Laser Printing Technology: Creating the Perfect Bioprinter

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Scientists from Russia, China, and the US have drawn the attention of the scientific community to one of the newest and most promising areas in bioprinting—laser-induced forward transfer (LIFT). The researchers have compared laser printing parameters, bioink composition, donor ribbons, and collector substrates for LIFT bioprinters, as well as post-printing treatments of fabricated materials—all of this may affect the properties of printed tissues and organs. The study will help scientists select the most appropriate techniques and materials, avoid many pitfalls in the process of bioprinting, and set the priorities for the development of this technology in the coming years. The details of the analysis were published in Bioprinting.

Tissue-engineering materials are increasingly used in medicine, mainly because they are created through mimicking the natural environment for cell development. The use of cell carriers (scaffolds) is a step forward compared to traditional cell therapy, which employs stem cells on their own. Bioprinting technologies allow to recreate tissues or organ models ("organs-on-chips") through layer by layer deposition of cells and biomolecules such as drugs or growth factors (compounds regulating cell growth and development) on a three-dimensional support structure.

LIFT technology transfers cells and biomolecules using laser pulse energy. The laser beam of a LIFT bioprinter focuses on the donor ribbon—a glass slide coated with an energy absorbing material (e.g. metal) and a layer of bioink (hydrogel with cells and biomolecules). Where the laser beam hits the surface, it heats and evaporates the energy absorbing layer, generating a gas bubble that propels a jet from the hydrogel layer. The resulting jet lands on another glass slide, the collector substrate, depositing a droplet.

LIFT technology provides a high print speed and cell survival rate, precise transfer of cells or molecules, and allows to work with various objects including microorganisms and whole cell structures such as spheroids. However, each hydrogel-cell combination requires a calculation of specific laser transfer parameters.

The authors of the paper analyzed 33 studies of bioprinting using LIFT. They systematically analyzed the descriptions of laser sources, energy absorbing materials, donor ribbons, and collectors substrates, as well as comparing the objectives and outcomes of the studies.

The most commonly used laser wavelengths were 193 and 1064 nanometers (short ultraviolet and near infrared ranges, respectively), although much longer and shorter wavelengths were successfully experimented with as well. Gold, titanium, gelatin and gelatin-containing mixtures were used as an energy absorbing material, while researchers in five studies did not use this layer at all.

Most of the studies used murine fibroblasts (connective tissue cells that synthesize extracellular matrix proteins) or mesenchymal stromal cells (cells that can differentiate into various connective tissue cells). The choice depended on cell availability.

The bioink used by many research teams contained glycerol and methylcellulose to help the bioink retain moisture, or blood plasma to support cell growth. Another common component was hyaluronic acid because it improved bioink viscosity as well as promoting cell growth. One of the best bioink materials was collagen, the main component of connective tissue. In some studies, the bioink also formed a "functional pair" with the collector substrate: for instance, if the donor ribbon was alginate-based, then the collector substrate contained calcium ions, while fibrinogen-containing donor ribbons were used with collector substrates containing thrombin. Such "functional pairs" allow to maintain the shape of the printed constructs effectively, because the substances in the collector substrate act as bioink fixatives.

The studies also used different types of printing: 2D, whereby the cells were arranged in a single layer (the researchers printed lines, shapes, letters, numbers, or the Olympic flag), or 3D, which allows to recreate complex cellular structures such as stem cell niches. Three-dimensional structures were created by depositing the bioink layer by layer.

The authors of the studies used various techniques to assess the impact of the bioprinting process on cells. Most researchers note that cell viability was fairly high, and there was no damage to the DNA despite the mechanical impact and the spike in temperature. There were no changes in either the proliferation rate of cells or the ability of stem cells to differentiate (transform into more specialized cells). In some of the studies, printed tissues were implanted into laboratory animals. The authors of the review believe that with the improvement of this technology in the next few years, there will be more studies involving animals.

"LIFT technology is quite new, and is only beginning to 'conquer' the world of biomedicine. Naturally, it will be improved and further used in tissue engineering, possibly even in clinical practice. In my opinion, however, its most promising application is in combination with other technologies, which will allow to create tissues and organs for transplantation", says Peter Timashev, one of the paper's authors, Director of the Institute for Regenerative Medicine, Sechenov University.



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