Evaluation of Thrombocyte-based extract in different human corneal cell types

Abstract

Purpose : The use of fetal calf serum (FCS) in biomedical sciences is controversially discussed since decades. Although the use of FCS is established and accepted within the scientific community the use of undefined animal sera leads to problems with the reproducibility of in vitro studies and to problems with immunogenic reactions especially in the field of stem cell or tissue transplantation. Platelet-derived growth media supplementation is therefore investigated as a suitable FCS replacement.

Methods : We used a new type of thrombocyte-based extract (Lindls TBE) from our collaborator (I-A-Z GmbH Prof. Toni Lindl) to establish animal-free cell culture media for in vitro studies of human corneal cell types and for a new type of storage medium for corneal grafts. We therefore analyzed the growth and vitality of human epithelial (HCE) and endothelial (HCEC12) cell suspensions as well as keratinocytes (HCK) derived from human corneal origin. All of them are used for 3-dimensional cell culture of the human cornea.

Results : Our research shows that all used human corneal cell lines respond in a very positive manner to the thrombocyte extract compared to FCS standard treatment although the Lindls TBE contains only 1% of the protein amount FCS normally contains. With a used protein yield of 0.3 mg/ mL in growth media human corneal keratinocyte cells showed no difference in growth speed and vitality compared to FCS control (FCS: 7,5x10⁵ +-3,6x10³ vs Lindls-TBE 5,4x10⁵+-8,3 x10³). The same applied to human corneal epithelial cells (FCS: 6.3x10⁵ +-4,4x10³ vs Lindls-TBE 6,7x10⁵+-3,0x10³) and human corneal endothelial cells (HCEC: 8,4x10⁴+-3,0x10² vs Lindls-TBE 9,0x10⁴+-1,3 x10³) if 0.4 mg/ mL final protein yield was used.

Conclusions : The Lindls TBE medium supplement is able to replace FCS in in vitro culture of human corneal cells in monoculture as in co-culture. The complete animal free approach will enable us to achieve a non-immunogenic, thus transplantable model of the human cornea in future using primary cells from patients or re-programmed stem cells. The low protein yield combined with excellent growth stimulation abilities make the Lindls-TBE a very promising supplement who’s further analysis via mass spectrometry (LC/MS/MS) will render a full defined media for cell culture possible in future.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.