LifeInk 200 is a high concentration collagen bioink useful for bioprinting structures with high shape fidelity. As the most common protein in many tissues’ ECMs, collagen-based bioinks are great for cell encapsulation because they demonstrate increased cell adhesion1. As a result of thermal crosslinking, the collagen molecules form fibrils and then more stable and stronger collagen fibers2. Many studies show collagen’s versatility to be used in many different tissue applications3-5. Here, we present a process for using the FRESH method as a support bath to print LifeInk 200, a high concentration collagen bioink. This process creates viable 3D structures of cell-laden collagen with repeatable, accurate geometries.
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When analyzing printed samples, a few parameters were found to affect construct viability, including the use of support material, print time, material concentration and incubation time.
Samples in FRESH Demonstrate Increased Viability
Prior to printing studies, viability was tested in pipetted thin films to create 3D controls. The viability of thin films pipetted into FRESH or no FRESH was first tested. Decreased viability was seen in films not printed into FRESH (Figure 1).
Crosslinking Time Affects Construct Viability
Next, a 24-hour bioprinting study was completed to analyze the effects of shear stress and crosslinking time on cell viability. After printing constructs, samples are crosslinked at 37 °C before adding media. Crosslink times of 0.5, 1 and 2 hours were analyzed. Crosslink times of up to 1 hour demonstrated high viability, while crosslinking times of 2 hours showed decreased viability (Figure 2).
Lower LifeInk200 Concentration Increases Viability
Next, a 7-day viability study was completed with printed thin films of LifeInk 200 concentrations of roughly 35 mg/ml and 20 mg/ml. 20 mg/ml thin films demonstrated higher viability over 7 days compared to 35 mg/ml concentrations (Figure 3).
LifeInk200 Produces Viable Bioprinted Constructs
With the above parameters taken into consideration, a final bioprint study was completed with thin films and printed geometries. Printed line geometries were successfully fabricated and imaged after 24 h of culture. Printed thin films demonstrated high viability up to 7 days of culture.
These results demonstrate the ability of LifeInk 200 and FRESH to support viable cultures and create consistent, high-resolution 3D geometries when used with an Allevi 2. When used together, these reagents can create cell-laden structures with specific, repeatable 3D geometries. Further, bioprinted thin films and pipetted thin films created in this study can be used as 3D controls for future experiments with these materials. Results highlighted a few parameters that can affect the viability of printed constructs, mainly material concentration, use of FRESH and incubation times.
Use of FRESH Support Material Increases Viability
The use of the FRESH bioprinting method keeps cells viable for longer. This result is likely due to the prevention of dehydration of samples during crosslinking.
Material Concentration Affects Viability
At higher concentrations (35mg/mL vs. 20mg/mL) of LifeInk 200, samples exhibited decreased viability. This result is likely due to lower material permeability, limiting nutrient delivery and waste export.
Crosslink Time Affects Viability
Incubation times of over 1 hour can lead to a significant decrease in viability. This decrease in viability is likely from sample dehydration or deprivation from nutrients for an extended period of time.
The results of this study show that use of a support material, the order of printing, the concentration of LifeInk 200, and the length of crosslinking time can all affect the viability of 3D bioprinted collagen constructs. By optimizing these parameters, viable cell-laden collagen structures with complex geometries were successfully fabricated on the commercially available Allevi platform. Future studies will include further investigation and optimization of these parameters. This work demonstrates the first commercially available collagen bioink compatible with a commercialized bioprinting platform. These standardized products, available to any researcher, allows for repeatable, reliable biofabrication with a versatile material useful for a variety of applications in tissue engineering and biology.
Primary Human Neonatal Dermal Fibroblasts (HNDFs) obtained from ATCC were cultured in monolayer cultures at 37 °C and 5% of CO2 using Dulbecco’s Modified Eagle Medium (Corning) supplemented with 10% fetal bovine serum (Hyclone) and 1% penicillin-streptomycin-amphotericin (Corning). Passage numbers under 10 were used.
Thin Film Fabrication
HDNFs and cell media were mixed with LifeInk 200 to result in a ~20mg/mL working concentration of collagen with a cellular concentration of 5 million/mL. Pipetted thin films were created by pipetting 10uL of solution into wellplates, half with FRESH and half without. Wellplates were then placed into the 37oC incubator for 0.5, 1, and 2 hours.
Bioprinted thin films were created with a custom gcode and suggested print parameters from Allevi. Volume of extruded bioprinted material was estimated with the volume test. Extrusion time in the printed gcode was set for extrusion of approximately 10 μl. All samples were crosslinked for 0.5, 1, or 2 hours in the incubator, then washed three times with phosphate buffered saline (PBS). All samples were bioprinted with an Allevi 2.
FRESH was prepared based on instructions included in the FRESH kit (Allevi). As described previously, the FRESH method was developed by the Feinburg Lab at Carnegie Mellon University to allow for the printing of complex structures using a gelatin slurry (6). For more information on FRESH preparation, email [email protected] for a detailed protocol.
Bioprinted Structure Fabrication
First, CAD files of matrices, rings, and lines were created with SolidWorks. Then, these STL files were loaded into Repetier Host and sliced with the print parameters suggested by Allevi. 3 mL of LifeInk 200 with a 5 million/mL HNDF concentration was loaded intro extruder 1 of the Allevi 2. 30 gauge, 1-inch needles were used, with a pressure of 15-20 (this can be a range) and print speed of 6mm/s. Designs were printed into FRESH support material, then crosslinked for 0.5 or 1 hours in the incubator. After crosslinking, samples were washed three times with phosphate buffered saline (PBS), as described in the Allevi collagen bioprinting protocol. All samples were bioprinted with an Allevi 2.
To assess cell viability, a LIVE/DEAD kit (Life Technologies) was used. Images were taken on a Nikon TE300 Inverted Fluorescent Microscope.
Many thanks to Advanced Biomatrix and Bowman Bagley for their help in making this protocol possible.
(1) Holzl, Katja, et. al. Bioink properties before, during, and after 3D bioprinting. IOPScience. 2016 September 23.
(2) Bagley, Bowman. Advanced Biomatrix. 2017.
(3) Lee, Yeong-Bae et. al. Bio-printing of collagen and VEGF-releasing fibrin gel scaffolds for neural stem cell culture.Experimental Neurology. 2010 March 6.
(4) Ren, Xiang et. al. Engineering zonal cartilage through bioprinting collagen type II hydrogel constructs with biomimetic chondrocyte density gradient. BMC Musculoskeletal Disorders. 2016 July 20.
(5) Rhee, Stephanie et. al. 3D Bioprinting of Spatially Heterogeneous Collagen Constructs for Cartilage Tissue Engineering. ACS Biomaterials. 2016 July 18.
(6) Hinton, Thomas J. et. al. Three-dimensional printing of complex biological structures by freeform reversible embedding of suspended hydrogels. Science Advances. 2015 October 23.