Introduction:
Mesenchymal stromal/stem cells (MSCs) possess unique biological properties, including self-renewal, differentiation, and secretory potentials. However, a standard 2D culture does not replicate MSCs' natural microenvironment, compromising their features. Engineering MSC-based constructs that support various 3D cell organizations and analyzing cell behavior under such conditions are crucial for biomedical applications, offering relevant model systems and aiding in the development of therapeutic agents. This study aimed to evaluate the impact of cultivating MSCs in spheroids, alginate microspheres (AMSs), and blood plasma scaffolds on viability and metabolic and functional activity.

Methods:
Human adipose tissue-derived MSCs (obtained with adult donors’ informed consent) were used. Spheroids were formed by the “hanging drop” method. AMSs were generated by electrospraying MSCs dispersed in 2% sodium alginate into 2% calcium chloride. Scaffolds were prepared through cryogelation and being seeded with cells. All constructs were cultured at 37 °C, 5% CO2, and 95% humidity in alpha-MEM supplemented with 10% fetal bovine serum, 50 μg/ml penicillin, and 50 μg/ml streptomycin. Viability (6-CFDA), metabolic activity (resazurin test), actin filaments (Phalloidin-FITC), cell spreading, and induced differentiation were examined.

Results and Discussion:
MSCs exhibited high viability in all constructs but displayed distinct morphologies (spindle-like in scaffolds, round in spheroids, and AMSs). Actin filament development was most pronounced in cells within scaffolds. Metabolic activity was reduced in spheroids and AMSs compared to the scaffolds. All groups demonstrated the ability for induced differentiation.

Conclusions:
The cultivation of MSCs within a macroporous adhesive scaffold promotes fibroblast-like morphology and high metabolic activity. A spheroid and AMS culture results in round-shaped cells with lower metabolic activity, which can reflect a natural-like quiescence state. This study highlights the importance of 3D culture systems in maintaining MSC properties and suggests that constructs' design significantly influences cell functionality, crucial for advancing biomedical applications and therapeutic strategies.

This study was supported by the National Research Foundation of Ukraine (project № 2021.01/0276).