The architecture of the human corneal stroma consists of a highly organized extracellular matrix (ECM) interspersed with keratocytes. Their progenitor cells; corneal stromal stem cells (CSSC) are located at the periphery, in the limbal stroma. A highly organized corneal ECM is critical for effective transmission of light but this structure may be compromised during injury or disease, resulting in loss of vision. Re-creating normal organization in engineered tissue equivalents for transplantation often involves lengthy culture times that are inappropriate for clinical use or utilisation of synthetic substrates that bring complications such as corneal melting. CSSC have great therapeutic potential owing to their ability to reorganize a disorganized matrix, restoring transparency in scarred corneas. We examined CSSC contractile behavior to assess whether this property could be exploited to rapidly generate cell and ECM organization in Real Architecture For 3D Tissues (RAFT) tissue equivalents (TE) for transplantation. Free-floating collagen gels were characterized to assess contractile behavior of CSSC and establish optimum cell density and culture times. To mediate cell and collagen organization, tethered collagen gels seeded with CSSC were cultured and subsequently stabilized with the RAFT process. We demonstrated rapid creation of biomimetic RAFT TE with tunable structural properties. These displayed three distinct regions of varying degrees of cellular and collagen organization. Interestingly, increased organization coincided with a dramatic loss of PAX6 expression in CSSC, indicating rapid differentiation into keratocytes. The organized RAFT TE system could be a useful bioengineering tool to rapidly create an organized ECM while simultaneously controlling cell phenotype.

Statement of significance

For the first time, we have demonstrated that human CSSC exhibit the phenomenon of cellular self-alignment in tethered collagen gels. We found this mediated rapid co-alignment of collagen fibrils and thus subsequently exploited this property in vitro to improve the architecture of engineered RAFT tissue equivalents of the corneal stroma. Existing techniques are extremely lengthy and carry significant risk and cost for GMP manufacture. This rapid and tunable technique takes just 8 hours of culture and is therefore ideal for clinical manufacture, creating biomimetic tissue equivalents with both cellular and ECM organization. Thus, cellular self-alignment can be a useful bioengineering tool for the development of aligned tissue equivalents in a variety of applications.

人角膜基质的结构由散布有角化细胞的高度组织化的细胞外基质（ECM）组成。他们的祖细胞；角膜基质干细胞（CSSC）位于角膜基质的外围。高度组织化的角膜ECM对于有效地透光是至关重要的，但是在受伤或患病期间，该结构可能会受损，从而导致视力下降。在工程组织等效物中重新建立正常组织以进行移植通常涉及漫长的培养时间，这对于临床使用或利用合成基质（例如角膜融化等并发症）而言，是不合适的。CSSC具有重组杂乱无章的基质，恢复疤痕角膜透明性的能力，因此具有巨大的治疗潜力。我们检查了CSSC的收缩行为，以评估是否可以利用此属性在Real Architecture for 3D组织（RAFT）组织等效物（TE）中快速生成细胞和ECM组织，以进行移植。自由浮动的胶原蛋白凝胶的特征在于评估CSSC的收缩行为并建立最佳的细胞密度和培养时间。为了介导细胞和胶原蛋白的组织，对接种有CSSC的系留胶原蛋白凝胶进行培养，然后通过RAFT工艺使其稳定。我们展示了具有可调结构特性的仿生RAFT TE的快速创造。这些显示了不同程度的细胞和胶原组织的三个不同区域。有趣的是，组织的增加与CSSC中PAX6表达的急剧丧失相吻合，表明其迅速分化为角膜细胞。有组织的RAFT TE系统可能是一个有用的生物工程工具，可以在同时控制细胞表型的同时快速创建有组织的ECM。

重要声明

首次，我们证明了人类CSSC在束缚的胶原蛋白凝胶中表现出细胞自对准现象。我们发现这种介导的胶原蛋白原纤维的快速共排列，因此随后在体外利用了该特性来改善角膜基质的工程RAFT组织等同物的结构。现有技术极其冗长，并且对GMP制造具有重大的风险和成本。这种快速且可调的技术仅需8小时的培养，因此非常适合临床制造，可通过细胞和ECM组织创建仿生组织的等效物。因此，细胞自对准可以是在各种应用中用于开发对准的组织等同物的有用的生物工程工具。