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New cell printing approach enables precise design of complex 3-D tissue structures

03/09/2010
Erin Podolak

A mechanical valve system that takes advantage of hydrogel droplets could improve the way cells are arranged for 3-D tissue engineering. Erin Podolak takes a look at the new printer making it possible.

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The ability to arrange and culture cells in three dimensions is a key feature of most tissue engineering applications. One of the difficulties in creating 3-D tissue scaffolds that mimic the microvasculature and microarchitecture of native tissue, however, comes from maintaining spatial and temporal control during cell seeding. The approach that some researchers have taken to control cell seeding is to modify traditional printing approaches, such as inkjet or laser printing, to deposit and pattern cells in precise locations for culturing in tissue engineering applications. The use of such ‘cell printers’ was first described in 2003 by a Clemson University team led by Thomas Boland (1-4); since then, groups around the globe have been advancing cell printing technology for tissue engineering applications.

Printing platform for 3D cell-laden droplet printing. (a) Cell-laden hydrogel droplets are generated by a mechanical valve that is operated by a controlled pulse width and a frequency to generate required volume and timed placement of droplets onto a substrate. (b) Image of a printed array of collagen droplets. Source: Tissue Engineering Part C: Methods
“Automated technologies give us more control over the amount and nature of cell cultures,” said Boland, who is now a researcher in the biomedical engineering department at the University of Texas, El Paso. “Fairly complex geometries can also be achieved with 3-D printing technology, allowing for more precise matching with a patient’s anatomy.”

While a variety of breakthroughs have pushed cell-printing technology forward, including scaffolds for tissue fabrication (5), heterogeneous 3-D patterns (6), designed seeding of individual cells (7), and tissue growth by layering (8), researchers have still been challenged with low cell viability, loss of cellular functionality, and clogging in many printing setups. Now, a team from Brigham and Women’s Hospital Center for Biomedical Engineering—in conjunction with the Harvard Medical School—have written in the journal Tissue Engineering about a new cell printing approach that addresses these drawbacks through the use of a unique microfluidic design and hydrogel droplets (9).

“The technology that we use is a valve-based system,” team leader Utkan Demirci told BioTechniques. “We avoid the heat and pressure that is generated by inkjet systems.” Heat and high pressure, while good for traditional ink-and-paper systems, are best avoided in cell printing since it requires viscous solutions like collagen in the cell droplet medium to mimic the extracellular matrix (ECM) found in natural tissues. This can be affected by the heat and high pressure, causing clogs and low cell viability. Demirci’s team’s new printer still uses cells encapsulated in collagen-based hydrogels and microgel droplets, but it bypasses the heat and high pressure issues through the use of a mechanical valve to deliver cell droplets to the substrate.

In the new system, cell-laden collagen medium was flowed into the valve and driven by constant and controlled air pressure, which did not disturb the density of the collagen. The medium was then loaded into a 10-mL syringe reservoir attached to the ejector mechanism, and controlled pressure from nitrogen gas was placed on the medium. The valve was then opened and closed at various time intervals to control the transfer of encapsulated cells to a microstage. The microstage platform spatially and temporally controlled the droplet placement. A computer managed the valve operation, applied pressure, and controlled cell concentration in the delivery system. The cell-laden collagen droplets were printed onto the collagen-coated substrate in three different cellular concentrations: 1 × 106, 5 × 106, and 10 × 106.The valve-based droplet ejector mechanism layered the cells on the substrate.

During the process, the ejector containing the medium and heat-sensitive collagen was kept cool with liquid nitrogen gas. Before each additional layer was printed, the tissue was gelled by incubation at 37°C for five minutes. The researchers found that the 1 × 106 concentration resulted in 6 ± 1 cells per droplet. The 5 × 106 concentration resulted in 29 ± 5 cells per droplet, and the 10 × 106 concentration resulted in 54 ± 8 cells per droplet. These results indicated that cell seeding density could be uniformly predicted.

By depositing cells in layers onto the engineered ECM, the authors were able to engineer a 3-D model tissue structure using smooth-muscle bladder cells from Sprague Dawley rats. Using the new valve-based delivery system, the researchers experienced no clogging in the delivery of the cells to the substrate using the viscous temperature- and pressure-sensitive collagen-based solution. Furthermore, the researchers were able to demonstrate a high cellular viability by calculating the number of cells that functioned pre-and post-printing. The platform averaged a total viability of 94% at day 14 post-printing and culturing, which, according to the researchers, is a successful rate of cell function.

According to Demirci, the new system also produced patterned cell cultures with more precision than those generated by previous technology, which could allow the new printer to be used to pattern multiple cell types, study cell-to-cell interactions, and enable high-throughput drug studies. “Achieving architectural complexity to mimic native tissues is a challenge,” said Demirci. “The control on cell seeding position and cell densities over a scaffold is also a challenge, which the current system addresses.”

Demirci believes his group’s cell-printing system will be applicable to a variety of areas in biological research. “We are working on cancer stromal cell interactions in collaboration with Tayyaba Hasan from Massachusetts General Hospital,” he said.

Boland predicted that within the next few years, cell printing technologies will be pushed to create 3-D cultures of increasing complexity. “The time of just showing that cells survive when squeezed out of a syringe should be over by now,” said Boland. “Long-term issues need to be addressed. For example, when using UV lasers to pattern cells, there is potential to cause DNA damage.” He said that finding ways to overcome this problem would be a beneficial route for researchers to explore.

Demirci echoed Boland’s opinion that the future rests with more complex 3-D tissue structures. “The ultimate goal is to mimic the cellular microenvironment as in vivo, taking into consideration all chemical, mechanical, and biological stimuli,” said Demirci. “The holy grail of cell patterning is to imagine one day that we could print out whole 3-D tissue constructs, and our 3-D layer-by-layer work is a baby step taken towards this challenge.”

References:

  1. Roth, E.A., T. Xu, M. Das, C. Gregory, J.J. Hickman, and T. Boland. 2003. Inkjet printing for high-throughput cell patterning. Biomaterials 25:3707-3715.
  2. Mironov, V., T. Boland, T. Trusk, G. Forgacs, and R.R. Markwald. 2003. Organ printing: computer-aided jet-based 3-D tissue engineering. Trends Biotechnol. 21:157–161.
  3. Boland, T., V. Mironov, A. Gutowska, E.A Roth., and R.R. Markwald. 2003. Cell and organ printing 2: fusion of cell aggregates in three-dimensional gels. Anat. Rec. 272A:497-502.
  4. Wilson, Jr., W.C., and T. Boland. 2003. Cell and organ printing 1: protein and cell printers. Anat. Rec. 272A:491-496.
  5. Ma, P.X. 2004. Scaffolds for tissue fabrication. Materials Today 7:30–40.
  6. Barron, J.A., P. Wu, H.D. Ladouceur, and B.R. Ringeisen. 2004. Biological laser printing: a novel technique for creating heterogeneous 3-dimensional cell patterns. Biomed Microdevices 6:139–147.
  7. Nakamura, M., A. Kobayashi, F. Takagi, A. Watanabe, Y. Hiruma, K. Ohuchi, Y. Iwasaki, M. Horie, et al. 2006. Biocompatible inkjet printing technique for designed seeding of individual living cells. Tissue Engineering 11:1658-1666.
  8. Kesari, P., T. Xu, T. Boland. 2005. Layer-by-layer printing of cells and its application to tissue engineering. Materials Research Society Symposium Proceedings. 845:111-118.
  9. S. Moon, S.K. Hasan, Y. S. Song, F. Xu, H.O. Keles, F. Manzur, S. Mikkilineni, J.W. Hong, et al. 2010. Layer by layer three-dimensional tissue epitaxy by cell-laden hydrogel droplets. Tissue Eng Part C Methods. 16:157-166.
  10. Demirci, U., G. Yaralioglu, E. Hæggström, and B.T. Khuri-Yakub. 2004. Acoustically actuated flextensional sixNy and single crystal silicon 2D micromachined ejector arrays. IEEE Trans. on Semiconductor Manufacturing 17:517-524.