Figure 2.

Subcultivation on a fibrin carrier and transplantation. (A) Diagram of the triple transgenic mouse model showing the inducible, tissue specific nature of this system. In this system, only in corneal epithelial cells can rtTA (reverse tetracycline transcriptional activator) be expressed. In the presence of doxycycline (dox), the rtTA/dox complex binds to the tetracycline operator elements (Tet-O) to drive the expression of Cre recombinase. Cre is then able to excise the tdTomato transgene encoding red fluorescent protein allowing for expression of EGFP. Following this recombination event EGFP expression is under the control of the chicken actin (pCA) promoter. Vibrissae hair follicles (HFs) were dissected from lips of whole body red triple transgenic mice. (B, C) The enzymatically isolated epithelial cells were seeded onto a 3T3 feeder cell layer to grow holoclones; white light (C); red light, 3-D imaging of a holoclone (D). (E–G) The clonal growth was monitored over the 2 week culture period and showed progressively growing red fluorescent holoclones, whereas EGFP expression was completely absent. The clones were photographed on culture day 3 (E), 7 (F), and 14 (G). (H, I) After two weeks of culture, the clones were transferred onto a biodegradable fibrin carrier and cultured for several days to form a confluent cell sheet appropriate for transplantation; white light (H), red and green light merged (I). (J) Prior to transplantation limbal stem cell deficiency was generated on murine eyes using an Algerbrush II corneal rust ring remover with a 0.5 mm burr. (K) The transplants were anchored to the conjunctiva with four sutures with the cells facing the intact recipient´s corneal epithelium basement membrane. (L, M) Examination of the sutured eye revealed that the HFSC were still intact and expressing the red fluorescent tdtomato protein, but as expected lacked EGFP. Magnification: B – 8X, E-I– 100X, J-M – 20X