular surface
• stem cells

Acknowledgments

The authors thank M.F. de la Paz, MD, and F.J. Iglesias, PhD, for providing human tissues, H.A. Martínez-Osorio, PhD, for his initial scientific advice, V. Sáez for technical-support, I. Fernández, PhD, and M.E. Mateo (statisticians) for statistical assistance, and B. Bromberg (Xenofile Editing) for his assistance in the editing of extensive parts of this manuscript.

This work was presented in part at the 86th ARVO May 2014 Orlando, FL, USA and at the TERMIS-EU June 2013 Istanbul, Turkey.

Declaration of interest

The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.

Funding

This work was supported by the Carlos III National Health Institute (CIBER-BBN and Spanish Network on Cell Therapy: ([grant number TerCel, RD12/0019/0036], Spain) and the Castilla-León Government (Regional Center for Regenerative Medicine and Cell Therapy: [grant numbers SAN126/VA11/09 and BIO/VA05/15). M. López-Paniagua and S. Galindo were supported by scholarships cofinanced by the Castilla-León Government and the European-Social-Fond.

Additional information

Funding

This work was supported by the Carlos III National Health Institute (CIBER-BBN and Spanish Network on Cell Therapy: ([grant number TerCel, RD12/0019/0036], Spain) and the Castilla-León Government (Regional Center for Regenerative Medicine and Cell Therapy: [grant numbers SAN126/VA11/09 and BIO/VA05/15). M. López-Paniagua and S. Galindo were supported by scholarships cofinanced by the Castilla-León Government and the European-Social-Fond.