School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney NSW 2052, Australia.
Biotechnol Prog. 2011 Jan-Feb;27(1):191-200. doi: 10.1002/btpr.509. Epub 2010 Nov 9.
Cell seeding and attachment in three-dimensional scaffolds is a key step in tissue engineering with implications for cell differentiation and tissue development. In this work, two new seeding methods were investigated using human chondrocytes and polyglycolic acid (PGA) fibrous mesh scaffolds. A simple semi-static seeding method using culture plates and tissue flasks was developed as an easy-to-perform modification of static seeding. An alginate-loading method was also studied, using alginate hydrogel as an adjuvant for entrapping cells within PGA scaffolds. Both the semi-static and PGA-alginate methods produced more homogeneous cell distributions than conventional static and dynamic seeding. Using 20 × 10(6) cells, whereas the seeding efficiency for static seeding was only 52%, all other techniques produced seeding efficiencies of ≥ 90%. With 40 × 10(6) cells, the efficiency of semi-static seeding declined to 74% while the dynamic and PGA-alginate methods retained their ability to accommodate high cell numbers. The seeded scaffolds were cultured in recirculation bioreactors to determine the effect of seeding method on cartilage production. Statically seeded scaffolds did not survive the 5-week cultivation period. Deposition of extracellular matrix in scaffolds seeded using the semi-static and PGA-alginate methods was more uniform compared with scaffolds seeded using the dynamic method. The new semi-static and PGA-alginate seeding methods developed in this work are recommended for tissue engineering because they provide substantial benefits compared with static seeding in terms of seeding efficiency, cell distribution, and cartilage deposition while remaining simple and easy to execute.
细胞接种和附着在三维支架中是组织工程的关键步骤，对细胞分化和组织发育有重要影响。在这项工作中，使用人软骨细胞和聚乙醇酸（PGA）纤维网支架研究了两种新的接种方法。开发了一种简单的半静态接种方法，使用培养板和组织培养瓶，作为静态接种的简单改进。还研究了一种藻酸盐加载方法，使用藻酸盐水凝胶作为将细胞包埋在 PGA 支架内的佐剂。半静态和 PGA-藻酸盐方法产生的细胞分布比传统的静态和动态接种更均匀。使用 20×10^6 个细胞，而静态接种的接种效率仅为 52%，所有其他技术的接种效率均≥90%。使用 40×10^6 个细胞时，半静态接种的效率下降到 74%，而动态和 PGA-藻酸盐方法仍然能够适应高细胞数量。接种后的支架在循环生物反应器中培养，以确定接种方法对软骨生成的影响。静态接种的支架在 5 周的培养期间未能存活。与使用动态方法接种的支架相比，使用半静态和 PGA-藻酸盐方法接种的支架中细胞外基质的沉积更均匀。本工作中开发的新的半静态和 PGA-藻酸盐接种方法推荐用于组织工程，因为与静态接种相比，它们在接种效率、细胞分布和软骨沉积方面具有显著优势，同时仍然简单易行。
