Cryopreservation technology plays an important role in conserving three dimensional (3D) constructs containing cells. Besides preserving the characteristics of the construct, it can also save a lot of resources in many aspects, such as cell culture space, culture vessel, culture medium etc. Otherwise, the biological properties of the cells and the 3D geometrical configuration will disappear with the death of cells and breakage of configuration. Consequently, a cryopreservation method for the 3D construct fabricated with a controlled cell assembling technology was studied. 10% dimethylsulfoxide (DMSO) was incorporated into the adipose-derived stem cell (ADSC)/gelatin/ alginate/fibrinogen mixture before assembling. Results indicate that the 3D construct containing cells can be preserved below —80°C for more than 1 week. After the construct underwent the thawing process, cell viability and proliferation ability were regained. This technique holds potential to be used widely in tissue engineering and organ manufacturing fields.

To overcome the weak mechanical properties of cell/hydrogel composites, a poly(DL-lactic-co-glycolic acid) sandwiched adipose-derived stem cell (ADSC)/fibrin construct was fabricated using a step-by-step mold/extraction method to generate the middle smooth muscle layer of natural blood vessels. A pulse bioreactor with an adjustable 0-0.2 MPa pressure, 0-7% pulse amplitude, and 0-80 times/min pulse frequency was developed to mimic the liquid movement in the natural blood vessels. This new type of pulse bioreactor is sterilizable and dismantles easily. A comparative study was conducted with static and dynamic in vitro cultures. Exogenous growth factors, such as hepatocyte growth factor, platelet-derived growth factor BB, transforming growth factor β1, and basic fibroblast growth factor were used as additives in the culture medium for inducing the ADSCs into smooth muscle cells. The dynamic training, integrated with the growth factor, induced the transformation of ADSCs into smooth muscle-like cells with regular arrangement. This strategy shows promise of being widely used in tissue engineering and complex organ manufacturing.

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A poly(DL-lactic-co-glycolic acid) (PLGA) sandwich fibrinogen/ adipose stem cell (ADSC) construct was fabricated to generate smooth muscle tissue. The mechanical properties and ADSC compatibility of PLGA, poly(ethylene glycol-1,6-hexamethyl diisocyanate-caprolactone) i.e. polyurethane (PU), gelatin, alginate, and fibrin composites were evaluated for vascular smooth muscle tissue generation. Synthetic PLGA and PU combined with natural gelatin, alginate, and fibrin for strength and cell compatibility were found to be effective. A trilayer construct was designed and built with a microporous inner PLGA layer to provide nutrient, oxygen, and metabolite transfer while the outer PLGA layer with no pores prevented leakage during in vitro culture and in vivo implantation. The fibrin layer suitably accommodated ADSC growth, migration, proliferation, and differentiation inside the construct. This design has the potential for wide use in tissue engineering and complex organ construction.

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