PrintAlive Bioprinter

PrintAlive Bioprinter

  • In flow organization of 3D soft materials
    In flow organization of 3D soft materials
  • Skin graft application and bioprinting system
    Skin graft application and bioprinting system
  • 2nd generation bioprinter
    2nd generation bioprinter
  • Rendered image of 3rd generation bioprinter
    Rendered image of 3rd generation bioprinter
  • Device with on-chip wells and pressure control
    Device with on-chip wells and pressure control
  • Devices to extrude varying sheet widths
    Devices to extrude varying sheet widths
  • Patterns in base biopolymer
    Patterns in base biopolymer
  • Different assembly methods after extrusion
    Different assembly methods after extrusion
  • Patterns and material combinations
    Patterns and material combinations
  • Patterned human keratinocytes and fibroblasts
    Patterned human keratinocytes and fibroblasts
Short introduction to our bioprinting technology and applications

Proprietary 3D bioprinting technology allowing human microtissue arrays to be routinely defined with unprecedented speed and resolution.


Many tissues in nature have unique 3-D and hierarchical architectures to organize multiple cell types and sub-structures. This spatial organization is critical to the biological function of the tissue, and is equally critical when mimicking the structure and function of human tissues in vitro. However, current commercially-available bioprinters rely on a top-down assembly approach, which drastically reduces the throughput of the printer, and limits the complexity of the 3-D structure and the resolution of individual features to the printer nozzle size and volume.

In high impact applications of bioprinting technology, such as the development of cellularized skin grafts for burn victims, the time available to produce the tissue is limited and large cross-sectional areas are required. In severe burn injuries where both the epidermal and dermal layers of the skin are destroyed, prompt wound closure is critical for favourable patient outcomes and reduced mortality rates; after burn injuries, patient survival is inversely proportional to the time required for wound stabilization and coverage, and the mortality rate increases by 10% for every additional 10% surface area burn [1]. The gold standard in current surgical practice is the use of split-surface autografts, allografts, acellular or cell-populated skin substitutes, and cell-spraying strategies. However, each of these methods have unique limitations [2-9]; autografts require substantial tissue donations from the patient themselves and limits the applicability in cases of severe burns where significant tissue donations are not possible, allografts can transmit pathogens from donor to recipient and can face immunologic rejection by the patient, skin substitutes are prohibitively expensive and can cause allergic reactions, and cell therapies require long cell expansion and culturing times (once cells have been expended to a sufficient number, additional culture time of 14 days at the minimum is required prior to implantation), with unreliable cell attachment to the wound in some cases [4].

Faced with this problem, we decided to apply our previously developed bioprinting technology to the formation of cell-populated wound dressings that accurately reproduce key features of human skin in a scalable and high-throughput format. Using our microfluidic cartridge-based technology, skin microtissue regions could be seamlessly incorporated within continuously produced hydrogel sheets with multiple cell types and precisely controlled graft thickness, structure, and composition.

[1] Miller, S. F. et al. National burn repository 2007 report: A synopsis of the 2007 call for data. Journal of Burn Care & Research 29, 862-870, doi:10.1097/BCR.0b013e31818cb046 (2008).
[2] Sheridan, R. Closure of the excised burn wound: autografts, semipermanent skin substitutes, and permanent skin substitutes. Clinics in Plastic Surgery 36, 643-+, doi:10.1016/j.cps.2009.05.010 (2009).
[3] Burke, J. F., Yannas, I. V., Quinby, W. C., Bondoc, C. C. & Jung, W. K. Successful use of a physiologically acceptable skin in the treatment of extensive burn injury. Annals of Surgery 194, 413-428, doi:10.1097/00000658-198110000-00005 (1981).
[4] Streit, M. & Braathen, L. R. Apligraf - a living human skin equivalent for the treatment of chronic wounds. International Journal of Artificial Organs 23, 831-833 (2000).
[5] Zaulyanov, L. & Kirsner, R. S. A review of a bi-layered living cell treatment (Apligraf (R)) in the treatment of venous leg ulcers and diabetic foot ulcers. Clinical Interventions in Aging 2, 93-98, doi:10.2147/ciia.2007.2.1.93 (2007).
[6] Wood, F. M., Stoner, M. L., Fowler, B. V. & Fear, M. W. The use of a non-cultured autologous cell suspension and Integra (R) dermal regeneration template to repair full-thickness skin wounds in a porcine model: A one-step process. Burns 33, 693-700, doi:10.1016/j.burns.2006.10.388 (2007).
[7] Cuono, C., Langdon, R. & McGuire, J. Use of cultured epidermal autografts and dermal allografts as skin replacement after burn injury. Lancet 1, 1123-1124 (1986).
[8]Jones, I., Currie, L. & Martin, R. A guide to biological skin substitutes. British Journal of Plastic Surgery 55, 185-193, doi:10.1054/bjps.2002.3800 (2002).
[9] Boyce, S. T. et al. Surface electrical capacitance as a noninvasive index of epidermal barrier in cultured skin substitutes in athymic mice. Journal of Investigative Dermatology 107, 82-87, doi:10.1111/1523-1747.ep12298286 (1996).


Each microfluidic device works based on the same principle: using arrays of microfabricated channels, uncrosslinked biopolymer is distributed and organized into the final hydrogel shape inside the microfluidic device. At this time, cell-laden printing solution can be controllably injected into the uncrosslinked bulk gel, forming discrete and well-defined cellularized regions in the main gel. During the extrusion of the gel from the microfluidic device, instantaneous crosslinking occurs at the outer surfaces of the gel and the complex spatial organization created inside the uncrosslinked fluid is maintained in the final solid gel. This extrusion process occurs in a liquid reservoir, and the extruded hydrogel is continually collected with an automated drum. The patterning is actuated via pneumatic control of on-chip fluid wells, which allows us to discretely incorporate multiple independent fluids or cell types in different configurations.

Using this approach, we developed custom devices to co-extrude viable human keratinocytes and fibroblasts in a structure which mimics the epidermal and dermal layers of human skin in both single and bilayered grafts. The local material composition in the cell-laden regions was optimized for each cell type, and we are currently moving from bench to animal testing in a murine wound model. The preliminary in vivo data obtained suggests an improved wound healing after implantation of our cell-populated patterned grafts compared to control which consisted of hydrogel sheets without cells.

In addition, our ability to localize high concentrations of human cells rather than homogeneously populating the entire sheet has the added advantage of reducing by up to 75% the number of cells required. This promises improvements in the time required for pre-operative graft preparation.

개발 단계

This project began in Axel Guenther’s lab at the University of Toronto as a MASc project for Lian Leng, and it continued as a MASc project for Arianna McAllister and a PhD project for Lian Leng. Initially, the project focus was on the development of microfluidic devices to continuously extrude hydrogel sheets with controllable incorporation of payloads and formation of 3-D patterns in material composition. These multi-layer micro-devices were made in a silicone material with partial curing and soft lithography techniques. Hundreds of design iterations were performed to optimize device design and accommodate new patterning abilities. Through continual experimentation and systematic testing, we increased the complexity of the microfluidic devices to increase the complexity of patterns that could be produced, the pattern density in the hydrogels, the hydrogel size, and the feature resolution. Concurrently, we also developed several generations of an automated collection reservoir to wind and collect the gels after formation. We currently have developed a second generation, pre-commercial prototype of the entire system. Our technology is protected by two strong patent families that are currently at the national or PCT filing stages. With this more mature prototype, we began modifying our existing approach to overcome the challenges of in flow skin graft formation.


The bioprinter and its inventors were awarded the University of Toronto Inventor of the Year title in 2013.

We have received significant media attention for our bioprinter technology:

- CBC The Nature of Things Interview. March 2014.
- Al Jazeera Interview. February 2014.
- Canada AM Interview. January 2014.
- CBC - Radio and News. October 2013.
- CNN interview. August 2013.
- TVO - The Agenda with Steve Paikin: 3D Printing, a Desktop Future. June 2013 (
- The Space Channel interview. June 2013.
- The Grid TO - Front cover - “Big Thinkers” article. April 24 2013 (
- CTV News - Live Interview. January 23, 2013.
- Global Toronto News coverage. January 21, 2013.
- The Globe and Mail, Health & Fitness. January 21, 2013
- U of T magazine. Vol. 40, No. 2. January 2013.
- CTV News coverage. September 14, 2012.
- OMNI News National Edition. August 10, 2012.