Processed Gelatin/Fibrin Bioprinted Viability

Wikis > Processed Gelatin/Fibrin Bioprinted Viability

Introduction

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 (1).

Gelatin has many properties useful in biological interactions, including its hydrophilicity, integrin binding motifs, and matrix metalloproteinase (MMP) degradation sites (2-4). 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 (3-5).

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 (5).

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.

Results

Gelatin Bioink Achieves High Resolution and Shape Fidelity

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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

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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

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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)

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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.

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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.

Conclusion

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.

Methods

Cell Culture

Primary Human Neonatal Dermal Fibroblasts (HNDFs) obtained from ATCC were cultured in monolayer cultures at 37 °C and 5% of COusing Dulbecco’s Modified Eagle Medium (Corning) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin-streptomycin-amphotericin (Corning). In some bioprint studies, medium was also supplemented with varying concentrations of ɛ-aminocaproic acid or tranexamic acid (6). The medium was changed every 2-3 days. Passage numbers under 10 were used.

Bioink Preparation 

Processed gelatin soft tissue kit was acquired from BioBots and prepared as described in the protocol provided. Briefly, processed gelatin was dissolved in phosphate-buffered saline (PBS) or cell culture medium, then sterile-filtered (0.45 μm Millipore). This solution was then mixed with a fibrinogen solution for a final gelatin concentration of 10 or 20% (w/v) and a final fibrinogen concentration of 10% (w/v). Next, this solution was mixed with cells then pipetted or loaded into a syringe for printing.

Pipetted thin films and bioprinted structures were crosslinked with a  PBS solution containing thrombin, calcium chloride and transglutaminase for 30 minutes, then washed with PBS before culturing.

Thin Film Fabrication 

To create pipetted thin films, the bioink prepared above was heated to 37˚C then pipetted in 10 μL volumes into well-plates. The pipetted solution was allowed time to cool to room temperature before adding a PBS solution containing thrombin, calcium chloride, and transglutaminase.

Bioprinted thin films were extruded from the BioBot 1 to created thin films with roughly 10 μL volumes. A PBS solution containing thrombin, calcium chloride, and transglutaminase was added immediately after printing.

Bioprinted Geometries Fabrication

Custom gcode files were used to fabricate bioprinted lines, lattices, circles and thin films. For spirals and “BWL” designs, an .STL file was first created then sliced with the print parameters suggested by BioBots to create gcode files. 1-3 ml of cell-laden bioink were loaded into extruder one and either 27 (for 20% (w/v) gelatin mixtures) or 28 (for 10% (w/v) gelatin mixtures) were used to print structures.

Sample Analyses

A Live/Dead kit (Life Technologies) was used to qualitatively assess the viability of samples. Images were taken on a Nikon TE300 Inverted Fluorescent Microscope or a Perkin Elmer Operetta Imaging System. For samples imaged on the Perkin Elmer Operetta system, an additional Hoechst nuclei stain was used in combination with the Live/Dead kit.

Supplementary Information

Supplementary Results

Gcode and STL Print Files

References

  1. M.E. Prendergast et al, “Bioinks for Biofabrication: Current State and Future Perspectives,” J. 3D Print. Med. 2016.
  2. J. Malda et al, “25th Anniversary Article: Engineering Hydrogels for Biofabrication,Adv. Mater., vol. 25, pp. 5011-5028, 2013.
  3. D. B. Kolesky et al, “3D Bioprinting of Vascularized, Heterogeneous Cell-laden Tissue Constructs,” Adv. Mater., vol. 26, pp. 3124-3130, 2014.
  4. B. D. Fairbanks et. al, “Photoinitiated Polymerization of PEG-diacrylate with lithium phenyl-2,4,6-trimethylbenzoylphosphinate: polymerization rate and cytocompatibility,Biomaterials, vol. 30, no. 35, pp. 6702-6707, Dec 2009.
  5. Kang, Hyun-Wook et al, “A 3D bioprinting system to produce human-scale tissue constructs with structural integrity,” Nature Biotechnology, vol. 3, no. 34, February 2016.
  6. A Koster et al, Antifibrinolytic Therapy for Cardiac Surgery: An Update” Anesthesiology, vol 123, no.1 July 2015.