Improving cell distribution on 3D additive manufactured scaffolds through engineered seeding media density and viscosity

Abstract

In order to ensure the long-term in vitro and in vivo functionality of cell-seeded 3D scaffolds, an effective and reliable method to control cell seeding efficiency and distribution is crucial. Static seeding on 3D additive manufactured scaffolds made of synthetic polymers still remains challenging, as it often results in poor cell attachment, high cell sedimentation and non-uniform cell distribution, due to gravity and to the intrinsic macroporosity and surface chemical properties of the scaffolds. In this study, the bio-inert macromolecules dextran and Ficoll were used for the first time as temporary supplements to alter the viscosity and density of the seeding media, respectively, and improve the static seeding output. The addition of these macromolecules drastically reduced the cell sedimentation velocities, allowing for homogeneous cell attachment to the scaffold filaments. Both dextran-and Ficoll-based seeding methods supported human mesenchymal stromal cells viability and osteogenic differentiation post-seeding. Interestingly, the improved cell distribution led to increased matrix production and mineralization compared to scaffolds seeded by conventional static method. These results suggest a simple and universal method for an efficient seeding of 3D additive manufactured scaffolds, independent of their material and geometrical properties, and applicable for bone and various other tissue regeneration.

Effect of Highly Loaded Nanohydroxyapatite Composite Scaffolds Prepared via Melt Extrusion Additive Manufacturing on the Osteogenic Differentiation of Human Mesenchymal Stromal Cells

“…Previously, it has been shown that cell distribution and density upon seeding can significantly influence hMSCs osteogenic differentiation in 3D scaffolds. [42, 58, 59] Accordingly, poor cell attachment and lack of confluency within the scaffold cross-section have been shown to result in poor differentiation and lack of matrix mineralization in vitro. Besides, some studies reported enhanced cell attachment on polymer-CaP composites, likely due to the hydrophilicity and protein adsorption capacity of the CaP [60, 61].…”

“…In the present study, in order to decouple the osteogenic potential of the PEOT/PBT-nHA composite scaffolds from the effect of attachment efficiency, a viscous solution was used as seeding media, thereby ensuring comparable cell attachment and homogeneity among all scaffolds regardless of the nHA content, as previously reported. [42] Due to scaffold surface saturation with cells upon seeding, no cell proliferation was observed in the first 7 days of culture, nor during the subsequent 28 days in BM on any scaffold type, as ECM production was limited to the scaffolds filaments surface. On the other hand, ECM was also produced within the pores when cultured in MM, which increased the growth surface area enabling cell migration and a slight, yet not statistically significantly different, increase of cell number.…”

“…Dextran is a temporary supplement and will be washed away during the first 24h. [42] A droplet of cell suspension (37 μl containing 200,000 cells) was placed on top of each scaffold before slowly filling the scaffolds’ pores. Seeded scaffolds were incubated for 4 hours at 37 °C and 5% CO 2 to allow cells to attach.…”

Effect of reduced graphene oxide (rGO) compaction degree and concentration on rGO-polymer composites printability and cell interactions

“…Subsequently, scaffolds were blotted on top of a sterile filter paper and placed in the wells of a non-treated wellplate. Trypsinized hMSCs were resuspended in a dextran solution (500 kDa, Farmacosmos) (10 wt% dextran in CM), to achieve uniform cell distribution, [33] and were seeded at a density of 2×10 5 cells with 37 μl of CM per scaffold. After 4h incubation for cell attachment, scaffolds were transferred to new wells containing 1.5 ml of basic media (BM) (CM supplemented with 200 μм L-Ascorbic acid 2-phosphate).…”

Influence of Stem-cell Size and Culture Media Flowing Modality on Cell’s Fate within a Microchannel; a Numerical Analysis

“…Based on Torres et al study result [35], four different densities and related viscosity were assigned to the culture media.…”