Bioengineering with utilization of cells as one of the components of devices has been expected to advance developments of medical and pharmaceutical technologies. When cells are engineered, it is ...important to establish means for maintaining the activity of the cells, enhancing cell functions, and controlling cell responses. This review summarizes researches for cell encapsulation using synthetic phospholipid polymers composed of 2-methacryloyloxyethyl phosphorylcholine unit, which make hydrogel spontaneously in a cell culture environment and then cells are preserved in situ. The phospholipid polymer hydrogels show no adverse effects on the cell culture process and the mechanical properties of the hydrogels can regulate for controlling the function of cells. It also introduces molecular designs that can be easily recovered from the hydrogel matrix after the encapsulated cells have differentiated. Furthermore, the application of these hydrogels to a microdevice also describes advanced utilization of cultured cells. Phospholipid polymer hydrogels can exhibit its function even when they are applied in vivo, and as one application, introduces the prevention of adhesion with other tissues in the tissue healing process. That is, the potential application of the phospholipid polymer hydrogels in cell engineering are described.
To develop a reversible forming polymer network system, a polymer complexation was conducted between water-soluble and cytocompatible polymers, namely poly(2-methacryloyloxyethyl ...phosphorylcholine-co-n-butyl methacrylate-co-p-vinylphenylboronic acid) (PMBV) and poly(vinyl alcohol) (PVA). The two polymers spontaneously form hydrogel upon mixing. The association constant depended on the chemical structure of the polyol compounds and the aggregation of the PMBV. Compared to the association constants of PVA to PMBV, d-glucose and D-sorbitol have smaller and larger association constants, respectively, which is in good accord with the dissociation phenomenon of PMBV/PVA hydrogels. In conclusion, consideration of the association constants between the PMBV and polyol compounds is important for obtaining good reversibility of a polymer hydrogel system.
In the nucleus of eukaryotic cells, chromatin is tethered to the nuclear envelope (NE), wherein inner nuclear membrane proteins (INMPs) play major roles. However, in Xenopus blastula, chromatin ...tethering to the NE depends on nuclear filamentous actin that develops in a blastula-specific manner. To investigate whether chromatin tethering operates in the blastula through INMPs, we experimentally introduced INMPs into Xenopus egg extracts that recapitulate nuclear formation in fertilized eggs. When expressed in extracts in which polymerization of actin is inhibited, only lamin B receptor (LBR), among the five INMPs tested, tethered chromatin to the NE, depending on its N2 and N3 domains responsible for chromatin-protein binding. N2-3-deleted LBR did not tether chromatin, although it was localized in the nuclei. We subsequently found that the LBR level was very low in the Xenopus blastula but was elevated after the blastula stage. When the LBR level was precociously elevated in the blastula by injecting LBR mRNA, it induced alterations in nuclear lamina architecture and nuclear morphology and caused DNA damage and abnormal mitotic spindles, depending on the N2-3 domains. These results suggest that LBR-mediated chromatin tethering is circumvented in the Xenopus blastula, as it is detrimental to embryonic development.
To expand the toolbox of imaging in living cells, we have engineered a single-chain variable fragment binding the linear HA epitope with high affinity and specificity in vivo. The resulting probe, ...called the HA frankenbody, can light up in multiple colors HA-tagged nuclear, cytoplasmic, membrane, and mitochondrial proteins in diverse cell types. The HA frankenbody also enables state-of-the-art single-molecule experiments in living cells, which we demonstrate by tracking single HA-tagged histones in U2OS cells and single mRNA translation dynamics in both U2OS cells and neurons. Together with the SunTag, we also track two mRNA species simultaneously to demonstrate comparative single-molecule studies of translation can now be done with genetically encoded tools alone. Finally, we use the HA frankenbody to precisely quantify the expression of HA-tagged proteins in developing zebrafish embryos. The versatility of the HA frankenbody makes it a powerful tool for imaging protein dynamics in vivo.
Abstract We considered that properties of the microenvironment surrounding cells are important for the control of the cell functions. Cytocompatible polymer hydrogels are good candidates to study ...such microenvironment. Here, we prepared spontaneously forming hydrogels composed of two polymer systems, namely poly(2-methacryloyloxyethyl phosphorylcholine -co-n- butyl methacrylate -co-p- vinylphenylboronic acid) (PMBV) and poly(vinyl alcohol) (PVA). The PMBV/PVA hydrogels could be reversibly dissociated by the addition of d -sorbitol. The storage modulus was measured for evaluating the mechanical properties of the PMBV/PVA hydrogels. The storage modulus could be controlled in the range 0.30–2.5 kPa by changing the cross-linking density of the hydrogels. After pluripotent stem cells were encapsulated within the PMBV/PVA hydrogels during the preparation of the hydrogel under normal cell-culturing conditions, the proliferation rate and the cell cycle of the encapsulated cells were observed. Cells lived for more than three days in every PMBV/PVA hydrogel. However, the proliferation significantly depended on the storage modulus of the hydrogels. Although the cell cycle of the initial cells was heterogenous, it developed uniformity toward the G1 phase when the cells were encapsulated within the PMBV/PVA hydrogel with a storage modulus of 1.1 kPa for three days. That is, the mechanical properties of the PMBV/PVA environment influenced the biological functions of the cells encapsulated in the hydrogels. From these results, we conclude that PMBV/PVA hydrogels are useful for adjusting cell cycles and proliferation, thus providing uniform cells for applications in the field of cell engineering.
The Xenopus oocyte is known to accumulate filamentous or F‐actin in the nucleus, but it is currently unknown whether F‐actin also accumulates in embryo nuclei. Using fluorescence‐labeled actin ...reporters, we examined the actin distribution in Xenopus embryonic cells and found that F‐actin accumulates in nuclei during the blastula stage but not during the gastrula stage. To further investigate nuclear F‐actin, we devised a Xenopus egg extract that reproduces the formation of nuclei in which F‐actin accumulates. Using this extract, we found that F‐actin accumulates primarily at the subnuclear membranous region and is essential to maintain chromatin binding to the nuclear envelope in well‐developed nuclei. We also provide evidence that nuclear F‐actin increases the structural stability of nuclei and contributes to chromosome alignment on the mitotic spindle at the following M phase. These results suggest the physiological importance of nuclear F‐actin accumulation in rapidly dividing large Xenopus blastula cells.
While nuclear F‐actin accumulates in Xenopus oocytes, it is unknown whether it does so in embryos. Using fluorescence‐labeled actin reporters and Xenopus egg extracts, we demonstrate that F‐actin accumulates in blastula nuclei, promoting chromatin–nuclear envelope binding, nuclear structural stability and M‐phase mitotic chromosome alignment.
Cell-imaging methods with functional fluorescent probes are an indispensable technique to evaluate physical parameters in cellular microenvironments. In particular, molecular rotors, which take ...advantage of the twisted intramolecular charge transfer (TICT) process, have helped evaluate microviscosity. However, the involvement of charge-separated species in the fluorescence process potentially limits the quantitative evaluation of viscosity. Herein, we developed viscosity-responsive fluorescent probes for cell imaging that are not dependent on the TICT process. We synthesized
and
, both of which contain 9,10-di(piperazinyl)anthracene, based on 9,10-bis(
,
-dialkylamino)anthracene that adopts a nonflat geometry at minimum energy conical intersection.
and
exhibited enhanced fluorescence as the viscosity increased, with sensitivities comparable to those of conventional molecular rotors. In living cell systems,
showed low cytotoxicity and, reflecting its viscosity-responsive property, allowed specific visualization of dense and acidic organelles such as lysosomes, secretory granules, and melanosomes under washout-free conditions. These results provide a new direction for developing functional fluorescent probes targeting dense organelles.
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The development of a compact and affordable fluorescence microscope can be a formidable challenge for growing needs in on-site testing and detection of fluorescent labeled biological ...systems, especially for those who specialize in biology rather than in engineering. In response to such a situation, we present an open-source miniature fluorescence microscope using Raspberry Pi. Our fluorescence microscope, with dimensions of 19.2 × 13.6 × 8.2 cm3 (including the display, computer, light-blocking case, and other operational requirements), not only offers cost-effectiveness (costing less than $500) but is also highly customizable to meet specific application needs. The 12.3-megapixel Raspberry Pi HQ Camera captures high-resolution imagery, while the equipped wide-angle lens provides a field of view measuring 21 × 15 mm2. The integrated wireless LAN in the Raspberry Pi, along with software-controllable high-powered fluorescence LEDs, holds potential for a wide range of applications. This open-source fluorescence microscope offers biohybrid sensor developers a versatile tool to streamline unfamiliar mechanical design tasks and open new opportunities for on-site fluorescence detections.
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