Progress and challenges in large-scale expansion of human pluripotent stem cells

The constant supply of high cell numbers generated by defined, robust, and economically viable culture processes is indispensable for the envisioned application of human pluripotent stem cells (hPSCs) and their progenies for drug discovery and regenerative medicine. To achieve required cell numbers and to reduce process-related risks such as cell transformation, relative short batch-like production processes at industry- and clinically-relevant scale(s) must be developed and optimized. Here, we will review recent progress in the large-scale expansion of hPSCs with particular focus on suspension culture, which represents a universal strategy for controlled mass cell production. Another focus of the paper relates to bioreactor-based approaches, including technical aspects of bioreactor technologies and operation modes. Lastly, we will discuss current challenges of hPSC process engineering for enabling the transition from early stage process development to fully optimized hPSC production scale operation, a mandatory step for hPSCs’ industrial and clinical translation

An electro-mechanical bioreactor providing physiological cardiac stimuli

In cardiac tissue engineering it has been widely demonstrated the fundamental role of physical stimuli in improving structural and functional properties of the engineered cardiac constructs. An electro-mechanical bioreactor has been designed and developed to provide physiological uniaxial stretching and electrical stimuli for inducing functional differentiation and promoting morphological and structural maturation of cultured cardiac constructs obtained from stem cell-seeded scaffolds. The bioreactor is composed of: a transparent and sterilizable culture chamber for housing four cell-seeded scaffolds and the culture medium (working volume = 70 ml); a mechanical stimulation system, with a dedicated grasping system, to provide cyclic stretching (strain up to 20%, cycling frequency up to 2 Hz); an electrical stimulation system to provide electrical monophasic square pulses (1-6 V/cm, 0.25-10 ms, 1-5 Hz); a recirculation system for the automated medium change; a control system for data acquisition and mechanical stimulation. Preliminary in-house tests confirmed the suitability and the performances of the bioreactor as regards fittingness of chamber isolation, grasping system, and physical stimulation systems. Cell culture tests are in progress for investigating the influence of stretching and electrical stimuli on development of engineered cardiac constructs. Due to its high versatility, this bioreactor is a multipurpose adaptable system for dynamic culture of cell-seeded scaffolds for tissue engineering research and application

An electro-mechanical bioreactor providing physiological cardiac stimuli

In cardiac tissue engineering it has been widely demonstrated the fundamental role of physical stimuli in improving structural and functional properties of the engineered cardiac constructs. An electro-mechanical bioreactor has been designed and developed to provide physiological uniaxial stretching and electrical stimuli for inducing functional differentiation and promoting morphological and structural maturation of cultured cardiac constructs obtained from stem cell-seeded scaffolds. The bioreactor is composed of: a transparent and sterilizable culture chamber for housing four cell-seeded scaffolds and the culture medium (working volume = 70 ml); a mechanical stimulation system, with a dedicated grasping system, to provide cyclic stretching (strain up to 20%, cycling frequency up to 2 Hz); an electrical stimulation system to provide electrical monophasic square pulses (1-6 V/cm, 0.25-10 ms, 1-5 Hz); a recirculation system for the automated medium change; a control system for data acquisition and mechanical stimulation. Preliminary in-house tests confirmed the suitability and the performances of the bioreactor as regards fittingness of chamber isolation, grasping system, and physical stimulation systems. Cell culture tests are in progress for investigating the influence of stretching and electrical stimuli on development of engineered cardiac constructs. Due to its high versatility, this bioreactor is a multipurpose adaptable system for dynamic culture of cell-seeded scaffolds for tissue engineering research and applications

Image-Based Three-Dimensional Analysis to Characterize the Texture of Porous Scaffolds

The aim of the present study is to characterize the microstructure of composite scaffolds for bone tissue regeneration containing different ratios of chitosan/gelatin blend and bioactive glasses. Starting from realistic 3D models of the scaffolds reconstructed from micro-CT images, the level of heterogeneity of scaffold architecture is evaluated performing a lacunarity analysis. The results demonstrate that the presence of the bioactive glass component affects not only macroscopic features such as porosity, but mainly scaffold microarchitecture giving rise to structural heterogeneity, which could have an impact on the local cell-scaffold interaction and scaffold performances. The adopted approach allows to investigate the scale-dependent pore distribution within the scaffold and the related structural heterogeneity features, providing a comprehensive characterization of the scaffold texture

A low-cost 3D-printed sample-holder for stirring-based decellularization of biological tissues

An innovative, low-cost, 3D-printed sample-holder is proposed for reproducible and effective stirring-based decellularization of biological tissues. The sample-holder was designed to be low-cost, easy to use with conventional laboratory equipment, and manufacturable through 3D printing. During stirring-based decellularization, the sample holder exposes the samples to convective flow, enhancing the reagent transport while protecting the samples from disruptive forces. Computational fluid dynamics analyses were carried out to elucidate the developing hydrodynamics. Explanatory tests, performed on human cardiac tissue samples, demonstrated the effectiveness of the presented device