static) also have an important influence around the 3D cultivation of primary hepatocytes (Fig.?8). CPN caused no significant changes in the morphology, size, and chemical structure of PNA microcapsules in cell culture media. Among four PNA microcapsule products (PNA-0, PNA-10, PNA-30, and PNA-50 with size 489??31?m, DBPR112 480??40?m, 473??51?m and 464??35?m, respectively), PNA-10 showed overall suitability for HepG2 growth with high cellular metabolic activity, indicating that the 3D PNA-10 microcapsule could be suitable to maintain better vitality and liver-specific metabolic functions. Overall, this novel design of PNA microcapsule and the one-step method DBPR112 of cell encapsulation can be a versatile 3D NIM system for spontaneous generation of organoids with like tissue architectures, and the system can be useful for numerous biomedical applications, especially for liver tissue engineering, cell preservation, and drug toxicity study. DBPR112 microenvironments2. Prolonged cell culture in 2D systems modifies the tissue-specific architecture (e.g. forced polarity, flattened cell shape, etc.), mechanical/biochemical signals and cell-to-cell communication, and eventually the response from 2D test system deviates from response3. To overcome these limitations and to better mimic conditions, different synthetic 3D cell culture platforms have been created using various methods: hanging\drop4, forced\floating5, matrices scaffolds6, and agitation-based approaches7. In native stage of living body, almost all the cells in tissues reside in a complex fibrous meshwork known as extracellular matrix (ECM). The remodeling of ECM is usually a key structural and biochemical support that accounts the cellular properties. Several recent studies have exhibited that changing the architecture DBPR112 of synthetic ECM around cells could enhance retention of tissue-specific functions. A synthetic, designed ECM in 3D systems can significantly impact cell proliferation, differentiation, and cell survival to reproduce tissue-drive component in platforms for drug discovery and toxicity screening12,13. This technique refers to immobilization of cells within a semipermeable hydrogel that allows bi-directional diffusion of nutrients, oxygen, wastes, and secretion of biomolecules. In cell therapy, the semi-permeable hydrogel avoids the foreign invaders, such as immune cells and antibodies which can destroy encapsulated cells14,15. In addition, the hydrogel microenvironment has other advantages particularly the ease of handling of cells in a highly hydrated environment that mimic the natural ECM in tissues2,14C16. Different extrusion methods have been used for cell encapsulation including electrostatic17, coaxial airflow18, vibrational nozzle19 and jet cutting20. Two main categories of hydrogels used extensively in cell encapsulation are: synthetic polymer-based hydrogels, such as poly(ethylene glycol) (PEG), 2-hydroxyethyl methacrylate (HEMA), poly(vinyl alcohol) (PVA), RAD26 polyvinylpyrrolidone (PVP), and PLGA-co-PEG21C24, and natural polymer-based hydrogel such as alginate, chitosan, collagen, gelatin, hyaluronic acid25C27. Although synthetic hydrogels have greater control over gelation time, macroscopic structure, and degradation kinetics, natural polymer-based hydrogels retain biological cues to guide cell and tissue growth16,28. Currently, a variety of hybrid hydrogels are developed to overcome the inherent limitation of both natural and synthetic hydrogels29,30. Designing of hybrid hydrogels by incorporating micro- and nanoscale features of both natural and synthetic polymers are emerging tools in tissue engineering to create biomimetic environments within the 3D system that enhances several cellular functions with high temporal and spatial resolution31C33. Alginate has been used extensively for 3D cell encapsulation because of its confirmed biocompatibility, relatively easy to prepare at physiological conditions in the presence of divalent cations, and easy to sterilize and storage34,35. However, alginate has poor biological properties in terms of cell adhesion, migration, and viability36,37. In addition, alginate hydrogel does not degrade functions69,70. Efforts have been given into increasing the mechanical strength of AHM either by adding multilayers of oppositely charged polymer coating71,72 or by covalently crosslinking with chemical brokers such as glutaraldehyde73,74. Both coating or covalent crosslinking techniques require either multi-step process or introduce toxic crosslinking agents into the microcapsules (e.g. glutaraldehyde) which can complicate the encapsulation process. Therefore, the present method of PNA microcapsules preparation provides.