Thin layer chromatography was performed on Merck TLC plates (60F254, 0.2 mm) using an appropriate solvent system. The chromatograms were visualized under UV light. Most of the products crystallized in ethanol. All solvents and liquid reagents were dried with appropriate reagents before use. Commercially available 2-furfural, Maldrum's acid, diethylamines, polyethyleneimine (branched), were purchased from Sigma Aldrich. Laboratory grade solvents like ethanol, THF, DCM and material polycarbonate were used without any pre-treatment.
Hematopoietic stem cell transplantation is successfully applied since the late 1950s; however, its efficacy still needs to be increased. A promising strategy is to transplant high numbers of pluripotent hematopoietic stem cells (HSCs). Therefore, an improved ex vivo culture system that supports proliferation and maintains HSC pluripotency would override possible limitations in cell numbers gained from donors. To model the natural HSC niche in vitro, we optimized the HSC medium composition with a panel of cytokines and valproic acid and used an artificial 3D bone marrow-like scaffold made of polydimethylsiloxane (PDMS). This 3D scaffold offered a suitable platform to amplify human HSCs in vitro and, simultaneously, to support their viability, multipotency and ability for self-renewal. Silicon oxide-covering of PDMS structures further improved amplification of CD34+ cells, although the conservation of naïve HSCs was better on non-covered 3D PDMS. Finally, we found that HSC cultivated on non-covered 3D PDMS generated most pluripotent colonies within colony forming unit assays. In conclusion, by combining biological and biotechnological approaches, we optimized in vitro HSCs culture conditions, resulting in improved amplification, multipotency maintenance and vitality of HSCs.
Combining modern methods in microsystem technology with the latest advancements in the life sciences, namely those in tissue engineering and advanced cell culturing, is promoting the development of a promising toolbox for modeling biological systems. The core problem to solve using this toolbox is the design of 3D artificial cellular environments, both in fluidic systems and on solid substrates. The construction of 3D biological fluidic environments involves the use of microfluidic devices where fluid direction and behavior can be tightly regulated in a geometrically constrained environment for advanced cell cultivation. This is used in modern cultivation devices, such as bioreactors and multicompartment systems, including systems with integrated multielectrode arrays in both 2D and 3D. The construction of 3D cell cultures on substrates involves various fabrication techniques that use different polymers and biopolymers processed by micromachining, chemical pattern guided cell cultivation, photopolymerization, and organ printing methods. These methods together have the potential to create an artificial system with the complete hierarchical, geometrical, and functional organization found in an actual biological system. In this review, we describe representative developments in this research area and the fusion of formerly unrelated disciplines that are generating new beneficial applications in life sciences.
A biocompatible cell culture environment that enables continued existence of three dimensionally aggregated cells in a polycarbonate-based scaffold structure was developed. A micro structured polymeric scaffold allows perfusion of cells due to a microporous structure generated by ion track etching and micro thermoforming. Biocompatibility and sterilizability was approved for the whole system. As oxygenation and mass transport within a closed system is most relevant for 3-D cell culture, two approaches of pumping systems were tested. The human hepatocarcinoma cell line HepG2 was used to examine basic cytological parameters in response to the enviroment. Our data indicate that an actively perfused 3-D cell culture induces a more differentiated phenotype in HepG2 cells than the 2-D setup. Thus, our results provide further support to the theory that 3-D-cultivated cells display a non-proliferative behavior. In this respect, 3-D cultures resemble in vivo conditions more closely. Microreactors are widely applied for organic syntheses, but can also be used for screening applications in drug discovery and medical research. The bioreactor versions presented here were equipped with active fluidic components.
Microsystems recently have been introduced as tools for screening in modern chemistry, biochemistry and biology. It has been shown that new microsystems can be implemented in the biomedical laboratory by using the microsystemic approach for the sample carrier -the miniaturized microtiter plate (''the nanotiter plate'') -or the production of nanodroplets with ink jetters and to integrate those systems in macrodevices like xyz tables and detection devices like CCD-cameras. We show in this paper that decisive problems of the approach -the evaporation problem and the problem of chemical/biochemical/biological compatibility of the assays and the used materials can be solved successfully. It is possible to realize chemical synthesis in miniaturized flow systems and to perform isothermal amplification of RNA in silicon wafers. Furthermore real high throughput screening with in vivo systems can be performed and all relevant parameters as evaporation, pipetting and detection can be controlled on reasonable time scales.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.