Development of Polyethylene Glycol-based Hydrogels Optimized for In Vitro 3D Culture of HepG2 Hepatocarcinoma Cells.

BACKGROUND/AIM: We report an in vitro three-dimensional (3D) culture system optimized for the growth of HepG2 hepatocarcinoma cells. MATERIALS AND METHODS: The 3D culture system was fabricated based on polyethylene glycol (PEG)-based hydrogels; their mechanical strength was controlled by differences in the arm number and concentration of PEG-vinylsulfone. Moreover, cellular growth was evaluated after culturing HepG2 cells in PEG-based hydrogels with various mechanical strengths. RESULTS: HepG2 cell culture in the 3D PEG-based hydrogels induced the formation of spherical colonies. Moreover, the highest number of spherical colonies formed from HepG2 cells at the single-cell level, and the formation of spherical colonies with a uniform size was observed in HepG2 cells cultured in 5% (w/v) 8-arm PEG-based hydrogels. CONCLUSION: 5% (w/v) 8-arm PEG-based hydrogels may be developed as a 3D culture system optimized for stimulating the in vitro growth of HepG2 cells.

Association of a single nucleotide polymorphism near the interleukin-28B gene with response to hepatitis C therapy in Asian patients.

BACKGROUND AND AIMS: A single nucleotide polymorphism near the interleukin-28B (IL28B) gene has been shown to predict hepatitis C virus (HCV) treatment response. We aim to determine the role of the IL28B genotype in Asian patients. METHODS: A total of 118 patients (all Korean, 55 patients with genotype 1 infection and 63 patients with genotype 2 infection) were consecutively enrolled and analyzed. RESULTS: The sustained virological response (SVR) rate was 74% (87/118), while 26 patients (22%) relapsed and five patients were non-responders (4%). For rs8099917, the frequencies of major homozygotes (TT), heterozygotes (GT), and minor homozygotes (GG) were 0.85, 0.14 and 0.01, respectively. Of the 55 patients with HCV genotype 1 infection, the SVR rate was 67% and 44% (P = 0.19) and the non-response rate was 2% and 22% (P = 0.015) for the major allele and minor or hetero allele, respectively. Of the 63 patients with HCV genotype 2 infection, the SVR rate was 80% and 100% (P = 0.13) and the non-response rate was 4% and 0% (P = 0.55) for major allele and hetero allele, respectively. CONCLUSIONS: The IL28B genotype may help identify non-responding patients in HCV genotype 1, but not in HCV genotype 2. Because of the high frequency of favorable alleles and the low frequency of non-response, the IL28B polymorphism may play a smaller role in Asian patients.

Cornelia de Lange Syndrome with NIPBL gene mutation: a case report.

Cornelia de Lange Syndrome (CdLS) is a multiple congenital anomaly characterized by distinctive facial features, upper limb malformations, growth and cognitive retardation. The diagnosis of the syndrome is based on the distinctive clinical features. The etiology is still not clear. Mutations in the sister chromatid cohesion factor genes NIPBL, SMC1A (also called SMC1L1) and SMC3 have been suggested as probable cause of this syndrome. We experienced a case of newborn with CdLS showing bushy eyebrows and synophrys, long curly eyelashes, long philtrum, downturned angles of the mouth and thin upper lips, cleft palate, micrognathia, excessive body hair, micromelia of both hands, flexion contracture of elbows and hypertonicity. We detected a NIPBL gene mutation in a present neonate with CdLS, the first report in Korea.

Difference in suitable mechanical properties of three-dimensional, synthetic scaffolds for self-renewing mouse embryonic stem cells of different genetic backgrounds.

We evaluated whether the genetic background of embryonic stem cells (ESCs) affects the properties suitable for three-dimensional (3D) synthetic scaffolds for cell self-renewal. Inbred R1 and hybrid B6D2F1 mouse ESC lines were cultured for 7 days in hydrogel scaffolds with different properties derived from conjugating 7.5, 10, 12.5, or 15% (wt/vol) vinylsulfone-functionalized three-, four-, or eight-arm polyethylene glycol (PEG) with dicysteine-containing crosslinkers with an intervening matrix metalloproteinase-specific cleavage sites. Cell proliferation and expression of self-renewal-related genes and proteins by ESCs cultured in feeder-free or containing 2D culture plate or 3D hydrogel were monitored. As a preliminary experiment, the E14 ESC-customized synthetic 3D microenvironment did not maintain self-renewal of either the R1 or B6D2F1 ESCs. The best R1 cell proliferation (10.04 vs. 0.16-4.39; p < 0.0001) was observed in the four-arm 7.5% PEG-based hydrogels than those with other properties, whereas the F1 ESCs showed better proliferation when they were embedded in the three-arm 10% hydrogels. Self-renewal-related gene and protein expression by ESCs after feeder-free 3D culture was generally maintained compared with the feeder-containing 2D culture, but expression patterns and quantities differed. However, the feeder-free 3D culture yielded better expression than the feeder-free 2D culture. In conclusion, genetic background determined the suitability of hydrogel scaffolds for self-renewal of ESCs, which requires customization for the mechanical properties of each cell line. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2261-2268, 2017.

Reconstruction with fibular osteocutaneous free flap in patients with mandibular osteoradionecrosis.

BACKGROUND: Osteoradionecrosis is a delayed complication from radiation therapy which causes chronic pain, infection and constant deformity after necrosis. Most of the osteoradionecrosis occurs spontaneously or after the primary oncologic surgery, dental extraction or by trauma of prosthesis. The treatment of osteoradionecrosis relies on both conservative measures and surgical measures. The fibular osteocutaneous free flap has become more popular choice for reconstruction of maxillofacial defects as a treatment of osteoradionecrosis. METHODS: We presented our experiences from 7 patients with osteoradionecrosis who have had reconstruction surgery with fibular osteocutaneous free flap at National Cancer Center during the recent 5 years. We performed segmental mandibular resection with fibular osteocutaneous free flap for all 7 patients of advanced osteoradionecrosis who were not controlled by conservative treatment such as wound irrigation, debridement, and antibiotics. RESULTS: A wide range of techniques were available for the reconstruction of composite defects resulted from the treatment of advanced mandibular osteoradionecrosis. Significant improvement was noted in relieving pain and treating trismus after the surgery however difficulty in swallowing and xerostomia showed less improvement. CONCLUSIONS: We concluded that fibular osteocutaneous free flap can be performed safely in patients with osteoradionecrosis and yields positive outcomes with significantly increased success rate. The fibular osteocutaneous free flap was our preferred choice for the mandibular reconstruction due to its versatility and predictability.

Culture of preantral follicles in poly(ethylene) glycol-based, three-dimensional hydrogel: a relationship between swelling ratio and follicular developments.

This study was undertaken to examine how the softness of poly(ethylene) glycol (PEG)-based hydrogels, creating a three-dimensional (3D) microenvironment, influences the in vitro growth of mouse ovarian follicles. Early secondary, preantral follicles of 2 week-old mice were cultured in a crosslinked four-arm PEG hydrogel. The hydrogel swelling ratio, which relates to softness, was modified within the range 25.7-15.5 by increasing the reactive PEG concentration in the precursor solution from 5% to 15% w/v, but it did not influence follicular growth to form the pseudoantrum (60-80%; p = 0.76). Significant (p < 0.04) model effects, however, were detected in the maturation and developmental competence of the follicle-derived oocytes. A swelling ratio of > 21.4 yielded better oocyte maturation than other levels, while the highest competence to develop pronuclear and blastocyst formation was detected at 20.6. In conclusion, gel softness, as reflected in swelling ratio, was one of the essential factors for supporting folliculogenesis in vivo within a hydrogel-based, 3D microenvironment.

Development of three dimensional culture and expression of integrin heterodimers in human embryonic stem cells.

Extensive effort has been made to develop a three-dimensional (3D) system for the culture of pluripotent stem cells in human and model animals, which yields lots of benefits for monitoring cell-to-cell or cell-to-environment interaction and for suggesting alternative materials for clinical cases. Initial study using animal model moved toward supporting embryonic stem cells (ESCs) self-renewal in a synthetic scaffold conjugated with suitable peptide motifs. As results, the feeder-free, 3D acellular niche consisting of vinyl sulfone (VS)-functionalized polyethylene glycol (PEG)-based hydrogel binding with extracellular matrix analogs could support ESC self-renewal, but main stemness signals were switched in the 3D environment. We employed this PEG-based hydrogel for 3D culture of human ESCs and further adjustment of hydrogel constituent made it possible to support self-renewal of three ESC lines. In this study, we examined transcriptional and translational activity of integrin heterodimers for optimizing the 3D system by using peptide motifs and subsequently elucidated that transcription and translation of integrin α 5ß 1, α 6ß 1 and αVß 5 were stronger than other heterodimers in a referenced human ESC line.

A feeder-free, defined three-dimensional polyethylene glycol-based extracellular matrix niche for culture of human embryonic stem cells.

We report optimization of a serum- and feeder-free, three-dimensional (3D) niche created with a synthetic polyethylene glycol (PEG)-based extracellular matrix for self-renewal of human embryonic stem cells (hESCs). Three hESC lines (H9, H1 and Novo) were cultured in hydrogels of different mechanical properties, and cellular morphology and activity were compared to culture in feeder-free or feeder-containing two-dimensional (2D) niches. Significant effects of PEG concentration (5, 7.5, 10, 12.5 or 15%) and vinyl sulfone-functionalized PEG multiarm number (3, 4 or 8) on hESC morphology were detected in the H9 line. Cell growth was maximal with an 8 multiarm architecture of any PEG concentration, which yielded the highest expression of stemness-related genes. Alkaline phosphatase activity in cultured H9 cells was similar between the optimized feeder-free 3D and the feeder-containing 2D systems. However, increased expression of the KLF4, CDH1, TERT, SOX2, and UTF1 genes and expression of pluripotency-specific SSEA-4, Oct3/4, Nanog, Tra-1-60 and Tra-1-81 were detected in the 3D-cultured hESC clumps. H1 and Novo cell lines also expanded in the optimized 3D system, which maintain stemness properties. Although different proliferation activities were detected among three lines, the difference was decreased after the 3D culture. These results demonstrate that chemically defined, acellular niches created using PEG-based hydrogels have the potential to support hESC self-renewal. Modulation of 3D properties can create various models for cell transformation and differentiation.