In the study of embryo development process, the morphological features at different stages are essential to evaluate developmental competence of the embryo, which can be used to optimize and improve the system for
Blastocyst ; Embryo Culture Techniques ; Embryonic Development ; Fertilization in Vitro

vitro are varied, and their effects are still inconclusive. In this study, we compared the effects of culture systems with undefined (foetal bovine serum) and defined (KnockOut Serum Replacement) materials on the in vitro culture of buffalo SSC-like cells. Significantly more DDX4- and UCHL1-positive cells (cultured for 2 days at passage 2) were observed in the defined materials culture system than in the undefined materials system (p < 0.01), and these cells were maintained for a longer period than those in the culture system with undefined materials (10 days vs. 6 days). Furthermore, NANOS2 (p < 0.05), DDX4 (p < 0.01) and UCHL1 (p < 0.05) were expressed at significantly higher levels in the culture system with defined materials than in that with undefined materials. Induction with retinoic acid was used to verify that the cultured cells maintained SSC characteristics, revealing an SCP3⁺ subset in the cells cultured in the defined materials system. The expression levels of Stra8 (p < 0.05) and Rec8 (p < 0.01) were significantly increased, and the expression levels of ZBTB16 (p < 0.01) and DDX4 (p < 0.05) were significantly decreased. These findings provided a clearer research platform for exploring the mechanism of buffalo SSCs in vitro.]]>
Buffaloes ; Cells, Cultured ; In Vitro Techniques ; Primary Cell Culture ; Stem Cells ; Tretinoin

BACKGROUND: Tissue engineering is a multidisciplinary field which attracted much attention in recent years. One of the most important issue in tissue engineering is how to obtain high cell numbers and tissue regeneration while maintaining appropriate cellular characteristics in vitro for restoring damaged or dysfunctional body tissues and organs. These demands can be achieved by the use of three dimensional (3D) dynamic cultures of cells combined with cell-adhesive micro-carriers. METHODS: In this study, human mesenchymal stem cells (hMSCs) were cultured in a wave-bioreactor system for up to 100 days, after seeding on Cultisphere-S porous gelatin micro-carriers. Cell counting was performed at the time points of 7, 12, 17, 31 days and compared to those of hMSCs cultured under static condition. Higher growth and proliferation rates was achieved in wave-type dynamic culture, when cell culture continued to day 31. A scanning electron microscope (SEM) photographs, both live and dead and MTT assays were taken to confirm the survival and distribution of cells on porous gelatin micro-carrier surfaces. The results of histological stains such as hematoxylin and eosin, Masson’s trichrome, Alcian blue and Alizarin red S also showed improved proliferation and tissue regeneration of hMSCs on porous gelatin micro-carriers. CONCLUSION: The experimental results demonstrated the effect and importance of both micro-carriers and bioreactor in hMSC expansion on cell proliferation and migration as well as extracellular matrix formation on the superficial and pore surfaces of the porous gelatin micro-carriers, and then their inter-connections, leading to tissue regeneration.
Alcian Blue ; Bioreactors ; Cell Count ; Cell Culture Techniques ; Cell Proliferation ; Coloring Agents ; Eosine Yellowish-(YS) ; Extracellular Matrix ; Gelatin ; Hematoxylin ; Humans ; In Vitro Techniques ; Mesenchymal Stromal Cells ; Regeneration ; Tissue Engineering

The development of long-term surviving fetal cell cultures from primary cell tissue from the developing brain is important for facilitating studies investigating neural development and for modelling neural disorders and brain congenital defects. The field faces current challenges in co-culturing both progenitors and neurons long-term. Here, we culture for the first time, porcine fetal cells from the dorsal telencephalon at embryonic day (E) 50 and E60 in conditions that promoted both the survival of progenitor cells and young neurons. We applied a novel protocol designed to collect, isolate and promote survival of both progenitors and young neurons. Herein, we used a combination of low amount of fetal bovine serum, together with pro-survival factors, including basic fibroblast growth factor and retinoic acid, together with arabinofuranosylcytosine and could maintain progenitors and facilitate in vitro differentiation into calbindin 1+ neurons and reelin+ interneurons for a period of 7 days. Further improvements to the protocol that might extend the survival of the fetal primary neural cells would be beneficial. The development of new porcine fetal culture methods is of value for the field, given the pig's neuroanatomical and developmental similarities to the human brain.
Brain ; Calbindins ; Cell Culture Techniques ; Congenital Abnormalities ; Cytarabine ; Fibroblast Growth Factor 2 ; Humans ; In Vitro Techniques ; Interneurons ; Neurons ; Stem Cells ; Telencephalon ; Tretinoin

BACKGROUND: AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that plays a pivotal role in the balance of cellular energy metabolism. Recent studies have reported that AMPK has numerous roles in physiological conditions, and dysregulation of AMPK induces pathological processes and diseases. However, the role of AMPK and its activators have not yet been studied in the context of hair growth regulation. OBJECTIVE: To investigate the effects of metformin on dermal papilla (DP) and outer root sheath (ORS) cells, as well as the role of the AMPK pathway in hair growth. METHODS: We evaluated whether metformin, a well-known AMPK activator, had any beneficial effects on hair growth. In addition, to evaluate the molecular and cellular mechanisms that were involved, protein levels of AMPK and β-catenin were analyzed. RESULTS: Metformin increased the cellular proliferation of human DP and ORS cells. Ki-67 expression was also significantly increased after metformin treatment in the ex vivo hair follicle organ culture. Furthermore, DP and ORS cells treated with metformin had a significant increase in AMPK phosphorylation, which in turn suppressed β-catenin degradation and enhanced its nuclear accumulation. CONCLUSION: We demonstrated that metformin promoted hair growth via the AMPK/β-catenin signaling pathway in vitro with DP and ORS cells. The hair-promoting effects of AMPK activators may potentially be used for the treatment of alopecia, and further investigation will be needed in the future.
Alopecia ; AMP-Activated Protein Kinases ; beta Catenin ; Cell Proliferation ; Energy Metabolism ; Hair Follicle ; Hair ; Humans ; In Vitro Techniques ; Metformin ; Organ Culture Techniques ; Pathologic Processes ; Phosphorylation ; Protein Kinases

BACKGROUND: Nano-hydroxyapatite/polyamide 66 (nHA/PA66) is a composite used widely in the repair of bone defects. However, this material is insufficient bioactivity. In contrast, D-RADA16-RGD self-assembling peptide (D-RADA16-RGD sequence containing all D-amino acids is Ac-RADARADARADARADARGDS-CONH2) shows admirable bioactivity for both cell culture and bone regeneration. Here, we describe the fabrication of a favorable biomaterial material (nHA/PA66/D-RADA16-RGD). METHODS: Proteinase K and circular dichroism spectroscopy were employed to test the stability and secondary structural properties of peptide D-RADA16-RGD respectively. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to characterize the surface of these materials. Confocal laser scanning (CLS), cell counting kit-8 tests (CCK-8), alizarin red S staining, cell immunofluorescence analysis and Western blotting were involved in vitro. Also biosafety and bioactivity of them have been evaluated in vivo. RESULTS: Proteinase K and circular dichroism spectroscopy demonstrated that D-RADA16-RGD in nHA/PA66 was able to form stable-sheet secondary structure. SEM and TEM showed that the D-RADA16-RGD material was 7–33 nm in width and 130–600 nm in length, and the interwoven pore size ranged from 40 to 200 nm. CLS suggests that cells in nHA/PA66/D-RADA16-RGD group were linked to adjacent cells with more actin filaments. CCK-8 analysis showed that nHA/PA66/D-RADA16-RGD revealed good biocompatibility. The results of Alizarin-red S staining and Western blotting as well as vivo osteogenesis suggest nHA/PA66/D-RADA16-RGD exhibits better bioactivity. CONCLUSION: This study demonstrates that our nHA/PA66/D-RADA16-RGD composite exhibits reasonable mechanical properties, biocompatibility and bioactivity with promotion of bone formation.
Actin Cytoskeleton ; Blotting, Western ; Bone Regeneration ; Cell Count ; Cell Culture Techniques ; Circular Dichroism ; Endopeptidase K ; Fluorescent Antibody Technique ; In Vitro Techniques ; Microscopy, Electron, Scanning ; Microscopy, Electron, Transmission ; Osteogenesis ; Sincalide ; Spectrum Analysis

PURPOSE: Almost all liver diseases are known to be accompanied by increased levels of reactive oxygen species (ROS), regardless of the cause of the liver disorder. However, little is known about the role of hypoxic conditioned media (HCM) in the view of pro-oxidative/antioxidative balance. METHODS: Normoxic conditioned media (NCM) and HCM were obtained after culturing adipose-derived stem cells in 20% O₂ or 1% O₂ for 24 hours, respectively. Their effects on the expression of various markers reflecting pro-oxidative/antioxidative balance were investigated in both in vitro (thioacetamide-treated AML12 cells) and in vivo (partially hepatectomized mice) models of liver injury, respectively. RESULTS: HCM treatment induced the higher expression of antioxidant enzymes, such as superoxide dismutase, glutathione peroxidase, and catalase than did NCM in the in vitro model of liver injury. We also found that HCM increased the expression of nuclear factor erythroid 2-related factor (NRF2). The in vivo models of liver injury consistently validated the phenomenon of upregulated expression of antioxidant enzymes by HCM. CONCLUSION: We thus could conclude that HCM provides protection against ROS-related toxicity by increasing the expression of antioxidant enzymes, in part by releasing NRF2 in the injured liver.
Antioxidants ; Catalase ; Culture Media, Conditioned ; Glutathione Peroxidase ; In Vitro Techniques ; Liver ; Liver Diseases ; Mesenchymal Stromal Cells ; Reactive Oxygen Species ; Stem Cells ; Superoxide Dismutase

BACKGROUND: The relationship between obstructive sleep apnoea (OSA) and metabolic disorders is complex and highly associated. The impairment of adipogenic capacity in pre-adipocytes may promote adipocyte hypertrophy and increase the risk of further metabolic dysfunction. We hypothesize that intermittent hypoxia (IH), as a pathophysiologic feature of OSA, may regulate adipogenesis by promoting macrophage polarization. METHODS: Male C57BL/6N mice were exposed to either IH (240 seconds of 10% O₂ followed by 120 seconds of 21% O₂, i.e., 10 cycles/hour) or intermittent normoxia (IN) for 6 weeks. Stromal-vascular fractions derived from subcutaneous (SUB-SVF) and visceral (VIS-SVF) adipose tissues were cultured and differentiated. Conditioned media from cultured RAW 264.7 macrophages after air (Raw) or IH exposure (Raw-IH) were incubated with SUB-SVF during adipogenic differentiation. RESULTS: Adipogenic differentiation of SUB-SVF but not VIS-SVF from IH-exposed mice was significantly downregulated in comparison with that derived from IN-exposed mice. IH-exposed mice compared to IN-exposed mice showed induction of hypertrophic adipocytes and increased preferential infiltration of M1 macrophages in subcutaneous adipose tissue (SAT) compared to visceral adipose tissue. Complementary in vitro analysis demonstrated that Raw-IH media significantly enhanced inhibition of adipogenesis of SUB-SVF compared to Raw media, in agreement with corresponding gene expression levels of differentiation-associated markers and adipogenic transcription factors. CONCLUSION: Low frequency IH exposure impaired adipogenesis of SAT in lean mice, and macrophage polarization may be a potential mechanism for the impaired adipogenesis.
Adipocytes ; Adipogenesis ; Animals ; Anoxia ; Culture Media, Conditioned ; Gene Expression ; Humans ; Hypertrophy ; In Vitro Techniques ; Inflammation ; Intra-Abdominal Fat ; Macrophages ; Male ; Mice ; Subcutaneous Fat ; Transcription Factors

The mammalian intestine contains many different cell types but is comprised of 2 main cell types: epithelial cells and smooth muscle cells. Recent in vivo and in vitro evidence has revealed that various alterations to the DNA methylation apparatus within both of these cell types can result in a variety of cellular phenotypes including modified differentiation status, apoptosis, and uncontrolled growth. Methyl groups added to cytosines in regulatory genomic regions typically act to repress associated gene transcription. Aberrant DNA methylation patterns are often found in cells with abnormal growth/differentiation patterns, including those cells involved in burdensome intestinal pathologies including inflammatory bowel diseases and intestinal pseudo-obstructions. The altered methylation patterns being observed in various cell cultures and DNA methyltransferase knockout models indicate an influential connection between DNA methylation and gastrointestinal cells' development and their response to environmental signaling. As these modified DNA methylation levels are found in a number of pathological gastrointestinal conditions, further investigations into uncovering the causative nature, and controlled regulation, of this epigenetic modification is of great interest.
Apoptosis ; Cell Culture Techniques ; Cell Differentiation ; DNA Methylation ; DNA ; Epigenomics ; Epithelial Cells ; In Vitro Techniques ; Inflammatory Bowel Diseases ; Intestinal Mucosa ; Intestinal Pseudo-Obstruction ; Intestines ; Methylation ; Muscle, Smooth ; Myocytes, Smooth Muscle ; Pathology ; Phenotype

PURPOSE: The reaction of cells to a titanium implant depends on the surface characteristics of the implant which are affected by decontamination. The aim of this study was to evaluate the cytocompatibility of titanium disks treated with various decontamination methods, using salivary bacterial contamination with dental pellicle formation as an in vitro model. METHODS: Sand-blasted and acid-etched (SA) titanium disks were used. Three control groups (pristine SA disks [SA group]; salivary pellicle-coated SA disks [pellicle group]; and biofilm-coated, untreated SA disks [NT group]) were not subjected to any decontamination treatments. Decontamination of the biofilm-coated disks was performed by 14 methods, including ultrasonic instruments, rotating instruments, an air-powder abrasive system, a laser, and chemical agents. MG63 cells were cultured in the presence of the treated disks. Cell proliferation assays were performed on days 2 and 5 of cell culture, and cell morphology was analyzed by immunofluorescence and scanning electron microscopy (SEM). A vascular endothelial growth factor (VEGF) assay was performed on day 5 of culture. RESULTS: The cell proliferation assay revealed that all decontaminated disks, except for the 2 groups treated using a plastic tip, showed significantly less cell proliferation than the SA group. The immunofluorescence and SEM analyses revealed that most groups showed comparable cell density, with the exception of the NT group, in which the cell density was lower and bacterial residue was observed. Furthermore, the cells grown with tetracycline-treated titanium disks showed significantly lower VEGF production than those in the SA group. CONCLUSIONS: None of the decontamination methods resulted in cytocompatibility similar to that of pristine SA titanium. However, many methods caused improvement in the biocompatibility of the titanium disks in comparison with the biofilm-coated, untreated titanium disks. This suggests that decontamination is indispensable for the treatment of peri-implantitis, even if the original biocompatibility cannot be restored.
Biocompatible Materials ; Cell Count ; Cell Culture Techniques ; Cell Proliferation ; Decontamination ; Dental Implants ; Dental Pellicle ; Fluorescent Antibody Technique ; In Vitro Techniques ; Methods ; Microscopy, Electron, Scanning ; Peri-Implantitis ; Plastics ; Titanium ; Ultrasonics ; Vascular Endothelial Growth Factor A