Making and Testing Alginate-Based Materials for 3D Printing Living Cell Structures

Fresh hydrogel mix must be prepared 1 day prior to bioprinting or rheological analyses to ensure appropriate cell viability, printability, and consistency in material properties. It is recommended to prepare the hydrogel mix at ∼70% higher concentration to account for subsequent dilution with the cell suspension in culture medium. For example, if the final desired concentration is 3% (w/v) alginate and 1% (w/v) gelatin, prepare the initial hydrogel at 4.28% (w/v) alginate and 1.43% (w/v) gelatin. Then, mix the hydrogel with the cell suspension (prepared in culture medium) in a 70:30 ratio to achieve the target final concentrations.

Polyethyleneimine (PEI) is applied as a coating on the culture plates for 3D‐printing alginate scaffolds to improve the adhesion of alginate filaments to the culture plate during printing (Mendoza García et al., 2017). This effect arises from the negatively charged surface of alginate and the positively charged nature of PEI, which generate strong ionic interactions that enhance scaffold stability and attachment.

As such, the printing speed is directly dependent on the printing pressure. Repeating the above for each operator‐set pressure will result in a list of printing pressures and corresponding printing speeds.

See Naghieh and Chen (2021); Ning, Sun, et al. (2018); Soltan et al. (2019); Zimmerling, Aubrey, et al. (2024); and Zimmerling, Zhou, et al. (2024).

Equation 5 assumes square pore geometries.

Determine the volume of solution required based on how many wells you are studying and the height of the cell‐laden constructs or cell‐seeded scaffolds. Typically, 0.5 ml per well is sufficient to fully cover constructs/scaffolds <1 mm in height.

It is important to maintain consistent cell passage numbers across all bioprinting experiments. Variation in cell passaging, especially primary cells can significantly affect cell growth rates, behavior, and response to post‐printing damage. Once printing and culture conditions are established, it is recommended to prepare sufficient stocks of cells at the same passage number to make sure all bioprinting experiments use cells with uniform passage history.

Determine the cell density based on Live/Dead assay in Basic Protocoland make sure the cells are consistently distributed throughout the printed constructs. Cell overcrowding can lead to nutrient depletion, hypoxia, and increased cell death after long‐term culture. As mentioned before, use cells of the same passage number to reduce variability at the determined seeding density. 4

Ideally, bioink temperature should be maintained within a physiological or material‐specific range (20° to 25°C for most cell compatible hydrogels, or 37°C for thermo‐responsive materials like gelatin) to preserve cell viability and material consistency during printing. It is not recommended to print for an extended period (3 to 4 hr) as cell suspended in the bioinks experience continuous shear and pressure stress within the extrusion system. Instead, keep printing sessions as short as possible or perform printing in batches.

The rheological properties of alginate‐based bioinks are influenced by factors, such as bioink concentration, cell density, and temperature. Using the rheological tests, as outlined in this protocol (via oscillation amplitude, flow sweep, and temperature ramp tests), one can iteratively tune and optimized bioink formulation for the high printing quality or fidelity and/or preserve cell survival. Figures 9A‐B show the storage and loss moduli, which indicate how “solid‐like” or how “liquid‐like” a bioink behaves, respectively. In oscillation amplitude tests, liquid‐like bioinks exhibit a dominant loss modulus, whereas solid‐like bioinks show a dominant storage modulus. These tests also give the information on loss angle (δ), with tan(δ) being the ratio of the loss modulus over the storage modulus. This ratio reflects the bioink ability to recover the deformation after force removal; tan(δ) <1 suggest that most deformation is recovered while tan(δ) >1 indicates that most deformation is permanent, manifesting as plastic deformation or flow. Bioink flow behavior or viscosity should be carefully adjusted to preserve the cell viability during the printing process, which can be achieved by modifying bioink composition or the cell density (Ning, Betancourt, et al., 2018). For example, Figure 9D show that adding both cells and collagen increase the viscosity of the alginate‐based bioink. Temperature also plays a critical role; as shown in Figure 9F, increasing temperature reduces viscosity. Overall, rheological tests simulate printing conditions and provide insights for refining bioink formulations to improve print quality and cell viability.

Printability of alginate‐based bioinks refers to its ability to form and maintain a 3D structure after printing and is essential for ensuring structural stability and supporting cellular functions. Printability of the bioink is dependent on rheological properties, which suggest the rheological behavior should be adjusted suitable for printing, as demonstrated in Figure 9. Scaffold design and printing parameters also influence printability and should be selected based on the intended application. Successfully fabricated scaffolds should demonstrate consistent filament deposition with interconnected pores and minimal breakages. Microscopic images as shown in Figure 8 combined with Equations 4 and 5 can be used to assess the filament diameter and pore sizes to determine printability. In addition to these quantitative measurements, factors including needle clogging, filament breakages, and printing consistency should be considered when determining printability. A filament printability value of 1 indicates the printed strands match the designed diameter; however, if this results in frequent breakages (as shown in Fig. 8A), a slightly larger value may be preferred for improved structural integrity. Similarly, a pore printability value of ∼1 is indicative of consistent pores with minimal irregularities and a stable structure. Notably, Equation 5 is only valid for square pore geometries, in cases where scaffolds have an angular design, printability may be assessed based on filament diameter and printing consistency.

Cell viability provides insight into the biocompatibility and functionality of the printed/bioprinted models. Fluorescent microscopy provides a visual representation of cell viability with live cells indicated by green fluorescence and dead cells shown in red fluorescence (Figs. 10 and 11). Subsequent analysis of these images with Fiji software provides a quantification of cell viability. Fluorescent images should indicate evenly distributed cells for both seeded and bioprinted cells as indicated by Figures 10 and 11. A recovery period after seeded cells is to be expected with later timepoints demonstrating enhanced viability as demonstrated by Figure 11. Bioprinted cells may demonstrate a similar trend or have more consistent viability across timepoints depending on cell type as shown in Figure 10 (Xu et al., 2022). Appropriate image analysis in Fiji software (Schindelin et al., 2012) is necessary to obtain an accurate quantification of viability. Correctly interpreted images used for viability quantification will demonstrate cells with clear boundaries, minimal noise, and few cells omitted. A viability of ≥70% can be achieved under appropriate conditions for 3D bioprinted constructs (Ning et al., 2021; Xu et al., 2022), and a viability of ∼90% is expected with cell‐seeded scaffolds (Ketabat et al., 2023; Ning et al., 2016).

Immunofluorescence is an important tool for detecting and localizing specific antigens within cells and tissue samples using fluorescently‐labeled antibodies, providing insights into the functionality of cells within constructs. Immunofluorescence can be performed using either the direct method (primary antibody only) or the indirect method (secondary antibody) (Im et al., 2019). In this protocol, we provided instructions for the indirect method. Staining can be performed either on the entire construct without embedding and freezing, or on embedded, frozen samples that are sectioned prior to staining (Fig. 12). The former allows for more integrated 3D localization of antigens within the construct, as it avoids potential damage caused by freezing and sectioning. This method offers higher spatial resolution in three dimensions but is limited by the fact that each whole construct can be used for only one staining, making it relatively expensive and unsuitable for repeated or multiplexed analysis. In contrast, frozen and sectioned samples may experience some signal loss or tissue damage, and the resulting images are limited to 2D slices. However, this approach enables multiple sections from the same sample, allowing for replication and the analysis of various antigens across different regions of the construct.

Cell thawing and expansion should be initiated at least 1 week prior to bioprinting to ensure cells reach a healthy state. Hydrogel mix can be prepared 1 to 2 days in advance and stored at 4°C, but prolonged storage is not recommended to maintain consistency and reproducibility.

Running rheology typically takes 1 to 3 hr, depending on the number of samples.

The printing solution and 0.1% (w/v) PEI coating of culture plates should be prepared 1 day before bioprinting. Cell‐laden bioink should be prepared within 1 hr before printing to preserve cell viability and function. It is recommended to bioprint within 3 to 4 hr per session to minimize cellular stress from prolonged shear exposure.

The cell viability assay typically takes up to 1 hr, depending on the number of samples. For immunofluorescence staining, allow up to 2 days as this includes incubation with both primary and secondary antibodies.