Strategies for 3D bioprinting of spheroids: A comprehensive review

Biofabricated tissues have found numerous applications in tissue engineering and regenerative medicine in addition to the promotion of disease modeling and drug development and screening. Although three-dimensional (3D) printing strategies for designing and developing customized tissue constructs have made significant progress, the complexity of innate multicellular tissues hinders the accurate evaluation of physiological responses in vitro. Cellular aggregates, such as spheroids, are 3D structures where multiple types of cells are co-cultured and organized with endogenously secreted extracellular matrix and are designed to recapitulate the key features of native tissues more realistically. 3D Bioprinting has emerged as a crucial tool for positioning of these spheroids to assemble and organize them into physiologically- and histologically-relevant tissues, mimicking their native counterparts. This has triggered the convergence of spheroid fabrication and bioprinting, leading to the investigation of novel engineering methods for successful assembly of spheroids while simultaneously enhancing tissue repair. This review provides an overview of the current state-of-the-art in spheroid bioprinting methods and elucidates the involved technologies, intensively discusses the recent tissue fabrication applications, outlines the crucial properties that influence the bioprinting of these spheroids and bioprinted tissue characteristics, and finally details the current challenges and future perspectives of spheroid bioprinting efforts in the growing field of biofabrication.

Introduction

Biofabrication encompasses designing and fabricating tissue and organ constructs to repair and regenerate defects caused by damage or loss of tissues by infection, trauma, excision, or diseases [1]. In recent years, biofabricated tissue and organ constructs have found prevalent usage in regenerative medicine, particularly in the repair and restoration of damaged tissues, and drug screening and discovery. Ideally, biofabricated constructs should be a custom-fit for each patient – additively manufactured and avoid any concerns related to biocompatibility and innate immune responses, which are crucial for in-vivo success [2]. In such regard, 3D printing technology has evolved from rapid prototyping techniques [3] and was initially used for developing 3D surgical models in the medical industry [4]. Since then, 3D printing has quickly ascended the ladder of the medical industry and found profound use in fabrication of customized patient- and defect-specific prosthesis, like bone implants [5], hearing aids [6], and many more. Until early 2000s, 3D printed constructs did not include any living component and lacked the functionality of human tissues [7], and were mainly used as templates to guide tissue formation [8].

The development of functional human tissues/organs was envisioned in the beginning through early embryonic development [9]. This was changed with the introduction of the concept of 3D printing with cells, referred to as “bioprinting” in 2003, where exposition of cells was demonstrated using an inkjet printer by Wilson and Boland [10]. Bioprinting can be defined as a computer-aided transfer process for simultaneous writing of living cells and biomaterials for various applications such as tissue engineering, regenerative medicine or other biological studies [11]. To a layman, 3D bioprinting may seem like a conglomeration of futuristic science fiction images; but in reality, it is built on several advancements across several fields of technologies and life sciences. Bioprinted tissue constructs imitate the complexity of natural tissues by providing an artificial microenvironment conducive to cell growth.

To date, two opposing strategies have been investigated to bioprint tissues for different applications: scaffold-based (biomaterials-based) and scaffold-free (cellular-based) methods. Conventionally, exogenous biomaterial matrices, like hydrogels [12], are premixed with appropriate monoculture or coculture of cells and 3D bioprinted to fabricate scaffold-based tissue constructs. These constructs are often supplemented with several biological growth factors [13] to create a 3D environment favorable for tissue growth. Tailored fabrication procedures, with precise control over engineering parameters, offer a wide range of possibilities over conventional tissue scaffold fabrication techniques with desired architecture, geometry, and increased reproducibility. Moreover, control over the composition of bioprinted materials (bioinks) helps achieve optimal structural properties, such as mechanical properties, porosity, and degradation profile alongside geometrical feasibility, leading to the reconstruction of target tissues in a consistent, automized and high-throughput manner [14]. Scaffold-based bioprinting techniques have been widely used due to their ease in tissue fabrication; however, further research needs to be conducted to fabricate suitable robust biomaterials and studying their interactions at the cellular level for functional tissue formation. Scaffold-free bioprinting approach [15], on the other hand, focuses on tissue fabrication using cell aggregates, without the need for scaffold support, triggering cells to secrete their own extracellular matrix (ECM). Cell aggregates can be formed into geometrical configurations, for example, strands, honeycomb, or spheroids, and then allowed to fuse into larger tissues [[16], [17], [18]]. Both approaches have pros and cons, and in cases of 3D bioprinting, they may complement each other in the pursuit of meeting the ever-increasing demand for fabrication of scalable physically-relevant tissues or organs.

In a discussion on bioprinting, it is pertinent to expound the advancements in bioprinting of induced-pluripotent stem cells (iPSCs) to develop tissue constructs, which mimic the function and anatomy of native tissues. Given the extreme sensitivity of iPSCs to stressors during bioprinting– particularly mechanical shear and bioink conditions (pH, viscosity, crosslinking method, etc.), the development of new bioinks for sustaining iPSC viability and differentiation is of utmost importance. Some success has been achieved by bioprinting of iPSC aggregates – also called embryonic bodies embedded in hydrogels [[19], [20], [21]]. The iPSC-laden bioinks undergo mechanical stress, light, ionic and temperature related stress during bioprinting and maintenance of the proliferation and pluripotency of these embryonic bodies after bioprinting is critical. iPSCs or iPSC-derived cells have also been bioprinted to fabricate different tissues – skin [22], bone [23], cartilage [24], cardiac [25] and liver tissues [26]. Although several challenges need to be addressed for iPSC-derived tissue bioprinting – like tendency of the constructs to form tumors after transplantation and low efficiency of their generation and differentiation, yet iPSC-derived transplantation have provided a paradigm shift to how disease treatment have been approached in the current times.

Achieving native-like cell density, complex vascular network and controlling tissue remodeling are some of the major impediments for successful fabrication of tissues – both scaffold-based and scaffold-free [27]. Even if it seems feasible to overcome these elementary concerns, bioprinting strategies are overwhelmed with the conservation of actual size and anatomy of tissues in tandem with their functionalities and shelf-life as well as the cost-effectiveness of the process [28]. Additionally, economic constraints and ethical considerations with engineered tissues make this even more challenging [29]. Towards a more realistic and technologically optimistic approach, vascularized cell aggregates offer a viable alternative for the repair and restoration of damaged tissues. Developmental biology forms the foundation and provides the template on which these engineered cell aggregates are based [30], and consequently assemble to form 3D tissues [31]. Successful integration of tissue engineering and developmental biology is still a work-in-progress; however, this biomimetic approach allows for insightful advancements. The understanding, mimicking and employment of the developmental mechanisms of embryonic histogenesis and organogenesis in tissue engineering serves as a paradigm shift towards scaffold-free applications of bioprinting. Such applications include the fabrication of anatomically-relevant constructs using cellular aggregates (like spheroids, honeycombs or strands) to imitate native tissues. Continuous co-developments in the fields of spheroid fabrication and 3D bioprinting technologies have led to the emergence of novel engineering methods enabling successful bioprinting and fusion of spheroids for tissue repair and regeneration. With the recent developments in several state-of-art strategies, there is a lacuna in available literature wherein no comprehensive review is available focusing on this domain. Towards bridging the gap, this paper reviews the bioprinting strategies involved to form self-assembled tissues from spheroids and highlights the recent progress in tissue fabrication applications using spheroids as building blocks for bioprinting as highlighted in Fig. 1.

Dishary Banerjee, Yogendra Pratap Singh, Pallab Datta, Veli Ozbolat, Aaron O’Donnell, Miji Yeo, Ibrahim T. Ozbolat, Strategies for 3D bioprinting of spheroids: A comprehensive review, Biomaterials, Volume 291, 2022, 121881, ISSN 0142-9612, https://doi.org/10.1016/j.biomaterials.2022.121881.