Cell-repellent polyampholyte for conformal coating on microstructures.

Repellent coatings are critical for the development of biomedical and analytical devices to prevent nonspecific protein and cell adhesion. In this study, prevelex (polyampholytes containing phosphate and amine units) was synthesized for the fine coating of microdevices for cell culture. The dip-coating of the prevelex on hydrophobic substrates altered their surfaces to be highly hydrophilic and electrically neutral. The range of prebake temperature (50-150 °C) after dip-coating was moderate and within a preferable range to treat typical materials for cell culture such as polystyrene and polydimethylsiloxane. Scanning electron microscopy revealed a conformal and ultra-thin film coating on the micro/nano structures. When compared with poly(2-hydroxyethyl methacrylate) and poly(2-methacryloyloxyethyl phosphorylcholine), prevelex exhibited better characteristics for coating on microwell array devices, thereby facilitating the formation of spheroids with uniform diameters using various cell types. Furthermore, to examine cellular functionalities, mouse embryonic epithelial and mesenchymal cells were seeded in a prevelex-coated microwell array device. The two types of cells formed hair follicle germ-like aggregates in the device. The aggregates were then transplanted to generate de novo hair follicles in nude mice. The coating material provided a robust and fine coating approach for the preparation of non-fouling surfaces for tissue engineering and biomedical applications.

Oriented immobilization of rBC2LCN lectin for highly sensitive detection of human pluripotent stem cells using cell culture supernatants.

Human pluripotent stem cells (hPSCs) are considered ideal cell sources for regenerative medicine, but their clinical and industrial applications are hindered by their tumorigenic potential. We previously identified an hPSC-specific lectin, rBC2LCN, that recognizes the podocalyxin glycoprotein secreted by undifferentiated hPSCs into the culture media. Using biotinylated rBC2LCN and a peroxidase-labeled R-10G antibody, we developed a sandwich assay for the detection of tumorigenic hPSCs. In this assay, the lectin is randomly immobilized on streptavidin-coated microplates to capture hPSC-derived podocalyxin. In the present study, rBC2LCN was genetically fused with polystyrene-binding peptides (PS-tags) for direct, site-specific, and oriented immobilization on polystyrene microplates. rBC2LCN lectins fused with PS-tags at the C-terminus were successfully overexpressed as a soluble form in Escherichia coli and then purified by affinity chromatography. We optimized the various parameters (protein and NaCl concentration, buffer pH, and blocking agents) of the sandwich assay by using PS-tagged rBC2LCN and the R-10G antibody. Finally, the lower limit of detection (LLOD) of the sandwich assay for hPSCs was examined. The LLOD was 2.2-fold lower than that achieved with the previous method. Considering that the developed method does not require the precoating of polystyrene microplates with streptavidin, it provides a cost-effective approach for the highly sensitive detection of hPSCs residing in hPSC-derived cell therapeutics.

Preservation of Memory Despite Unresected Contralateral Hippocampal Volume Loss After Resection of Hippocampal Sclerosis in Seizure-Free Patients.

OBJECTIVE: To determine postoperative long-term changes of hippocampal volume (HV) correlating with cognitive functions in patients who underwent surgery for hippocampal sclerosis with postoperative freedom from seizures. METHODS: We studied 1.5T magnetic resonance imaging before and after surgery in 24 patients (mean ± SD age, 36.9 ± 11.0 years) with hippocampal sclerosis. We performed serial magnetic resonance imaging at 6 months to 1 year, 1-2 years, 2-3 years, and 3-5 years postoperatively. We compared HVs of 24 patients with HVs of 14 age-matched control subjects. We analyzed correlations between consecutive HVs and seizure duration and age at surgery. We compared consecutive changes in HVs between dominant and nondominant hemispheres with concurrent cognitive functions. RESULTS: Preoperative HVs of unresected contralateral hippocampus were significantly smaller than HVs of control subjects (P < 0.01). Unresected contralateral HV changes compared with preoperative HVs were -3.6% ± 6.9%, -2.3% ± 8.5%, -3.6% ± 10.2% (P < 0.05), and -5.0% ± 9.5% (P < 0.05) at consecutive postoperative periods. Largest change in HVs at 3-5 years was significantly correlated with older age at surgery (P < 0.05). Unresected contralateral dominant 14 HVs remained consistently smaller than nondominant 10 HVs up to 5 years with statistical significance (P < 0.05). Verbal memory was preserved in 14 patients with unresected contralateral smaller dominant hippocampus. CONCLUSIONS: In seizure-free patients after hippocampal sclerosis resection , unresected contralateral HV significantly declined with older age at surgery. Visual memory was preserved regardless of side and volume loss. Despite significantly reduced HVs, verbal memory was preserved with the unresected contralateral dominant hippocampus. Earlier surgical intervention may have lower potential risk for memory decline secondary to postoperative HV loss.

Identification and characterization of polydimethylsiloxane-binding peptides (PDMS-tag) for oriented immobilization of functional protein on a PDMS surface.

In this study we focused on identifying and characterizing polydimethylsiloxane-binding peptides (PDMS-tags) that show a strong binding affinity towards a PDMS surface. Three kinds of E. coli host proteins (ELN, OMC and TPA) that were preferentially adsorbed onto a PDMS surface were identified from the E. coli cell lysate via 2-D electrophoresis and MALDI TOF MS. Digestion of these PDMS-binding proteins by 3 types of proteases (trypsin, chymotrypsin and V8 protease) resulted in the production of a wide variety of peptide fragments with different amino acid biases. Nine types of peptide fragments showing binding affinities to a PDMS surface were identified, and they were genetically fused at the C-terminal region of glutathione S-transferase (GST). The adsorption kinetics of peptide-fused GSTs to a PDMS surface were evaluated using a quartz crystal microbalance (QCM) sensor equipped with a sensor chip coated with a PDMS thin film. Consequently, all GSTs fused with the peptides adsorbed at a level higher than that of wild-type GST. In particular, the adsorption levels of GSTs fused with ELN-V81, TPA-V81, and OMC-V81 peptides were 8- to 10-fold higher than that of the wild-type GST. These results indicated that the selected peptides possessed a strong binding affinity towards a PDMS surface even in cases where they were introduced to the C-terminal region of a model protein. The remaining activities of GSTs with PDMS-binding peptides were also greater than that of the wild-type GST. Almost a third (30%) of enzymatic activity was maintained by genetic fusion of the peptide ELN-V81, compared with only 1.5% of wild-type GST in the adsorption state. Thus, the PDMS-binding peptides (PDMS-tags) identified in this study will be considerably useful for the site-specific immobilization of functional proteins to a PDMS surface, which will be a powerful tool in the fabrication of protein-based micro-reactors and biosearation chips.