3D printing in biotechnology—An insight into miniaturized and microfluidic systems for applications from cell culture to bioanalytics

Since its invention in the 1980s, 3D printing has evolved into a versatile technique for the additive manufacturing of diverse objects and tools, using various materials. The relative flexibility, straightforwardness, and ability to enable rapid prototyping are tremendous advantages offered by this technique compared to conventional methods for miniaturized and microfluidic systems fabrication (such as soft lithography). The development of 3D printers exhibiting high printer resolution has enabled the fabrication of accurate miniaturized and microfluidic systems—which have, in turn, substantially reduced both device sizes and required sample volumes. Moreover, the continuing development of translucent, heat resistant, and biocompatible materials will make 3D printing more and more useful for applications in biotechnology in the coming years. Today, a wide variety of 3D‐printed objects in biotechnology—ranging from miniaturized cultivation chambers to microfluidic lab‐on‐a‐chip devices for diagnostics—are already being deployed in labs across the world. This review explains the 3D printing technologies that are currently used to fabricate such miniaturized microfluidic devices, and also seeks to offer some insight into recent developments demonstrating the use of these tools for biotechnological applications such as cell culture, separation techniques, and biosensors.     

Excretion of cytoplasmic proteins (ECP) in Staphylococcus aureus

E xcretion of c ytoplasmic p roteins (ECP) is a common physiological feature in bacteria and eukaryotes. However, how these proteins without a typical signal peptide are excreted in bacteria is poorly understood. We studied the excretion pattern of cytoplasmic proteins using two glycolytic model enzymes, aldolase and enolase, and show that their excretion takes place mainly during the exponential growth phase in Staphylococcus aureus very similar to that of Sbi, an IgG‐binding protein, which is secreted via the Sec‐pathway. The amount of excreted enolase is substantial and is comparable with that of Sbi. For localization of the exit site, we fused aldolase and enolase with the peptidoglycan‐binding motif, LysM, to trap the enzymes at the cell wall. With both immune fluorescence labeling and immunogold localization on electron microscopic thin sections aldolase and enolase were found apart from the cytoplasmic area particularly in the cross wall and at the septal cleft of dividing cells, whereas the non‐excreted Ndh2, a soluble NADH:quinone oxidoreductase, is only seen attached to the inner side of the cytoplasmic membrane. The selectivity, the timing and the localization suggest that ECP is not a result of unspecific cell lysis but is mediated by an as yet unknown mechanism.     

Estimating the 3D Pore Size Distribution of Biopolymer Networks from Directionally Biased Data

The pore size of biopolymer networks governs their mechanical properties and strongly impacts the behavior of embedded cells. Confocal reflection microscopy and second harmonic generation microscopy are widely used to image biopolymer networks; however, both techniques fail to resolve vertically oriented fibers. Here, we describe how such directionally biased data can be used to estimate the network pore size. We first determine the distribution of distances from random points in the fluid phase to the nearest fiber. This distribution follows a Rayleigh distribution, regardless of isotropy and data bias, and is fully described by a single parameter—the characteristic pore size of the network. The bias of the pore size estimate due to the missing fibers can be corrected by multiplication with the square root of the visible network fraction. We experimentally verify the validity of this approach by comparing our estimates with data obtained using confocal fluorescence microscopy, which represents the full structure of the network. As an important application, we investigate the pore size dependence of collagen and fibrin networks on protein concentration. We find that the pore size decreases with the square root of the concentration, consistent with a total fiber length that scales linearly with concentration.     

Use of benthic diatom communities to evaluate water quality in rivers of southern Poland

Biological and chemical data were processed to estimate trophic stage and degree of pollution in several streams and rivers in southern Poland. The majority were eutrophic and some of them heavily polluted; only a few were oligo-mesotrophic. The differences in the water quality of the rivers were reflected by different types of diatom community and also by the values for some diatom indices, which were calculated using the latest version of the 'Omnidia' database software. Except for the Sládeček's index, all diatom indices correlated significantly with organic load (COD), oxygen concentration, conductivity and most of the measured ions. Some indices showed a significant negative correlation with trophic level (expressed by NH₄-N and PO₄-P). In general, IPS (Specific Pollution Sensitivity Index) and GDI (Generic Diatom Index) indices gave the best results. Among the investigated diatom communities, only a few taxa indicated oligo-mesotrophy and oligo-β-mesosaprobity. Most of the sites were characterised by a greater relative contribution of eutraphent and tolerant ones as well as α-mesosaprobic and polysaprobic diatoms. This study suggests that the structure of benthic diatom communities and diatom indices, especially GDI, can be applied for monitoring rivers in Poland. This revised version was published online in June 2006 with corrections to the Cover Date.     

Serological relationship between three varieties of Bacillus popilliae

The serological relationship between three varieties of Bacillus popilliae was investigated by double diffusion and immunoelectrophoresis, using antigens prepared from homogenized vegetative cells in the logarithmic growth phase. The results showed the existence of a close serological relationship among the three varieties in agreement with a recently proposed taxonomic scheme for the milky disease bacteria (Wyss, 1971).     

Genetical changes in rhizobium bacteria and in their bacteriophages during coexistence

Summary Three systems, each consisting of a virulent phage and a susceptible bacterium, were incubated for 24 weeks in liquid culture and the processes of interaction and genetical change in both partners investigated. The initial stage of interaction in all systems was identical; all susceptible bacterial cells were lysed and only cells that originally were resistant or mutated to resistance, remained alive. In two systems the multiplication of resistant cells was not restricted by large concentration of phage; in the third there was no bacterial multiplication. Phage concentration was maintained at a high titre for the duration of the experiment by the presence of the resistant mutant bacteria. Phage resistant mutants were genetically less stable than the parent forms; some also had changed colony morphology and symbiotic properties. Mutations in these different characteristics were independent. In two systems virulent phage mutated into temperate form; hence some of the phage-resistant mutant cells were genetically suitable for establishment of lysogeny.     

Antioxidant activity of anthocyanin glycoside derivatives evaluated by the inhibition of liposome oxidation

Cyanidin-3-glycosides (arabinoside, rutinoside, galactoside and glucoside) and delphinidin-3-rutinoside were examined for their ability to inhibit lipid peroxidation induced either by Fe(II) ions, UV irradiation or 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) peroxyl radicals in a liposomal membrane system. The antioxidant abilities of anthocyanins were compared with a water-soluble tocopherol derivative, trolox. The antioxidant efficacies of these compounds were evaluated by their ability to inhibit the fluorescence intensity decay of the extrinsic probe 3-[p-(6-phenyl)-1,3,5,-hexatrienyl] phenylpropionic acid, caused by the free radicals generated during peroxidation. All the anthocyanins tested (at concentrations of 15-20 microM) exhibited higher antioxidant activities against Fe(II)-induced peroxidation than UV- and AAPH-induced peroxidation, suggesting that metal chelation may play an important role in determining the antioxidant potency of these compounds. It was also found that delphinidin-3-rutinoside had a higher antioxidant activity against Fe(II)-induced liposome oxidation than cyanidin-3-rutinoside, which indicates an important role of the OH group in the B ring of delphinidin-3-rutinoside in its antioxidant action. The antioxidant activity of all the anthocyanins studied was higher than that of trolox in the case of Fe(II)-induced liposome oxidation and was comparable with the action of trolox in the case of UV- and AAPH-induced liposome membrane oxidation.