Effects of cell seeding technique and cell density on BMP‐2 production in transduced human mesenchymal stem cells

Abstract

Small animal models have demonstrated the efficacy of ex vivo regional gene therapy using scaffolds loaded with BMP‐2‐expressing mesenchymal stem cells (MSCs). Prior to clinical translation, optimization of seeding techniques of the transduced cells will be important to minimize time and resource expenditure, while maximizing cell delivery and BMP‐2 production. No prior studies have investigated cell‐seeding techniques in the setting of transduced cells for gene therapy applications. Using BMP‐2‐expressing transduced adipose‐derived MSCs and a porous ceramic scaffold, this study compared previously described static and dynamic seeding techniques with respect to cell seeding efficiency, uniformity of cell distribution, and in vitro BMP‐2 production. Static and negative pressure seeding techniques demonstrated the highest seeding efficiency, while orbital shaking was associated with the greatest increases in BMP‐2 production per cell. Low density cell suspensions were associated with the highest seeding efficiency and uniformity of cell distribution, and the greatest increases in BMP‐2 production from 2 to 7 days after seeding. Our results highlight the potential for development of an optimized cell density and seeding technique that could greatly reduce the number of MSCs needed to produce therapeutic BMP‐2 levels in clinical situations. Further studies are needed to investigate in vivo effects of cell seeding techniques on bone healing.

In vivo effects of cell seeding technique in an ex vivo regional gene therapy model for bone regeneration

“…Cell seeding on to osteoconductive scaffolds remains a critical step for any cell‐based therapy for bone healing with numerous factors contributing to optimal cell dispersion including cell number and seeding technique. Previously, our in vitro study demonstrated that transduced ASCs had improved seeding efficiency and homogeneity of cell distribution with lower density of cells, and that dynamic seeding with the orbital shaker technique had a higher BMP‐2 production per cell despite lower cell seeding efficiency 21 . We hypothesized that fewer cells loaded with the orbital shaker technique could have similar total BMP‐2 production and therefore fewer cells could be used to heal a segmental bone defect when subsequently adapting this model for use in patients.…”

“…Human adipose tissue was harvested from the fat pad of healthy patients undergoing total knee arthroplasty at our institution. The stromal vascular fraction (SVF) was obtained according to previously published protocols 21,22 . Briefly, the fat pad was extensively washed with Dulbecco's PBS (DPBS, Lonza, Basel, CH) to remove red blood cells and debris.…”

“…In a recent in vitro study, our lab evaluated the effects of cell loading density and cell seeding methods on cell distribution and BMP‐2 production 21 . Cell density was noted to have an inverse relationship with seeding efficiency in both static and dynamic seeding models.…”

Advances in 3D printing techniques for cartilage regeneration of temporomandibular joint disc and mandibular condyle

“…Therefore, seed cells are required to have the ideal ability to proliferate and secrete extracellular matrix (ECM). Seeding techniques such as static seeding, negative pressure seeding, centrifugal seeding, spinner flask, orbital shaker, and perfused bioreactor can be used to seed cells onto the scaffolds to promote articular cartilage regeneration [ 42 - 44 ] . The commonly used seed cells in cartilage tissue engineering and the effects of cell density in the scaffolds will be discussed below.…”

Modelling, design, fabrication and characterization of engineered human myocardium made with melt electrowriting and cardiac cells derived from hiPSCs

“…Other studies focus on modifying the surface of the scaffolds, which can be done by chemical etching [515], plasma treatment [356], or immobilizing cell binding motifs such as the peptide sequence arginineglycine-aspartate [516]. More complex dynamic seeding methods such as negative pressure, orbital shaking or perfusion have also been described, but those are more relevant for constructs with remarkably inaccessible surfaces, such as constructs displaying intricate interconnected pores or the inside of tubular scaffolds [254,517]. Another way in which we could improve our approach would be by implementing a way to directly measure the contractile force of the cardiac minitissues, instead of having to rely on parameters extracted from video analysis.…”