The stromal cell line HS-5 is an alternative mesenchymal stem cell source for the development of a 3D bone marrow model

Developing a novel three-dimensional (3D) in vitro model of the bone marrow microenvironment using bone mar- row mesenchymal stem cells (BM-MSCs) in commercial matrices involves a series of distinct processes. This starts with selecting an appropriate cell source and growth envi- ronment. Primary cells isolated from healthy donors have low proliferation rate and lack homogeneity that can result in experimental variation. This study evaluated the immor- talized stromal cell line HS-5 as an alternative cell source and evaluated their behaviour in the 3D culture. This study compared cell morphology, plastic adherence and expres- sion of surface markers of HS-5 vs. primary cells. Also it evaluated culture medium to improve cell growth and preserve cell characteristics to as close as possible to the in vivo conditions. Inoculation techniques into the matrix (Biomerix, Nano ber and Alvetex) were evaluated, as well as cell retrieval from the 3D system for further investiga- tion. The results indicate that both HS-5 and primary cells meet the criteria for MSC demonstrating plastic adherence, correct cluster of differentiation marker (CD) expression and broblastic morphology; however cells are rounded in 3D culture. BM-MSCs were not able to penetrate into the nano bre; however, HS-5 cells penetrated and grew in- side the nano bre but with different morphology to cells in other scaffolds. HS-5 grows best in high glucose DMEM medium supplemented with 11% heat inactivated FBS and 2mM L-glutamine. Under these conditions HS-5 adhere well to the scaffolds at room temperature and are easily retrieved using trypsin solution whereas primary cells need up to 120 and 90 minutes to adhere to Nano ber and Biomerix, respectively. HS-5 proliferation rate, size and CD expression decreased in the 3D compared to the 2D. It was noted that some cells escaped the scaffolds and formed a 2D monolayer. The data con rm HS-5 as a suitable alter- native cell source for 3D culture