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Heli Skottman, Anni Sorkio, Lothar Koch, Laura Koivusalo, Andrea Deiwick, Susanna Miettinen, Boris Chichkov; 3D laser-assisted bioprinting of tissue engineered cornea using human stem cells. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3451.
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© ARVO (1962-2015); The Authors (2016-present)
There is an increased demand for developing methods for producing more native-like 3D corneal structures. In the present study, we produced 3D cornea-mimicking tissues using human stem cells and laser-assisted bioprinting (LaBP). Human embryonic stem cell differentiated limbal epithelial stem cells (hESC-LESC) were used as a cell source for printing epithelium-mimicking structures, whereas human adipose tissue stem cells (hASCs) were used for constructing layered stroma-mimicking structures.
The culture of hESC-LESC and hASC were conducted with previously established methods. We chose recombinant human laminin and human sourced collagen I as bases for the functional bioinks. Two previously established LaBP setups based on laser induced forward transfer, with different laser wavelengths and appropriate absorption layer materials, gold and MatrigelTM, were used. For 3D structures, acellular layers of bioink were printed between layers with hASCs in order to establish structures mimicking the corneal stroma. The printed structures were cultured up to 14 days. Cell viability and proliferation of both cell types after LaBP were assessed with LIVE/DEAD® Viability/Cytotoxicity and PrestoBlue™cell viability assays. The cell migration, morphology, expression of cell specific markers and tissue structure were analyzed with immunofluorescence (Ki67, p63a, p40, CK3, CK15, collagen type I, VWF). The 3D printed stromal constructs were also implanted into porcine corneal organ cultures. One week after implantation, the corneal organ cultures were histologically analyzed.
Both cell types had good viability after printing. Laser-printed hESC-LESCs showed epithelial cell morphology, expression of Ki67 proliferation marker and co-expression of corneal progenitor markers p63α/p40. Importantly, the printed hESC-LESCs formed a stratified epithelium with apical expression of CK3 and basal expression of the progenitor markers. The structure of the 3D bioprinted stroma demonstrated that the hASCs had organized horizontally as in the native corneal stroma and showed positive labeling for collagen I. After 7 days in porcine organ cultures, the 3D bioprinted structures attached to the host tissue with migration of hASCs from the printed structure.
In this study, we demonstrate for the first time, the feasibility of LaBP for fabrication of layered 3D bioprinted structure mimicking the native corneal tissues.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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