1. Introduction
Over the past ten years, 3D bioprinting has emerged as a groundbreaking technology in tissue engineering, making remarkable progress in the reconstruction of transplantable tissues and also intricate organs, such as the human ear, bones, skin, and nose. This approach, which integrates additive manufacturing (AM), biology, and material science, entails the deposition of bioinks (viscous substances infused with living cells and supplementary matrix elements) via 3D printers to accomplish layer-by-layer assembly for fabricating three-dimensional structures through computer-aided design (CAD).
Several bioprocessing strategies, including selective laser sintering (SLS), inkjet printing, and extrusion/deposition-based methodologies, have been devised to facilitate the 3D printing of viable cells and biological scaffolds [
1]. While significant advancements have been achieved in producing clinically sized hard tissues, such as bones, the fabrication of scaffolds for soft tissues is still constrained to small-scale medical applications with limited structural intricacy [
2].
Recent advancements in 3D bioprinting research have delivered encouraging results, particularly in generating skin substitutes for individuals suffering extensive skin injuries caused by burns, chronic ulcers, cancer, or surgical procedures. While conventional tissue-engineering approaches encounter difficulties in replicating biomimetic and heterogeneous tissue structures, emerging 3D bioprinting technologies present a promising alternative.
Bioinks, composed of diverse blends of biomaterials, biomolecules, and cells, influence printability, biocompatibility, and mechanical stability. After bioprinting, constructs can undergo crosslinking to reinforce their form and structure, ensuring consistency, reproducibility, and precise modulation. These multi-component bioinks have been designed to facilitate the fabrication of biomimetic and intricate tissue structures [
3].
Recently, bioinks have been characterized as material formulations compatible with automated biofabrication techniques, which must incorporate living cells [
4]. Bioink is a blend of cells, biopolymers, and biologically active compounds that influence cell viability during printing, support cell multiplication and expansion, and promote tissue formation [
5]. Bioinks designed for printing living cells are typically scaffold-based, where cells are encapsulated in hydrogels or comparable materials, offering adhesion sites to enable cell spreading.
The selection of an appropriate bioink is essential for achieving optimal bioprinting conditions [
6]. Hydrogels are essential to maintaining the structural integrity and three-dimensional stability of printed tissues. A fundamental characteristic of hydrogels as bioinks is their ability to transition between liquid and solid states under specific conditions, enabling the printing process, as these transition parameters directly influence 3D bioprinting results.
Recent studies have concentrated on developing materials and bioink formulations with adequate rheological properties to preserve both dimensional stability and biocompatibility, fostering cell proliferation. Cells are essential components of bioinks, while carrier biomaterials are equally vital, as they assist in establishing a stable spatial arrangement, sustaining cellular viability during printing, and supporting post-printing proliferation and differentiation [
7].
Despite the promising outputs, the industrial application of 3D bioprinting to produce customized, functional living constructs faces ethical barriers [
1] and technical challenges, such as achieving high-resolution cell deposition and controlled cell distribution [
3], that need exploration and identification before therapeutic 3D bioprinting can be widely adopted in human patients.
Bioinks pose another critical challenge due to their high cost and limited accessibility. In addition, the intricate nature of bioinks further contributes to the complexity and cost of the bioprinting process. Although many bioinks are commercially available, it may be necessary to obtain a personalized composition. Currently, to obtain the materials, it is necessary to purchase them from the same companies that supply the bioprinters. This fact greatly limits accessibility, as the purchase of materials is tied to specific suppliers, often associated with the biological printers owned by the same companies.
The small quantity and high prices of these materials are an additional obstacle, especially in the research field, where budgets can be tight. The purchase of high-cost bioinks can become difficult to sustain, limiting the scope of scientific experiments and investigations in the field of 3D bioprinting.
Addressing these issues is crucial to the implementation of 3D bioprinting in both research and therapeutic applications. Efforts to simplify and/or customize the manufacturing process, reduce costs, and establish more accessible bioink resources are critical to unlocking the potential of this technology. Overcoming these challenges will not only improve the viability of 3D bioprinting but also increase its impact in various fields, from research to regenerative medicine, and more.
In this study, we introduce lab-formulated alginate hydrogel structures. Alginate hydrogels are selected due to their extensive research and application in the medical field [
8]. This choice is attributed to their known biocompatibility, controllable stiffness, and ability to form highly porous structures conducive to cell regeneration [
2]. These properties make alginate suitable to produce biodegradable hydrogels utilized in the manufacturing of skin scaffolds.
Furthermore, the crosslinker steps performed by immersing the printed hydrogel in 80 mM calcium chloride significantly extended the stability of the printed polymeric system without compromising cell viability. The material has been chosen for its cost-effectiveness, widespread availability, and ease of processing.
To carry out this first study, we used cancer cells as they grow easily and are prone to form three-dimensional aggregates. Specifically, we used the leiomyosarcoma cell line (SK-LMS-1), which represents a malignant tumor of soft tissues and belongs to the sarcoma family. We chose these cells because we have already used them in previous biological studies and have already developed the method with the printer we currently have in our laboratory [
9].
The rheological characteristics of hydrogels are analyzed before the printing process, and additionally, cell viability after printing is monitored over a defined timeframe. This research aims to design three-dimensional printable hydrogels derived from biocompatible natural polymers and assess their mechanical behavior and ability to support cell growth for potential applications in scientific studies.
4. Discussion
The rheological data obtained clearly underscore the suitability of the proposed bioink for bioprinting applications The detailed evaluation of its handling, elasticity, and viscosity characteristics demonstrated that the alginate-based bioink exhibits both liquid and solid behaviors. While its liquid form presents challenges during the bioprinting process, making it unsuitable for immediate printing, the use of a crosslinking agent in the post-printing stage effectively mitigates this issue, allowing the material to solidify and acquire the desired structural integrity. These findings suggest that a 10% w/v alginate-based gel is a promising candidate for bioprinting, offering a balanced combination of viscosity and elasticity.
In particular, regarding the rheological tests, all the formulated hydrogels show non-Newtonian behavior; in fact, their apparent viscosity (η) is dependent on shear rate (
). By increasing the shear rates, a clear shear-thinning behavior can be detected, with the viscosity value decreasing at increasing shear rates (
Figure 1d). The shear-thinning ability is an important requirement for bioinks to be printable [
14] as the reduction in η values improves the bioink process. The hydrogels can be easily extruded as soon as the shear stress exceeds the yield stress, σ
y (
Table 2). The yield stress parameter reveals the resistance of the fluid to flow during the extrusion process and the capability to support subsequent 3D-printed layers without squeezing [
15]. The use of CaCl
2 improves alginate-based hydrogel mechanical properties, due to a crosslinking reaction between the carboxyl groups of sodium alginate and Ca
2+ ions. Alginate-10% exhibits solid-like behavior for a wider range of angular frequencies than Alginate-8% ((10 < ω < 100) rad/s and (13 < ω < 100) rad/s, respectively) as can be seen in
Figure 2b,c. This evidence suggests selecting Alginate-10% as a more promising candidate bioink to realize scaffolds through the 3D bioprinting process.
Regarding the morphological investigations of the scaffolds, phase-contrast inverted microscopy was employed to monitor and quantify spheroid growth on the 10% alginate scaffolds. On the other hand, observation of the Cellink alginate scaffold was not possible due to its dense structure, which interfered with both visual inspection and image acquisition. The panels shown in
Figure 6a illustrate spheroid growth from the day following printing up to day 6, with spheroid areas quantified for analysis. As shown in
Figure 6b, statistical analysis revealed a significant increase in spheroid area on days 5 and 6 compared to day 1 for the 10%
w/
v alginate. This indicates a positive cellular response and supports the hydrogel’s biocompatibility.
Following DAPI staining, the cells’ nuclei were clearly highlighted due to their fluorescence, confirming the cellular viability of the 3D aggregates. H&E staining revealed that each spheroid was composed of multiple cells in both matrix types (
Figure 7c,d), with the nuclei clearly highlighted in purple in the top left corner of the images.
With reference to the FE-SEM observations, the abundant mitochondria observed are consistent with the known ultrastructural features of leiomyosarcoma spheroids, which are typically rich in mitochondria. Leiomyosarcoma, a highly malignant tumor with metastatic potential, is characterized by its smooth muscle tissue origin. The literature reports that smooth muscle tumors, including leiomyosarcoma, frequently show a high density of mitochondria, a feature that has been linked to their energetic demands and aggressive nature. This mitochondrial enrichment is sometimes utilized as a histopathological marker for identifying smooth muscle differentiation in tumor cells [
16]. Moreover, the observed disruption of the spheroid surface likely reflects the high metabolic activity and potentially altered cell morphology associated with the malignant nature of the tumor, providing further insight into the tissue architecture at the ultrastructural level. The FE-SEM images of the Cellink bioink show filamentous structures that may resemble collagen fibers. As is well-known, collagen constitutes the extracellular matrix and plays a crucial role in tumor formation and progression [
5].
As for the FE-SEM images of the 10% w/v alginate scaffold, they reveal the presence of subcellular structures that may correspond, on the basis of their small size, to mitochondria, as identified in the commercial matrix. Additionally, the surface of the spheroid shows fibril-like structures, potentially collagen fibers. The collagen fibers are especially noticeable in the regions surrounding the spheroid, suggesting a possible interaction between the cells and the extracellular matrix.
Furthermore, the comparison between the two scaffolds highlights significant differences in their mesh structures. The 10% w/v alginate scaffold presents a more tightly organized mesh, while the commercial matrix displays a relatively looser, more interconnected network. These differences may influence the mechanical properties and the behavior of the encapsulated cells, potentially affecting their proliferation and interaction with the scaffold.
In comparison to previous alginate-based bioprinting studies, especially those by Tabriz et al. [
2] and Siviello et al. [
8], the present work introduces several distinctive aspects. While Tabriz et al. focused on fabricating complex 3D alginate structures using a multi-step crosslinking strategy optimized for tissue engineering and vascular constructs, our study emphasizes the development of a simplified, lab-formulated alginate bioink tailored for efficient tumor spheroid formation in vitro. Furthermore, Siviello et al. investigated the viscoelastic aging behavior of alginate gels, whereas our work integrates rheological optimization with practical bioprinting performance and biological validation in the context of 3D cancer cell culture. Primarily, our approach leverages a cost-effective and customizable formulation, avoiding the constraints of commercial bioinks and enabling on-demand preparation for specific research needs. In addition, by using a single post-printing crosslinking step and a commercially available pneumatic extrusion bioprinter, we propose a reproducible and accessible workflow suitable for standard laboratory settings. Finally, we demonstrate that the proposed bioink effectively supports sustained spheroid growth and biological activity, which is essential for modeling tumor cell behavior and was not a primary focus of previous studies.
5. Conclusions
5.1. Cost-Effectiveness and Ethical Benefit
The use of low-cost, in-house-formulated bioinks presents substantial advantages for modeling tumor cell growth in vitro. These bioinks facilitate the creation of models that more accurately replicate the cellular microenvironment, eliminating the need for expensive materials typically used in tissue engineering applications. This approach is particularly advantageous for tumor cell studies, where the focus lies on understanding fundamental cellular behaviors rather than on regenerating tissues. By enabling the production of relevant experimental models at a reduced cost, this strategy enhances research efficiency without compromising scientific rigor.
In addition, using bioinks free from animal-derived components offers important ethical benefits. By circumventing the need for animal tissues, this approach aligns with the growing commitment to reduce animal testing in research, providing a more ethical and sustainable alternative for in vitro studies.
5.2. Biological Validation of the Lab-Prepared Bioink
In this study, our lab-prepared alginate hydrogel demonstrated favorable rheological and morphological properties and effectively supported cell viability and proliferation over time, as confirmed by phase-contrast microscopy and DAPI staining. After 72 h, microscopy revealed consistent and increasing cell presence, validating the bioink’s capacity to sustain viable and metabolically active cells within the 3D matrix. These observations confirm the biological compatibility of the formulation and its potential to support the development of functional tissue models.
5.3. Flexibility and Reproducibility
Economically, the lab-prepared bioink offers substantial advantages over commercial alternatives. Producing the bioink from accessible raw materials provides greater control over formulation, significantly reducing costs while enabling on-demand customization. Additionally, this flexibility allows researchers to adapt the bioink composition to specific experimental needs. However, it should be acknowledged that achieving batch-to-batch reproducibility may require further optimization and standardization.
In terms of economic comparison, the raw material cost for preparing the lab-formulated alginate bioink is approximately €0.05–0.10 per milliliter, including sodium alginate and crosslinker. The commercial Cellink Bioink is approximately 400 to 800 times more expensive per milliliter than the lab-prepared sodium alginate-based bioink, further underscoring the significant economic advantage of the proposed lab-formulated bioink for research applications. This represents a cost reduction of over 99%, highlighting the substantial economic advantage of the in-house formulation. Such a difference is particularly relevant for research contexts that require large volumes of bioink or iterative experimental work, where material costs can become a limiting factor.
5.4. Comparison with Commercial Bioinks
It is also important to note that the commercial bioink used for comparison is proprietary, and its exact composition is not disclosed. Unknown additives, viscosity modifiers, or extracellular matrix components in the commercial product may significantly influence its mechanical behavior and cellular response. This lack of transparency limits the possibility of making a fully direct and quantitative comparison between the two materials. In contrast, our fully characterized, lab-prepared alginate hydrogel offers a customizable, low-cost, and reproducible platform that can be tailored to specific research needs and biomedical applications.
5.5. Study Limitations and Future Directions
Some limitations of this study must be acknowledged. The biological validation was performed using a single cancer cell line (SK-LMS-1), which restricts the generalizability of the findings. Future studies should aim to test the bioink with additional cell types, including non-cancerous and primary cells, to further demonstrate its versatility and relevance across different biological contexts.
5.6. Conclusion
In summary, although further validation is required, the developed bioink demonstrates strong potential as a cost-effective, ethically conscious, and customizable material for 3D bioprinting applications in cancer research. It represents a viable and sustainable alternative to commercial bioinks, particularly in research settings focused on studying cellular behavior rather than tissue engineering or regeneration.