Many cell-encapsulating bioinks require choosing between high print fidelity or high biocompatibility. BioBots, manufacturer of the first affordable desktop bioprinter and commercial bioprinting materials, has developed a new gelatin-based bioink which allows for bioprinting of complex 3D structures with high cell survivability rate. The company has also release a full report on the studies it conducted.
Matrix bioinks, or cell-encapsulating bioinks, are arguably the most important reagents used in the bioprinting processes. These materials must shield cells from shear stresses during the printing process, closely mimic the extracellular matrix and offer quick, nontoxic gelation for optimal print resolution. Types of matrix bioinks range from simple cell slurries suspended in media to cell-laden synthetic or naturally derived hydrogels such as gelatin.
Gelatin has many properties useful in biological interactions, including its hydrophilicity, integrin binding motifs, and matrix metalloproteinase (MMP) degradation sites. Because of these advantages, it is one of the most common materials used in matrix bioinks, including formulations such as gelatin methacrylate and gelatin/fibrin mixtures.
While gelatin-based bioinks offer many advantages over other hydrogels, these formulations are far from perfect. Photocrosslinkable formulations that utilize free radical polymerization provide high printability but sacrifice viability due to exposure to free radicals. Fibrin/gelatin mixtures that solidify through enzymatic crosslinking provide a much more biocompatible biofabrication process but lack the shape fidelity provided through photocrosslinking inks.
Below, we describe a fibrin/gelatin mixture created with a proprietary processed gelatin formulation, designed to have a specific range of particle sizes for improved extrusion and shape fidelity, that demonstrates improved printability over previous gelatin-based bioinks and maintains high viability up to 7 days of culture.
Gelatin Bioink Achieves High Resolution and Shape Fidelity
Figure 1: Printability Testing with Processed and Unprocessed Gelatin. All geometries were printed 5 times, with 3 images taken of each printed structure and 3 measurements taken from each image. The printed ear geometry (1A) and lattice (1B) were fabricated with 10% processed gelatin at 23 °C room temperature. Average line width and coefficient of variation are provided for Unprocessed gelatin 10% (1C), Unprocessed Gelatin 5% (1D), and Processed Gelatin 10% (1E) at 23 °C room temperature. Average line width and coefficient of variation are provided for processed gelatin 20% at 23 °C ( 1F) and 28 °C (1G). Scale bars 1 mm.
First, processed gelatin was tested for printability (Figure 1). Once pressure, speed and needle tip were optimized on the BioBot 1, printed line geometries were used to compare resolution and consistency of the reagents at 10% and 20% (w/v) to unprocessed gelatin at 10% and 5% (w/v) concentration (see previous printability testing of unprocessed gelatin here). 5 line geometries were printed and analyzed for each formulation (Figure 1C-1G). Line widths were analyzed with brightfield imaging and ImageJ software.
At ambient room temperatures of 23 °C, processed gelatin solutions of 10% (w/v) achieved the best resolution, with an average line width of 0.35 ± 0.03 mm and a coefficient of variation of 0.09 (Figure 1B,1E). However, in some later bioprinting studies, at higher room temperatures around 28 °C, 10% processed gelatin began to melt, creating a viscosity too low to create 3D structures. Increasing the concentration of processed gelatin to 20% solved this issue, with an average line width of 0.40 ± 0.05 mm and a coefficient of variation of 0.09 (Figure 1F). Even at 23 °C room temperature, 20% processed gelatin had a smaller average line width and coefficient of variation than unprocessed gelatin at 10% or 5% (Figure 1G).
Additionally, larger structures were fabricated to test the shape fidelity of the material (Figure 1A). An ear structure with dimensions of 64 x 32 x 14 mm was successfully fabricated with both 10% and 20% processed gelatin at 23 °C. This structure, which took about 1 hour and 40 minutes to print, was fabricated without changing the needle tip, demonstrating consistent extrusion of the material.
Processed Gelatin Does Not Affect Viability
Figure 2: Viability testing with Processed and Unprocessed Gelatin. Pipetted thin films were fabricated from processed (2 E,F) and unprocessed gelatin (10%) (2C,D) mixed with fibrinogen and compared to 2D controls (2A,B). No significant differences were observed in live/dead images taken over 72 hours. Scale bars 1 mm.
Next, encapsulated cell viability in 10% processed gelatin/fibrin pipetted thin films was compared to 10% unprocessed gelatin/fibrin pipetted thin films and 2D controls (Figure 2). Over 72 hours, processed gelatin showed no significant difference in viability compared to unprocessed gelatin/fibrin pipetted thin films or 2D controls. Cell concentrations of 5 million/ml were used. These processed gelatin/fibrin pipetted thin films were used as 3D controls in all future bioprinting studies.
Bioink Produces Viable Complex Geometries
Figure 3: Bioprinted sprial (3B), circle (3C), lines (3D) and “BWL” (3E) were compared to bioprinted thin filsm (3A) via Live/Dead imaging after 24 hours of culture. No significant differences were observed. Scale bars 1 mm.
A 24-hour bioprint study compared the viability of printed geometries to bioprinted thin films. Bioprinted lines demonstrated similar viability to bioprinted thin films. In addition to bioprinted thin films and lines, a variety of complex geometries were fabricated with the material and imaged. Spirals, circles and the letters “BWL” (standing for “Build with Life” ), were successfully fabricated with high viability.
Complex Bioprinted Structures Viable for up to 7 Days
A final 7-day bioprint study was completed comparing the viability of pipetted and printed thin films to bioprinted line structures over a period of 7 days. A 20% (w/v) processed gelatin mixture with a 2.5 million cells/ml concentration was used in this final study. Bioprinted lines maintained high viability for up to 7 days of culture, with improved viability compared to bioprinted and pipetted thin film controls. Samples were cultured in medium containing 0.01 L/ml ɛ-Aminocaproic Acid (see Supplementary Information)
Figure 4: Day 7 Live/Dead images of pipetted thin films (4A-C), bioprinted thin films (4D-F) and bioprinted line structures (4G-J). All structures maintained high viability and structural integrity up to 7 days of culture.
A 3D rendering of the bioprinted lines and thin films is visualized in figure 5 with surface plots fabricated from confocal stacks taken on a Perkin Elmer Operetta imaging system. Thin films had a decreased viability closer to the center of the structures, while line structures maintained high viability and structural integrity.
Figure 5: Day 7 surface plot renderings of 3D bioprinted lines (5A) and bioprinted thin film (5B) control. Images were taken with a Perkin Elmer Operetta System, and plots were created via ImageJ software. X and Y axes are in mm and Z axes are in um.
As one of the most common biomaterials used in bioinks, gelatin has many useful properties and wide-ranging applications in regenerative medicine. This study demonstrates this novel processed gelatin and fibrin bioink is able to create high-resolution, viable 3D structures with tunable degradation rates. This bioink has a wide range of potential applications, while processed gelatin itself has the potential to replace unprocessed gelatin in other gelatin-based bioinks for improved printability and viability.