BioMediTech Institute and Faculty of Medicine and Life Sciences, University of Tampere, Arvo Ylpön katu 34, FI-33520 Tampere, Finland; Laser Zentrum Hannover e.V., Hollerithallee 8, 30419 Hannover, Germany.
Laser Zentrum Hannover e.V., Hollerithallee 8, 30419 Hannover, Germany.
Biomaterials. 2018 Jul;171:57-71. doi: 10.1016/j.biomaterials.2018.04.034. Epub 2018 Apr 16.
There is a high demand for developing methods to produce 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 derived limbal epithelial stem cells (hESC-LESC) were used as a cell source for printing epithelium-mimicking structures, whereas human adipose tissue derived stem cells (hASCs) were used for constructing layered stroma-mimicking structures. The development and optimization of functional bioinks was a crucial step towards successful bioprinting of 3D corneal structures. Recombinant human laminin and human sourced collagen I served as the bases for the functional bioinks. We used two previously established LaBP setups based on laser induced forward transfer, with different laser wavelengths and appropriate absorption layers. We bioprinted three types of corneal structures: stratified corneal epithelium using hESC-LESCs, lamellar corneal stroma using alternating acellular layers of bioink and layers with hASCs, and finally structures with both a stromal and epithelial part. The printed constructs were evaluated for their microstructure, cell viability and proliferation, and key protein expression (Ki67, p63α, p40, CK3, CK15, collagen type I, VWF). The 3D printed stromal constructs were also implanted into porcine corneal organ cultures. Both cell types maintained 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 and 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 stromal structures attached to the host tissue with signs of hASCs migration from the printed structure. This is the first study to demonstrate the feasibility of 3D LaBP for corneal applications using human stem cells and successful fabrication of layered 3D bioprinted tissues mimicking the structure of the native corneal tissue.
人们对于开发更接近天然的 3D 角膜结构的方法有很高的需求。在本研究中，我们使用人类干细胞和激光辅助生物打印（LaBP）来生产 3D 角膜模拟组织。人类胚胎干细胞衍生的角膜缘上皮干细胞（hESC-LESC）被用作打印上皮模拟结构的细胞来源，而人类脂肪组织衍生的干细胞（hASCs）则用于构建分层基质模拟结构。功能性生物墨水的开发和优化是成功生物打印 3D 角膜结构的关键步骤。重组人层粘连蛋白和人源胶原蛋白 I 用作功能性生物墨水的基础。我们使用了两种先前建立的基于激光诱导前向转移的 LaBP 设备，使用不同的激光波长和适当的吸收层。我们生物打印了三种类型的角膜结构：使用 hESC-LESCs 打印分层角膜上皮，使用无细胞生物墨水层和带有 hASCs 的层交替的层状角膜基质，最后是具有基质和上皮部分的结构。评估了打印结构的微观结构、细胞活力和增殖以及关键蛋白表达（Ki67、p63α、p40、CK3、CK15、胶原蛋白 I、VWF）。打印的基质结构也被植入猪角膜器官培养物中。两种细胞类型在打印后都保持良好的活力。激光打印的 hESC-LESCs 显示出上皮细胞形态，Ki67 增殖标志物的表达以及角膜祖细胞标志物 p63α 和 p40 的共表达。重要的是，打印的 hESC-LESCs 形成了具有 CK3 顶端表达和祖细胞标志物基底表达的分层上皮。3D 生物打印基质的结构表明，hASCs 已水平组织化，类似于天然角膜基质，并对胶原蛋白 I 呈阳性标记。在猪器官培养物中培养 7 天后，3D 生物打印的基质结构与宿主组织附着，并出现 hASCs 从打印结构迁移的迹象。这是第一项使用人类干细胞和成功制造模拟天然角膜组织结构的分层 3D 生物打印组织的 3D LaBP 用于角膜应用的可行性研究。
