In this article we examine printing a gelatin/alginate blend with incorporated cells using the BioBot Basic.
What are Gelatin/Alginate Blends?
Gelatin and alginate are used for many biomedical applications, including as scaffolds for tissue engineered constructs (1). Gelatin is a naturally occurring, water soluble protein derived from collagen, and is widely used for 3D bioprinting. Gelatin forms a gel at room temperature, but melts in a 37°C incubator, making its printability temperature dependent. Therefore, gelatin is often combined with other biomaterials, such as silk, agarose, or alginate, to enhance its printability and stability in culture. Alginate is a polysaccharide naturally found in seaweed, which is also frequently blended with other hydrogels for bioprinting applications. Alginate, prior to crosslinking, has poor mechanical properties and does not hold its shape when printed. Additionally, alginate is not cell-adhesive. For these reasons, neither gelatin nor alginate alone is optimal for biological printing. However, when blended, gelatin/alginate can be readily printed, and provides an environment conducive to 3D cell culture (2). The gelatin component enables the material to hold its shape after printing at room temperature. Alginate improves gelatin printability by altering its viscosity, creating a smoother print. After printing the alginate is crosslinked, which allows the construct to maintain its shape after the majority of gelatin has dissolved in a 37°C incubator. Residual gelatin in the construct also improves cellular adhesion.
Gelatin/alginate blends are a well-established bioink used for printing cells in a 3D environment. Concentrations ranging from 4% gelatin/3% alginate to 10% gelatin/9% alginate have been reported for various applications (1,2). Here, we used a 6% gelatin and 5% alginate blend in phosphate buffered saline (PBS). Gelatin was first dissolved in hot PBS, then sterile filtered. Alginate was added to the sterile solution in a biosafety cabinet. Once dissolved, the material was brought to 37°C before combining with human mesenchymal stem cells (Rooster Bio) at 400K cells/ml (3).
Prints were performed on a BioBot Basic inside a biosafety cabinet with a 22GA conical needle, at a pressure of 25 PSI and speed of 15 mm/sec (pressure and speed may vary slightly between batches). Structures printed included a cube, a tube, and a rectangular box (Fig 1).
After printing, constructs were submerged in a 0.75% calcium chloride solution to crosslink the alginate. After crosslinking the structures were rinsed thoroughly with PBS, flooded with culture medium containing DMEM, 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin, and placed in the incubator for two days. To measure cell viability, a Calcein-AM (Thermo Fisher) stain, which stains live cells, and a Hoechst stain (Invitrogen) for total nuclei.
Results & Discussion
Printed constructs ranged from between 4mm – 8mm in height, width, and depth, with a wall thickness of ~1mm. This demonstrates the printer’s ability to print fine, stable constructs and the bioinks ability to adequately adhere to itself and maintain its structure. Calcein and Hoechst staining indicated an 80% cell viability two days after printing.
The small footprint and utility of the BioBot™ Basic is ideal for use in a tissue culture hood for aseptic bioprinting of tissue constructs. Here, we demonstrate using the BioBot™ Basic to print a commonly used gelatin/alginate bioink containing live cells resulting in a viable cell culture.
Notes: Other needle sizes and types may be used, although when using non-conical needles, the material printability becomes more temperature sensitive. The optimal printing temperature for non-conical needles is 28°C. Repeated heating cycles should be avoided. Autoclaving gelatin blends should be avoided, as this degrades the gelatin and reduces printability. Coating cell culture plates with alginate or other ECM proteins can be used to prevent constructs from lifting off plates during culture, if needed.
Axpe, E.; Oyen, M.L. “Applications of Alginate-Based Bioinks in 3D Bioprinting. Int. J. Mol. Sci. 2016, 17, 1976
Gao, Teng, et al. “Optimization of Gelatin–Alginate Composite Bioink Printability Using Rheological Parameters: a Systematic Approach.” Biofabrication, 2018, 10, p. 034106.