Page 476 - IJB-9-6

International Journal of Bioprinting Bioprinting cell-laden protein-based hydrogel

are known for their biocompatibility and ability to support are deposited within each filament or droplet. Within
cell adhesion and proliferation, but their mechanical cell aggregate bioprinting, preformed cell aggregates are
strength is low and they degrade rapidly. Although fibrin embedded into bioinks and then printed (Figure 1).
[9]
hydrogels have a higher mechanical strength and a slower The goal of single-cell bioprinting is to distribute single
degradation rate than collagen hydrogels, they may not cells in a controlled manner in order to fabricate delicate
promote cell adhesion and proliferation as well as collagen tissues and study single cells genetically [47,48] . Particularly,
hydrogels [37-39] . The sequence and structure of proteins the arrangement of cells and the microenvironment play
determine the stiffness, swelling, and degradation rate of a crucial role in stem cell research. There are several
hydrogels. As a consequence, it can influence the behavior methods available for isolating and manipulating single
of cells and the regeneration of tissue within bioprinted cells as the first phase in the single-cell characterization ;
[49]
constructs [40-42] . Furthermore, specific functional groups to separate single cells, fluorescent-activated cell sorting
or peptides may be introduced into protein sequences (FACS) and limiting dilution are two widely
[50]
[51]
to enhance cell adhesion, differentiation, or tissue [52]
regeneration . For example, arginine-glycine-aspartic used approaches . Nevertheless, the inefficiency of
[43]
acid (RGD) peptides are known to enhance cell adhesion limiting dilutions, together with the need for specialized
and can be incorporated into the protein sequence of instruments, as well as requiring professional expertise to
[49,52]
hydrogels in order to enhance biocompatibility and cell employ FACS restrict their use . Recent innovations
behavior . Thus, it is necessary to carefully consider the in the field of single-cell printing have contributed
[44]
protein sequence and structure of the hydrogel in order to to circumventing these limitations, one of which is
optimize the mechanical and biological properties of the utilizing a variety of microfluidic approaches for the
final tissue constructs. encapsulation and examination of single cells, comprising
droplet microfluidics [53-55] , microwell arrays [56,57] , and
This study aims to provide a comprehensive review of hydrodynamic traps [58,59] . Single-cell bioprinting offers
innovative bioprinting technologies, which are classified multiple benefits; firstly, it enables precise and effective cell
based on the cell format and the number of cells generated distribution at high throughput , and second, each cell
[60]
during the bioprinting procedure. The study focuses on or colony can be recovered readily with addressability for
PBHs utilized in cartilage and bone TE. Additionally, the further analysis. Another advantage of single-cell printing
critical role of microenvironmental factors, including is that it can easily be integrated with other methods, such
biophysical and biochemical parameters, in bioprinting as imaging systems [61,62] , electric fields [63-65] , and acoustic
cell-laden PBHs is examined in detail, with reference fields [66,67] , with the encapsulation efficiency exceeding
to relevant research. The subsequent section provides 90%. Based on Poisson’s distribution, the broadly employed
a thorough explanation of the process compatibility droplet-based microfluidics’ theoretical limit is only 37%.
considerations for PBHs, including mechano-rheological Furthermore, single-cell printing can create both high-
properties, biocompatibility, and process factors, through resolution two-dimensional (2D) structures as well as 3D
an overview of recent experiments on PBHs bioprinting. tissue matrices for TE, drug delivery, and toxicology [68,69] .
Finally, the challenges and perspectives associated with Barron et al. first proposed the concept of single-cell
[70]
PBHs that must be carefully addressed to advance this bioprinting in 2005, when they described the bioprinting
fascinating field are highlighted. of human osteosarcoma cells, shortly after laser-assisted
bioprinting was invented. To control the number of cells
2. Bioprinting strategies in a droplet, they developed bioinks with varying cell
There have been numerous developments within the concentrations. Although the droplets were homogeneous
bioprinting field to meet the needs of different research and of small size, identifying only one cell per droplet
fields in terms of manufacturing capabilities, such as was difficult, and the number of cells within the droplet
printing resolution, speed, or throughput , as well as generally followed the Poisson’s distribution regardless
[45]
cell requirements, including cell viability, proliferation, or of their concentration. Since then, single-cell bioprinting
differentiation . It is possible to categorize the bioprinting was not invented until 2012, when a camera with high
[46]
technology into two distinct groups, which are not speed was employed in order to monitor cells in the nozzle
mutually exclusive. In essence, they can be classified based tip . In this regard, droplets containing more than one
[71]
on the format of the cells (distributed cells or aggregated cell or not incorporating any cells were discarded. It was
cells) and the number of cells produced during printing in 2014 that Zhang et al. developed a method to block
[72]
(single-cell or multi-cell). Single-cell bioprinting involves cell-print breast cancer cells, utilizing traps with hook-
printing one cell at a time, while in multi-cell bioprinting, shaped ends arranged in a prescribed pattern to capture
the cells are suspended in a bioink, and a number of cells and print singular cells. Currently, a variety of techniques

Volume 9 Issue 6 (2023) 468 https://doi.org/10.36922/ijb.1089