Bosworth Lucy A, Rathbone Sarah R, Cartmell Sarah H
School of Materials, The University of Manchester.
School of Materials, The University of Manchester;
J Vis Exp. 2015 Apr 10(98):52135. doi: 10.3791/52135.
Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(ε-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37 (o)C, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged - the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.
生物材料和组织工程的研究通常包括基于细胞的体外研究，这需要了解起始细胞数量的初始信息。虽然研究人员通常会提及他们的接种密度，但这并不一定表明附着在所研究材料上的实际细胞数量。对于那些没有覆盖标准细胞培养孔板底部的材料或支架来说，情况尤其如此。本研究调查了在培养4小时后，以已知数量接种到电纺聚（ε-己内酯）纱线上的人间充质干细胞的初始附着情况。电纺纱线被放置在几种不同的装置中，包括以9转/分钟旋转的生物反应器容器、置于低结合力孔板中的细胞培养插入物以及放置在培养皿内的聚四氟乙烯（PTFE）槽。后两者分别处于静态条件下或放置在摇床上（30转/分钟）。在37℃、5%二氧化碳条件下孵育4小时后，通过细胞DNA检测确定接种细胞的位置。将支架从其容器中取出并置于裂解缓冲液中。同样地，取出培养基部分并进行离心——弃去上清液，用裂解缓冲液将沉淀打散。向每个容器或孔中加入裂解缓冲液，并刮擦以释放可能存在的任何细胞。细胞DNA检测确定了支架、培养基和孔部分中存在的细胞百分比。所有实验装置中的细胞附着率都很低，置于细胞培养插入物中并进行摇动的纱线附着率最高（30%）。本研究提高了人们对附着在支架上的实际细胞数量的认识，而不考虑所述的细胞接种密度。
