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Cheptsov VS, et al.
focused through the donor slide on the absorbent surface soil or sediment, require pretreatment to remove living
(60 nm of gold). The conditions used for printing of cells from their solid-phase carrier, creating a liquid
S. bayanus and C. vulgaris were droplet volume (180 pl) phase sample. This process destroys close relationships
and the cell concentration of 200 cells per a droplet. The that can be crucial for the cultivation and study of the
growth and development of microcolonies were studied isolated microorganisms. In Ringeisen et al. study ,
[68]
by confocal microscopy, and the growth rates of colonies a high-performance automated method based on laser
were determined by the image analysis. The developed printing that isolates pure microbial cultures and spatially
protocols for printing of microorganisms and determining bound microbial consortia directly from solid-phase
the growth rate of microcolonies are very promising for complex environmental samples is described. A mixture
future studies of the growth and development of colonies. of soil with water or water and glycerol was applied to
In Koch et al. study , skin cell lines (fibroblasts/ a quartz tape coated with titanium dioxide, 85 nm thick,
[66]
keratinocytes) and human mesenchymal stem cells producing a donor slide. Adjustable amounts of soil
(hMSCs) were selected for laser printing experiments were transferred to different substrates using a pulsed
because of their high potential in human skin regeneration excimer laser (wavelength 248 nm, pulse duration
and new applications of stem cell therapy. The effect of in the range of 2–10 ns, and pulse energy varied from
laser printing on cell survival, proliferation, apoptotic 7 to 23 μJ), including 96-well plates filled with broth
activity, and DNA damage has been investigated. at the rate exceeding 20 microparticles per second or
Approximately 98% of skin cells and about 90% of more than a thousand microparticles per minute. After
hMSC cells survived after the laser printing procedure. printing, the viability of microbial cultures, culture
All used cell types kept their ability to reproduce after value, and significant morphological diversity have
laser printing. In addition, skin cells and hMSC showed been demonstrated. However, it is not clear whether it
no increase in apoptosis or the DNA fragmentation. exceeds the diversity obtained by cultivation with the use
The hMSCs also have maintained their phenotype, as of traditional methods. Nevertheless, the results showed
confirmed by the analysis of sorting with the help of that single-stage soil printing could be used to (a) produce
FACS. This study declares laser printing as a suitable pure microbial cultures (isolates) and (b) isolate consortia
method for computer positioning of different cell types from the micro-ecological system. The study, described
and a promising tool for future applications in ex vivo here, is the first extension of bioprinting to solid-phase
tissue generation. environmental samples for the isolation and cultivation
In Deng et al. study , the influences of laser pulse of individual microorganisms or consortia.
[67]
energy, laser spot size, distance to the acceptor substrate The LEMS method uses 8 ns, 24 μJ, 1.06 µm laser
on the number, size, and proliferation of laser printed pulses [70-72] . The donor plate is a glass coated with a
HELA cells are analyzed. It is shown that the laser 50–100 nm layer of gold, titanium, or chromium. To print
power and the thickness of the titanium film are the biological objects, bacterial cells or soil are mixed with
main factors affecting the survival of the isolated cells. a gel (2% hyaluronic acid), which prevents rapid drying
To provide a sufficient working distance and increase of the sample and spraying of microdroplets during laser
the viscosity of the culture medium, glycerin was used. printing. This technology allows obtaining a large number
To soften the landing of the cell on the acceptor plate a of separate bacterial colonies. It was also demonstrated that
layer of alginate was used. It was found that the optimal the LEMS technology allows cultivating a significantly
parameters to obtain a viable cell are pulse energy - 9 higher bacterial diversity in comparison with traditional
µJ, spot size - 60 µm, the thickness of the titanium film methods of cultivation (Figures 5 and 6). In particular, with
- 12 nm, working distance - 700 µm, the concentration of the use of this method, a strain of a rare Nonomuraea [70,71]
glycerin in the culture medium 2–4%, and the thickness genus was isolated from the soil (Figure 5), while the
of the alginate is more than 1 µm. To avoid contamination isolation of bacteria of this kind by traditional methods
and increase humidity, the process of cell shooting was requires the use of a number of special techniques, for
carried out in a special chamber made from PDMS. example, the addition of antibiotics and vitamins to the
It should be noted that very few works devoted to the nutrient media [73,74] . When using the LEMS method, in
isolation of microorganisms from complex heterogeneous addition to increase of the microorganisms diversity, an
systems with the use of laser printing are existing in the increase in the number of cultivated microorganisms
literature. Nowadays, two modifications of the laser was observed [70,72] . This effect was demonstrated both for
printing method - biological laser printing [68,69] and laser natural samples and for pure cultures of bacteria.
engineering of microbial systems (LEMS) - have been The reasons for the high efficiency of laser printing
proposed for the cultivation of microorganisms from methods in the isolation of bacteria from heterogeneous
natural environments [70-72] . environments are not yet sufficiently investigated.
As it was noted above, traditional methods to isolate Probably, one of the reasons is that, unlike traditional
microorganisms from environmental samples, such as cultivation methods, these methods do not use
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