Figure 1: BioLP schematic showing orifice-free bioprinting.

NRL has developed the Biological laser printer, or BioLP, a non-contact, orifice-free bioprinter with the demonstrated ability to create micron-scale patterns of living mammalian cells and biomaterials. 3D cellular patterns can be routinely created with single-cell resolution and no deleterious effects to the printed cells. These technologies have direct application to tissue engineering by enabling the direct and controlled deposition of living mammalian cells in both 2D and 3D patterns with micron-scale resolution, opening the possibility to bio-fabricate tissue and cellular structures at the scale of nature. 1, 2

BioLP uses a thin laser absorption layer (usually nm-scale thickness of titanium or titania) to optimize the printing process. This improves the spot-to-spot reproducibility of the printer while protecting the bioink from potentially damaging UV laser light (see Figures 1 and 2).

Cellular patterns generated using BioLP include primary rat olfactory ensheathing cells for spinal cord repair, human umbilical vein endothelial and smooth muscle cells for modeling vasculature (Figure 2), and primary human astrocytes and microvascular endothelial cells for a blood-brain-barrier model. 3, 4, 5

Figure 2: Demonstration of high fidelity, large area printing (protein microarray)
Figure 3: Printed human umbilical vein endothelial cells in a vascular tree pattern showing lumen-like growth after 24 hours.
Figure 3: (a) Printed human umbilical vein endothelial cells (HUVECs) in a vascular tree pattern showing lumen-like growth after 24 hours. Cells were printed onto Matrigel via BioLP, a LIFT-based process. (b-c) Crisscross HUVECs pattern printed onto a PLGA biopaper via BioLP. Lumen-like growth is observed with multiple crossings after 24 hours. Spacing between printed lines is 150 μm.

Advantages

  • Versatile bioprinting instrument
  • Creates heterogeneous 3D cellular constructs with higher resolution than commercially available extrusion bioprinters
  • Single-cell resolution
  • Spot to spot reproducibility
  • No deleterious effects to the printed cells

Applications

  • Creation of engineered tissues and organs with the potential to provide replacements for diseased or damaged organs, solving the shortage of donor organs.
  • High throughput in vitro engineered tissues (blood-brain-barrier, liver, lung, gastrointestinal, etc.) for drug discovery and/or toxicity screening.

Status and Opportunity

  • U.S. Patent Nos. 6,805,918; 6,815,015; and 7,294,367 are available for license
  • Potential for collaboration with NRL researchers

References

  • 1Barron, J.A., Wu, P., Ladouceur, H.D., and Ringeisen, B.R. (2004). Biological Laser Printing: A Novel Technique for Creating Heterogeneous 3-dimensional Cell Patterns. Biomed. Microdevices 6, 139–147.
  • 2Chen, C.Y., Barron, J.A., and Ringeisen, B.R. (2006). Cell patterning without chemical surface modification: cell-cell interactions between bovine aortic endothelial cells (BAEC) on a homogeneous cell-adherent hydrogel. Appl. Surf. Sci. 252, 8641–8645.
  • 3Othon, C.M., Wu, X., Anders, J.J., and Ringeisen, B.R. (2008). Single-cell printing to form three-dimensional lines of olfactory ensheathing cells. Biomed Mater 3, 034101.
  • 4Pirlo, R.K., Wu, P.K., and Ringeisen, B.R. (2014). Computer Aided Design and Manufacturing of Soft, Three-Dimensional, Multilayer, Biological Constructs via Laser Printing onto Laser Machined Composite Biopapers. J. Imaging Sci. Technol. 58, 404011–404017.
  • 5Wu, P.K., and Ringeisen, B.R. (2010). Development of human umbilical vein endothelial cell (HUVEC) and human umbilical vein smooth muscle cell (HUVSMC) branch/stem structures on hydrogel layers via biological laser printing (BioLP). Biofabrication 2, 014111.

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