*3.4. Cell Aggregates*

Bioprinting of cell aggregates has been used as a sca ffold-free methodology of creating tissue engineered constructs. These cell aggregates consist of spheroid structures, which can then be specifically positioned, creating for instance tubular or ring-like structures [52,53]. Although the constructs are primarily sca ffold-free, the cells are usually encapsulated with a hydrogel material that is biocompatible and biodegradable, for cell survival and for mechanical support of the cell construct. The hydrogel also helps to prevent tissue fusion while the cells are maintained in the suspension reservoir of the 3D printer [54]. The use of cross-linking solutions, such as those containing CaCl2 or gelatin, can help to further minimize cell aggregation [55,56]. As the pH of the bioink is important for cellular survival and sca ffold integrity, a study by Lozano and colleagues used the addition of NaOH to stabilize the pH of a modified bio-polymer hydrogel [57].

The advantages of using sca ffold-free constructs for tissue engineering include the absence of potentially toxic or immunogenic sca ffold materials, as well as the ability to create high cell density constructs [37,58]. Limitations of the cell aggregate approach include the relatively time-consuming cellular fusion of the spheroids to create larger tissue structures (which may also create non-uniform structures) [54]. Certain advances have been made to minimize this limitation such as the development of multicellular cylinders as an alternative structure, which require up to four days to create the appropriate shape [53]. While most 3D printing of cell aggregate studies have been performed in vitro, there is a limited understanding of its potential in vivo and further studies must be performed to demonstrate its safety and feasibility as a sca ffold-free construct for tissue engineering. A summary of the 3D printing materials and their potential applications can be found in Table 2.


**Table 2.** Summary of 3D printing materials for tissue engineering.

### **4. Pre-Clinical and Clinical Applications**
