Jason A. Barron

Laser Printing of Pluripotent Embryonal Carcinoma Cells

A technique by which to print patterns and multilayers of scaffolding and living cells could be used in tissue engineering to fabricate tissue constructs with cells, materials, and chemical diversity at the micron scale. We describe here studies using a laser forward transfer technology to print single-layer patterns of pluripotent murine embryonal carcinoma cells. This report focuses on verifying cell viability and functionality as well as the ability to differentiate cells after laser transfer. We find that when cells are printed onto model tissue scaffolding such as a layer of hydrogel, greater than 95% of the cells survive the transfer process and remain viable. In addition, alkaline comet assays were performed on transferred cells, showing minimal single-strand DNA damage from potential ultraviolet-cell interaction. We also find that laser-transferred cells express microtubular associated protein 2 after retinoic acid stimulus and myosin heavy chain protein after dimethyl sulfoxide stimulus, indicating successful neural and muscular pathway differentiation. These studies provide a foundation so that laser printing may next be used to build heterogeneous multilayer cellular structures, enabling cell growth and differentiation in heterogeneous three-dimensional environments to be uniquely studied.

Laser printing of single cells: statistical analysis, cell viability, and stress.

Methods to print patterns of mammalian cells to various substrates with high resolution offer unique possibilities to contribute to a wide range of fields including tissue engineering, cell separation, and functional genomics. This manuscript details experiments demonstrating that BioLP TM Biological Laser Printing, can be used to rapidly and accurately print patterns of single cells in a noncontact manner. Human osteosarcoma cells were deposited into a biopolymer matrix, and after 6 days of incubation, the printed cells are shown to be 100% viable. Printing low numbers of cells per spot by BioLPTM is shown to follow a Poisson distribution, indicating that the reproducibility for the number of cells per spot is therefore determined not by the variance in printed volume per drop but by random sampling statistics. Potential cell damage during the laser printing process is also investigated via immunocytochemical studies that demonstrate minimal expression of heat shock proteins by printed cells. Overall, we find that BioLPTM is able to print patterns of osteosarcoma cells with high viability, little to no heat or shear damage to the cells, and at the ultimate single cell resolution.

The Evolution of Cell Printing

Tissue and organs are highly complex systems with innate heterogeneous components, each with their own structure and function. Many facets of this structure have micron-scale features (e.g., capillaries, sinuses, cell–cell contacts, extracellular matrix). Traditional approaches in tissue engineering and regenerative medicine attempt to recreate this structure and function in vitro by randomly seeding cells onto 3D scaffolds. These 3D scaffolds provide the structure and an environment for seeded cells to differentiate into tissue-like materials [17, 67, 82]. The structure and function of natural tissue is replicated by using either multifunction stem cells (e.g., pluripotent, mesenchymal, embryonic) or highly sophisticated scaffolds (e.g., micro-/nanostructured, chemically/biologically functionalized), or both [14, 24, 36, 37, 41–43, 49, 52, 71, 78, 89]. There are tissue-engineering success stories, but most are for simple, homogeneous systems such as skin and thin membrane (bladder) replacements.

Biological laser printing as an alternative to traditional protein arrayers

Current proteomics experiments often rely upon printing techniques such as ink jet, pin, or quill arrayers that were developed for the creation of cDNA microarrays. These techniques often do not meet the spotting requirements needed for successful high throughput protein identification and profiling. The Naval Research Laboratory has developed an alternative to these commercially available arrayers that does not rely upon a solid pin or capillary-based fluidics. This presentation describes experiments demonstrating that biological laser printing, or BioLP, is capable of depositing microarrays of proteins rapidly and efficiently. This technique utilizes a focused laser pulse to obtain micron-scale resolution rather than a pin or orifice, thereby eliminating clogging and protein loss commonly encountered in commercially available printers. The speed and spot-to-spot reproducibility of the printer is comparable to other techniques, while the minimum spot diameter and volume per printed droplet is significantly less at 30 microns and ~500 fL, respectively. The transfer of fluid by BioLP occurs through a fluid jetting mechanism, as observed by high-speed images of the printing process. In addition, printed biotinylated bovine serum albumin is identified through immunoassay and observed by fluorescent detection. These results indicate that BioLP holds promise as a novel protein printer for use in a wide range of applications in the proteomics field.