The in vitro fabrication of wholly vascularized millimeter-sized engineered tissues is still a key challenge in the tissue engineering field. Recently we reported a unique approach ‘sedimentary culture’ using a collagen microfiber (CMF) to fabricate large-scale engineered tissues. The millimeter-sized tissues with high extracellular matrix (ECM) density were easily obtained by centrifugation of cells and CMFs and subsequent cultivation because the CMFs acted as a micrometer-sized scaffold. However, cell distribution in the obtained tissues was not homogeneous because of the different sedimentation velocity of the cells and CMFs because of their size difference.

Here we report the fabrication of wholly vascularized millimeter-sized engineered tissues using cell-sized CMFs. To avoid dissolving, vacuum drying was performed at 200 °C for 24 h for thermal crosslinking of primary amine groups of type I collagen. The 200- and 20-μm-sized CMFs (CMF-200 and CMF-20) were obtained by homogenization and subsequent sonication of the crosslinked collagen. Interestingly, the CMF-20 indicated a similar sedimentation velocity with cells because of their same size range, thus uniform millimeter-sized tissue with homogeneous cell distribution was fabricated by the sedimentary culture method. To form a whole blood capillary structure in the tissues, fibronectin (FN) was adsorbed on the surface of CMF-20 to stimulate endothelial cell migration. The distribution of the blood capillary network in 1.6-mm-sized tissues was markedly improved by FN-adsorbed CMF-20 (FN-CMF-20). Sedimentary culture using FN-CMF-20 will create new opportunities in tissue engineering for the in vitro fabrication of wholly vascularized millimeter-sized engineered tissues.

全血管化毫米级工程组织的体外制造仍然是组织工程领域的一个关键挑战。最近，我们报道了一种独特的“沉积培养”方法，使用胶原微纤维（CMF）来制造大规模工程组织。由于 CMF 可充当微米级支架，因此通过细胞和 CMF 的离心以及随后的培养，可以轻松获得具有高细胞外基质 (ECM) 密度的毫米级组织。然而，由于细胞和 CMF 的沉降速度不同，因此获得的组织中的细胞分布不均匀。

在这里，我们报告了使用细胞大小的 CMF 制造全血管化的毫米大小的工程组织。为了避免溶解，在 200°C 下真空干燥 24 小时，以实现 I 型胶原伯胺基团的热交联。通过对交联胶原蛋白进行均质化和随后的超声处理，获得了 200 微米和 20 微米大小的 CMF（CMF-200 和 CMF-20）。有趣的是，由于细胞尺寸范围相同，CMF-20 与细胞表现出相似的沉降速度，因此通过沉降培养方法可以制备出细胞分布均匀的毫米级组织。为了在组织中形成全血毛细血管结构，纤连蛋白（FN）被吸附在CMF-20表面以刺激内皮细胞迁移。吸附 FN 的 CMF-20 (FN-CMF-20) 显着改善了 1.6 毫米大小组织中毛细血管网络的分布。使用 FN-CMF-20 进行沉积培养将为组织工程领域创造新的机会，用于体外制造全血管化的毫米级工程组织。