Concentration Of Fetal Bovine Serum Research Articles

Administration of growth factors promotes salisphere formation from irradiated parotid salivary glands.

Worldwide, 500,000 cases of head and neck cancer (HNC) are reported each year and the primary treatment for HNC is radiotherapy. Although the goal of radiotherapy is to target the tumor, secondary exposure occurs in surrounding normal tissues, such as the salivary glands. As a result, despite successful treatment of the cancer, patients are left with long-term side effects due to direct damage to the salivary glands. The effect is chronic and currently there is no treatment. Stem cells are an attractive therapeutic option for treatment of radiation-induced glandular dysfunction because of the potential to regenerate damaged cell populations and restore salivary gland function. However, limited knowledge about the endogenous stem cell population post irradiation hinders the development for stem cell-based therapies. In this study, an ex vivo sphere formation cell culture system was utilized to assess the self-renewal capacity of cells derived from parotid salivary glands at a chronic time point following radiation. Salivary glands from irradiated mice generate significantly fewer salispheres, but can be stimulated with fetal bovine serum (FBS) to generate an equivalent number of salispheres as unirradiated salivary glands. Interestingly, the number and size of salispheres formed is dependent on the concentration of FBS supplemented into the media. Salispheres derived from irradiated glands and cultured in FBS media were found to contain cells that proliferate and express progenitor and acinar cell markers such as Keratin 5, Keratin 14, Aquaporin 5, and NKCC1. Utilization of insulin-like growth factor (IGF1) injections following radiation treatment restores salivary gland function and improves salisphere generation. These findings indicate that stimulation of these cellular populations may provide a promising avenue for the development of cell-based therapies for radiation-induced salivary gland damage.

Cell-loaded carboxymethylcellulose microspheres sustain viability and proliferation of ATDC5 cells

Cell-loaded carboxymethylcellulose (CMC) microspheres were generated via a flow focusing microfluidic device, with a final aim to obtain viable ATDC5 aggregates with sustained proliferation capacity. We synthesized various CMC with phenolic groups (CMC-Ph) and demonstrated that high CMC-Ph molecular weight, high CMC-Ph concentration (>0.8 g/ml) or long culturing period had obvious inhibition effect on ATDC5 proliferation, but low horseradish peroxidase concentration (HRP, <0.4 mg/ml) did not. CMC-Ph gels being obtained through HRP/H2O2 reaction showed an enhancing strength and decreasing break stain as the molecular weight of CMC-Ph increased, along with a decreasing gelation time. The microfluidics-based synthesis of cell-loaded microspheres with great design flexibilities was achieved using CMC-Ph with weight-average molecular weight of 1.0 × 105. ATDC5 cells were viable up to 41 days of culture and proliferated gradually with increasing culture time. High cell density in CMC-Ph solution and high fetal bovine serum concentration in culture medium were prone to forming cell aggregates. Isolated cells from the microspheres showed the typical spherical morphology of undifferentiated ATDC5. Therefore, CMC-Ph microspheres might be used as cell aggregates depots to study cell-cell or cell-biomaterials functions for tissue engineering applications.

Development of image analysis software for quantification of viable cells in microchips.

Over the past few years, image analysis has emerged as a powerful tool for analyzing various cell biology parameters in an unprecedented and highly specific manner. The amount of data that is generated requires automated methods for the processing and analysis of all the resulting information. The software available so far are suitable for the processing of fluorescence and phase contrast images, but often do not provide good results from transmission light microscopy images, due to the intrinsic variation of the acquisition of images technique itself (adjustment of brightness / contrast, for instance) and the variability between image acquisition introduced by operators / equipment. In this contribution, it has been presented an image processing software, Python based image analysis for cell growth (PIACG), that is able to calculate the total area of the well occupied by cells with fusiform and rounded morphology in response to different concentrations of fetal bovine serum in microfluidic chips, from microscopy images in transmission light, in a highly efficient way.

Effect of serum on the differentiation of neural stem cells

To investigate the effect of serum on the differentiation of neural stem cells. The neural stem cells were isolated from the embryonic hippocampus tissues of Sprague Dawley rats at 14 day of pregnancy. After culturing and passaging, the 3rd generation cells were identified by immunocytochemical staining. Then, the cells were divided into 3 groups according to the concentrations of fetal bovine serum (FBS) used in the differentiation cell culture medium: 5% (group A), 1% (group B), 0 (group C), respectively. The other components of the culture media in 3 groups were the same. Cell viability was determined by using the Live/Dead cell staining at 8 days; the expressions of glial cell marker [glial fibrillary acidic protein (GFAP)] and neuronal marker (β-Ⅲ Tubulin) were determined and analyzed by immunocytochemical staining and real-time fluorescent PCR at 4 and 8 days of culture. Based on cell morphology and immunocytochemical staining, neural stem cells were identified. Cells were growing well with no death in all groups. With decreasing FBS concentration, the expression of GFAP was significantly decreased on both protein and mRNA level, whereas the expression of β-Ⅲ Tubulin was evidently increased. The staining of each group at 8 days was more obvious than that at 4 days. There were significant differences in mRNA expressions of GFAP and β-Ⅲ Tubulin at 4 and 8 days between groups ( P<0.05). Serum can promote the differentiation of neural stem cells into glial cells. At the same time, it inhibits the differentiation of neural stem cells into neurons, the lower the serum concentration, the smaller the effect.

Effect of photobiomodulation on viability and proliferation of stem cells from exfoliated deciduous teeth under different nutritional conditions

This study aimed to evaluate the effect of different doses of low-level laser irradiation on the viability and proliferation of stem cells from exfoliated deciduous teeth (SHED) cultured under nutritional deficit (cellular stress) or regular nutritional conditions. SHED underwent irradiation by a red laser between 1.2 and 6.2 J cm−2. Prior to the irradiation, all groups received culture medium (MEMα, Eagle’s minimum essential medium alpha modification) supplemented with 1% of fetal bovine serum (FBS) for 1 h. After the irradiation, cells received MEMα supplemented with 10% of FBS (regular nutrition) or 1% of FBS (nutritional deficit). Cell viability and proliferation were respectively determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and crystal violet assays 6 and 24 h after irradiation (P < 0.05). At 24 h, SHED under nutritional deficit showed lower viability and proliferation after 1.2 J cm−2 irradiation. All of the irradiated groups revealed significantly higher viability and proliferation in SHED maintained under nutritional deficit than in regular nutritional conditions, except in the 3.7 and 6.2 J cm−2 groups by MTT assay. In the crystal violet assay, SHED irradiated with 1.2 J cm−2 showed no difference between the different nutritional conditions. Decrease of FBS concentration in the culture medium seems to enhance the sensitivity of SHED to the effects of photobiomodulation therapy. Nutritional stress conditions improved cell viability and proliferation of SHED after laser irradiation, except for 1.2 J cm−2.

Resveratrol Can Be Stable in a Medium Containing Fetal Bovine Serum with Pyruvate but Shortens the Lifespan of Human Fibroblastic Hs68 Cells.

This study is aimed at developing a method that can inhibit resveratrol (Res) degradation in Dulbecco's modified Eagle medium (DMEM) and at evaluating the effects of Res on the replicative lifespan of Hs68 cells. We hypothesized that Res can extend the lifespan of Hs68 cells if we can inhibit the oxidative degradation of Res in the medium. We found that the addition of ≥5 U/mL SOD to the medium could completely inhibit Res degradation in DMEM. Fetal bovine serum (FBS) contained 29.3 ± 1.1 U/mL of SOD activity. FBS could prevent Res degradation in the medium through SOD activity and Res–FBS interaction, but the regular FBS concentration (i.e., 10% FBS) exhibited an insufficient ability to completely inhibit Res degradation. We found that pyruvate (1 mM) could potentiate SOD to scavenge superoxide at approximately 2.2-fold. Thus, 10% FBS combined with pyruvate (1 mM) could completely inhibit Res degradation. When Res was not degraded, it still shortened the lifespan of Hs68 cells. Overall, the proposed method involving 10% FBS combined with pyruvate (1 mM) could completely prevent Res degradation. However, in contrast to our hypothesis, Res could induce the shortening of the lifespan of Hs68 cells. The stability of Res analogs (i.e., oxy-Res and acetyl-Res) in the medium and their effects on the lifespan of Hs68 cells were also investigated.

Effects of Fetal Bovine Serum Concentration on the Propagation of Korean Viral Haemorrhagic Septicaemia Virus in an Epithelioma Papulosum Cyprini Cell Line

Effects of Fetal Bovine Serum Concentration on the Propagation of Korean Viral Haemorrhagic Septicaemia Virus in an Epithelioma Papulosum Cyprini Cell Line

Determination of minimum serum concentration to develop scaffold free micro-tissue

Objective. Formation of three-dimensional (3D) micro-tissues without scaffolds are widely used not only to define in vivo tissue formation mechanisms but also the development of different tissue-specific drugs. However, depending on high serum and growth factor concentrations, it would be hard to identify major effective biological cues on micro-tissue formation. The aim of the study is to determine the effect of different serum concentrations on Human Umbilicial Vein Endothelial Cells (HUVECs) micro-tissue formation. Methods. Micro-tissue of HUVEC line was formed by using 3D petri dish technique with medium containing 0%, 1%, 5% and 10% fetal bovine serum (FBS). On the 7 th day after micro-tissue formation, live/dead cells analysis was conducted. Micrograph taken on days 1, 3, 5 and 7 th of micro-tissue formation were determined by image analysis with ImageJ. Results. Sizes of micro-tissue formed with 0% FBS on day 1 and 3 determined as 277 ± 12 µm and 279 ± 20 µm, respectively; however, especially on day 7 micro-tissue size significantly decreased to 229 ± 6 µm. When live/dead analysis results were examined, high cell viability was observed in 5% and 10% FBS concentration. Although micro-tissue like structures were observed in 0% and 1% FBS concentrations dead cell ratio considerably increased compared to 5% and 10% FBS concentration. Conclusions. It has been determined that 0% and 1% serum are appropriate for determining the efficacy of biomimetic peptides and different extracellular matrix proteins on micro-tissue formation parameters of HUVEC. High cell viability in micro-tissues was observed with 5% and 10% serum concentrations.

A novel method of neural differentiation of PC12 cells by using Opti-MEM as a basic induction medium.

The PC12 cell line is a classical neuronal cell model due to its ability to acquire the sympathetic neurons features when deal with nerve growth factor (NGF). In the present study, the authors used a variety of different methods to induce PC12 cells, such as Opti-MEM medium containing different concentrations of fetal bovine serum (FBS) and horse serum compared with RPMI-1640 medium, and then observed the neurite length, differentiation, adhesion, cell proliferation and action potential, as well as the protein levels of axonal growth-associated protein 43 (GAP-43) and synaptic protein synapsin-1, among other differences. Compared with the conventional RPMI-1640 medium induction method, the new approach significantly improved the neurite length of induced cells (2.7 times longer), differentiation rate (30% increase), adhesion rate (21% increase) and expression of GAP-43 and synapsin-1 (three times), as well as reduced cell proliferation. The morphology of induced cells in Opti-MEM medium containing 0.5% FBS was more like that of neurons. Additionally, induced cells were also able to motivate the action potential after treatment for 6 days. Therefore, the research provided a novel, improved induction method of neural differentiation of PC12 cells using Opti-MEM medium containing 0.5% FBS, resulting in a better neuronal model cell line that can be widely used in neurobiology and neuropharmacology research.

Induction of interdigitating cell processes in podocyte culture

Highly organized cell processes characterize glomerular podocytes invivo. However, podocytes in culture have a simple morphology lacking cell processes, especially upon reaching confluence. Here, we aimed to establish culture conditions under which cultured podocytes extend cell processes at confluence. Among various culture conditions that could possibly cause phenotypic changes in podocytes, we examined the effects of heparin, all-trans retinoic acid, fetal bovine serum, and extracellular matrices on the morphology of podocytes in rat primary culture. Consequently, long arborized cell processes were observed to radiate extensively from the cell body only when cells were cultured in the presence of heparin and all-trans retinoic acid on laminin-coated dishes with decreasing concentrations of fetal bovine serum. Primary processes branching repeatedly into terminal processes and cell process insertion under adjacent cell bodies were evident by electron microscopy-based analysis. Immunostaining for podocin showed conspicuous elongations of intercellular junctions. Under these conditions, the expression levels of podocyte-specific proteins and genes were markedly upregulated. Thus, we succeeded in establishing culture conditions in which the cultured podocytes exhibit phenotypes similar to those under invivo conditions.

Human Umbilical Cord Blood Serum Has Higher Potential in Inducing Proliferation of Fibroblast than Fetal Bovine Serum

Background: Cytokines and growth factors were reported to play an important role in stimulating fibroblast proliferation. In vitro culture, fibroblast is mostly culture in medium containing fetal bovine serum (FBS). Human umbilical cord blood (hUCB) has been reported to have low immunogenic property and potential in wound healing, so therefore hUCB serum (hUCBS) could be potential and were investigated in current study.Materials and Methods: Five hUCBs were collected from healthy volunteers with normal delivering procedure. hUCB was ex utero immediately collected from umbilical vein in vacutainers and processed. NIH3T3 cells were cultured in DMEM with 10% FBS or 5-20% hUCBS for 48 hours. Cells were then quantified using MTT assay. Protein concentration of FBS and hUCBS were quantified using Bradford assay.Results: NIH3T3 cells density grown in DMEM with 10% FBS was the lowest. NIH3T3 cells densities were increased along with the increment of hUCBS concentrations. MTT results showed that average number of NIH3T3 cells grown in DMEM with 10% FBS was 6,185±1,243. Meanwhile average numbers of NIH3T3 cells grown in DMEM with 5%, 10% and 20% hUCBS were 8,126±628, 9,685±313 and 12,200±304, respectively. Average numbers of NIH3T3 cells grown in DMEM with 5% hUCBS were significantly higher than the ones with 10% FBS (p=0.000). Bradford results showed that concentration of hUCBS was significantly higher than the one of FBS (p=0.000).Conclusion: hUCBS could induce higher proliferation rate of NIH3T3 cells than FBS. Hence hUCBS could be suggested as an alternate of FBS in inducing fibroblast.Keywords: NIH3T3, fibroblast, UCB, serum, FBS, proliferation

Establishment of a cell line with high transfection efficiency from zebrafish Danio rerio embryos and its susceptibility to fish viruses.

A cell line ZBE3 isolated from a continuous cell culture derived from zebrafish Danio rerio blastomeres by clonal growth was characterized. ZBE3 cells had been subcultured for >120 passages since the initial primary culture of the blastomeres. The ZBE3 cells grow stably at temperature from 20 to 32° C with an optimum temperature of 28° C in ESM2 or ESM4 medium with 15% foetal bovine serum (FBS). The optimum FBS concentration for ZBE3 cell growth ranged from 15 to 20%. Cytogenetical analysis indicated that the modal chromosome number of ZBE3 cells was 50, the same as the diploid chromosome number of D. rerio. Significant cytopathic effect was observed in ZBE3 cells after infection with redspotted grouper nervous necrosis virus, Singapore grouper iridovirus and grass carp reovirus, and the viral replication in the cells was confirmed by real-time quantitative PCR and transmission electron microscopy, indicating the susceptibility of ZBE3 cells to the three fish viruses. After transfected with pEGFP-N3 plasmid, ZBE3 cells showed a transfection efficiency of about 40% which was indicated by the percentage of cells expressing green fluorescence protein. The stable growth, susceptibility to fish viruses as well as high transfection efficiency make ZBE3 cells be a useful tool in transgenic manipulation, fish virus-host cell interaction and immune response in fish.

Abstract 2234: Efatutazone reduces mammosphere formation in Brca1WT/fl11/Cre/p53+/- and Brca1fl11/fl11/Cre/p53+/- mice

Abstract Introduction: Efatutazone, a PPAR gamma agonist, may affect tumor growth through the induction of terminal cell differentiation. Thus, we hypothesize that efatutazone could affect progenitor cell number. Here, we evaluate progenitor cell number by measuring mammospheres, which is considered one way to measure potential progenitor cells. Methods: Two month old Brca1WT/fl11/Cre/p53+/- (n= 14) and Brca1fl11/fl11/Cre/p53+/- (n= 10) C57Bl/6 mice were randomly placed on either control or treated with efatutazone through the diet (30-mg/kg concentration, F3028, rodent diet, grain-based, 1/2-in pellets; Bio-Serv, Frenchtown, NJ) with necropsy at four months and isolation of primary mammary epithelial cells from thoracic glands for studies of mammosphere formation using SCIVAX 96-well low adhesion nanoculture plates (Organogenix, Inc., Japan) using EpiCult-B Mouse Media (Stem Cell Technology, Inc., Vancouver, Canada) with 0,1,5, and 10% fetal bovine serum (FBS) added. Thoracic mammary gland tissue was frozen at -20°C followed by isolation of RNA and evaluation of PPAR gamma pathway gene expression by real-time RT-PCR using TaqMan® Array Mouse Lipid Regulated Genes (Thermo Fisher Scientific, Inc., Waltham MA). One inguinal gland was fixed for mammary gland whole mount and the other inguinal gland was formalin-fixed and paraffin-embedded for histology. Results: SCIVAX nanoculture plates showed reproducible increases in mammosphere numbers with increasing FBS concentrations (p &amp;lt;0.05 one-way, Kruskal-Wallis). A significant increase in sphere number was seen in Brca1fl11/fl11/Cre/p53+/- compared to Brca1WT/fl11/Cre/p53+/- mice (p&amp;lt;0.05 one-way, Kruskall Wallis). Efatutazone treatment significantly decreased sphere numbers in Brca1WT/fl11/Cre/p53+/- and in Brca1fl11/fl11/Cre/p53+/- mice (p&amp;lt;0.05 one-way, Kruskall Wallis). Expression of PPAR gamma pathway genes were increased at the RNA level with statistically significant increases in Acadvl, Tnf, Alox5 (p&amp;lt;0.05) and Il1B, Srebf2, Hmgcs1, Hmgcr (p&amp;lt;0.01) genes (BootsRatio, http://rht.iconcologia.net/stats/br/several.html) in mice on efatutazone as compared to control diet. Discussion: SCIVAX nanoculture plates can be used to quantitatively evaluate and compare mammosphere numbers between different genotypes and treatment groups. The higher numbers of mammospheres found with loss of both Brca1 copies as compared to one copy is consistent with previously published literature. The decrease in mammosphere numbers with efatutazone treatment could be secondary to its differentiating impact on mammary epithelial cells. Conclusion: While efatutazone statistically significantly reduced mammosphere numbers, the absolute reduction was less the 25%. Citation Format: Sahar J. Alothman, Shan Chao, Weisheng Wang, Priscilla A. Furth. Efatutazone reduces mammosphere formation in Brca1WT/fl11/Cre/p53+/- and Brca1fl11/fl11/Cre/p53+/- mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2234. doi:10.1158/1538-7445.AM2017-2234

Effect of human autologous serum and fetal bovine serum on human corneal epithelial cell viability, migration and proliferation in vitro.

To analyze the concentration-dependent effects of autologous serum (AS) and fetal bovine serum (FBS) on human corneal epithelial cell (HCEC) viability, migration and proliferation. AS was prepared from 13 patients with non-healing epithelial defects Dulbecco's modified eagle medium/Ham's F12 (DMEM/F12) with 5% FBS, 0.5% dimethyl sulphoxide (DMSO), 10 ng/mL human epidermal growth factor, 1% insulin-transferrin-selenium, then were incubated in serum media: DMEM/F12 supplemented by 5%, 10%, 15% or 30% AS or FBS. HCEC viability was analyzed using cell proliferation kit XTT, migration using a wound healing assay, proliferation by the cell proliferation enzyme-linked immunosorbent assay (ELISA) BrdU kit. Statistical analysis was performed using the generalized linear model, the values at 30% AS or 30% FBS were used as the baselines. HCEC viability was the highest at 30% AS or 15% FBS and the lowest at 10% AS or 30% FBS application. HCEC migration was the quickest through 30% AS or 30% FBS and the slowest through 5% AS or 5% FBS concentrations. Proliferation was the most increased through 15% AS or 5% FBS and the least increased through 30% AS or 30% FBS concentrations. HCEC viability at 10% and 15% AS was significantly worse (P=0.001, P=0.023) compared to baseline and significantly better at 15% FBS (P=0.003) concentrations. HCEC migration was significantly worse (P≤0.007) and HCEC proliferation significantly better (P<0.001) in all concentration groups compared to baseline. For the best viability of HCEC 30% AS or 15% FBS, for HCEC migration 30% AS or 30% FBS, for proliferation 15% AS or 5% FBS should be used. Therefore, we suggest the use of 30% AS in clinical practice.

Xeno-Free Cultivation of Mesenchymal Stem Cells From the Corneal Stroma.

The human cornea has recently been described as a source of corneal stroma-derived mesenchymal stem cells (hMSCs). In vitro expansion of these cells involves basal medium supplemented with fetal bovine serum (FBS). As animal-derived serum can confer a risk of disease transmission and can be subject to considerable lot-to-lot variability, it does not comply with newer Good Manufacturing Practice (GMP)-required animal component-free culture protocols for clinical translation. This study investigated animal-free alternatives to FBS for cultivation of human corneal stromal MSCs. Proliferative capacity was studied for cultures supplemented with different concentrations (2.5%, 5%, and 10%) of FBS, human AB serum, human platelet lysate (HPL), and XerumFree. Unsupplemented basal medium was used as a control. The expression of specific hMSC markers (CD73+, CD90+, CD105+, CD19-, CD34-, CD79α-, CD11b-, CD14-, CD45-, and HLA-DR-) and trilineage differentiation (adipogenesis, osteogenesis, and chondrogenesis) were compared for the two outperforming supplements: 10% FBS and HPL. HPL is the only consistent non-xeno supplement where hMSC cultures show significantly higher proliferation than the 10% FBS-supplemented cultures. Both FBS- and HPL-supplemented hMSC cultures showed plastic adherence and trilineage differentiation, and no significant differences were found in the expression of the hMSC marker panel. No significant differences in stemness were detected between FBS and HPL cultures. We conclude that HPL is the best supplement for expansion of human corneal stromal MSCs. HPL significantly outperforms human AB serum, the chemically defined XerumFree, and even the gold standard, FBS. The xeno-free nature of HPL additionally confers preferred standing for use in GMP-regulated clinical trials using human corneal stromal MSCs.

Establishment of an in vitro culture model of theca cells from hierarchical follicles in ducks.

Theca cells, including theca interna cells and theca externa cells, are vital components of ovarian follicles. The aim of the present study is to identify a reliable method for the in vitro culture of theca cells from duck ovarian hierarchical (F4-F2) follicles. We improved the method for cell separation by using trypsin to further remove granular cells, and we increased the concentration of fetal bovine serum used in in vitro culture to improve cytoactivity. Cell antibody immunofluorescence (IF) showed that all inoculated cells could be stained by the CYP17A1/19A1 antibody but not by the FSHR antibody, which could stain granulosa cells. Furthermore, morphological differences were observed between the outlines of theca interna and externa cells and in their nuclei. Growth curve and CYP17A1/19A1 mRNA relative expression analyses suggested that the growth profile of theca interna cells may have been significantly different from that of theca externa cells in vitro. Theca interna cells experienced the logarithmic phase on d1–d2, the plateau phase on d2–d3, and the senescence phase after d3, while theca externa cells experienced the logarithmic phase on d1–d3, the plateau phase on d3–d5, and the senescence phase after d5. Taken together, these results suggested that we have successfully established a reliable theca cell culture model and further defined theca cell characteristics in vitro.

Establishment and characterization of a cell line from the head kidney of golden pompano Trachinotus ovatus and its application in toxicology and virus susceptibility.

A cell line derived from the head kidney of golden pompano Trachinotus ovatus (TOHK) was established and characterized in this study. The TOHK cells grew most rapidly at 28° C and the optimum foetal bovine serum concentration in L-15 medium was 10%. The TOHK cells have a diploid chromosome number of 2N = 54. The transfection efficiency of TOHK cells was 7·5% at the 15th passage and 72% at the 40th passage. The transfection efficiency in TOHK cells was high, so these cells are suitable for foreign gene expression. The cytotoxic effects of heavy metals and extracellular products from Vibrio anguillarum and Vibrio alginolyticus were demonstrated in TOHK cells, so this TOHK cell line could also be applied in environmental monitoring of heavy metals and pathogenic bacteria. TOHK cell line showed high virus susceptibility, such as grouper nervous necrosis virus (GNNV) and Singapore grouper iridovirus (SGIV). Then, TOHK cell line could be used for the study of viral pathogenesis and the development of antiviral strategies.

Serum from plasma rich in growth factors regenerates rabbit corneas by promoting cell proliferation, migration, differentiation, adhesion and limbal stemness.

To evaluate the regenerating potential and the mechanisms through which the autologous serum derived from plasma rich in growth factors (s-PRGF) favours corneal wound healing invitro and invivo. We compared the effect of various concentrations of s-PRGF versus fetal bovine serum (FBS) and control treatment in rabbit primary corneal epithelial and stromal cells and wounded rabbit corneas. Cell proliferation was measured using an enzymatic colorimetric assay. In vitro and invivo wound-healing progression was assessed by image-analysis software. Migration and invasion were evaluated using transfilter assays. Histological structure was analysed in stained sections. Protein expression was evaluated by immunohistochemistry. s-PRGF promoted the robust proliferation of epithelial cultures at any concentration, similar to FBS. Likewise, s-PRGF and FBS produced similar re-epithelialization rates in invitro wound-healing assays. In vivo, s-PRGF treatment accelerated corneal wound healing in comparison with control treatment. This difference was significant only for 100% s-PRGF treatment in our healthy rabbit model. Histological analysis confirmed normal epithelialization in all cases. Immunohistochemistry showed a higher expression of cytokeratins 3/76 and 15, zonula occludens-1 and alpha-smooth muscle actin proteins as a function of s-PRGF concentration. Notably, keratocyte density in the anterior third of the stroma increased with increase in s-PRGF concentration, suggesting an invivo chemotactic effect of s-PRGF on keratocytes that was further confirmed invitro. s-PRGF promotes proliferation and migration and influences limbal stemness, adhesion and fibrosis during corneal healing.

Sperm cryopreservation of Japanese eel, Anguilla japonica

In the present study, methods for the cryopreservation of Anguilla japonica spermatozoa were examined. Spermatozoa were collected from artificially matured males and incubated in Japanese eel K30 artificial seminal plasma (K30 ASP) before experiments. Sperm motility was investigated using computer-assisted sperm analysis (CASA). 10% and 15% MeOH as cryoprotectant was the most successful cryoprotectant with percentage of the initial motility of 59.7±12.1%; the combination of 5% MeOH and 5% DMA was also viable. DMSO was unsuitable as a cryoprotectant with K30 ASP as it showed no cryopreservation properties and was toxic to sperm, causing sperm to be immotile immediately after dilution. Japanese eel spermatozoa had a narrow range of optimal cooling rates (6.3–28.6°Cmin−1) and immersion temperatures of −40 to −70°C were effective. The different fetal bovine serum (FBS) concentration in extender, temperature of milt before cooling, dilution rates (3–100 times) and equilibrium time showed no significant difference. The results indicate that the type of extender media greatly influenced the suitability of cryoprotectant. The establishment of a protocol to cryopreserved Japanese eel sperm will benefit the artificial seed production of Japanese eel and provide an important tool for genetic and breeding studies. Statement of relevanceThe development of the sperm cryopreservation protocol for Japanese eel will ensure a stable supply of high quality seed for experimental and commercial purposes.

Optimization of culture medium for scarless healing of fetal mouse wounds and analysis of regions with active cell proliferation

Objective To optimize the culture medium for scarless wound healing in outbred fetal Kunming (KM) mice, and to locate and preliminarily analyze the region with active cell proliferation. Methods After 6 pregnant mice were sacrificed at the gestational age of 15 days, a total of 180 skin grafts were obtained from the back of 60 fetal mice, and wounds on the skin grafts were made uniformly with minimally invasive sterile device. Then, these skin grafts with wounds were randomly and equally divided into 6 groups to be treated with high-glucose Dulbecco′s modified Eagle′s medium (DMEM) , DMEM+ 5% fetal bovine serum (FBS) , DMEM+ 10% FBS, low-glucose Eagle′s minimum essential medium (MEM) , MEM+ 5% FBS, and MEM+ 10% FBS for 3 days, respectively. Then, these skin grafts were embedded in optimal cutting temperature (OCT) compound, and subjected to hematoxylin and eosin (HE) staining to select the optimal culture condition for wound healing. Under the optimal culture condition, the region with active cell proliferation was located by labeling and tracking cutaneous stem cells with 5-ethynyl-2-deoxyuridine (EdU) , and activator protein-1 (AP-1) expression was detected by immunofluorescent staining. Results After 3-day cultivation, wound healing rates significantly differed among the DMEM group, DMEM+ 5% FBS group, DMEM+ 10% FBS group, MEM group, MEM+ 5% FBS group and MEM+ 10% FBS group (0, 3.33%, 6.67%, 3.33%, 46.67% and 26.67%, respectively, χ2= 41.39, P < 0.05) . Additionally, the MEM+ 5% FBS group showed a significantly higher wound healing rate than the other groups except the MEM+ 10% FBS group (all P < 0.01) . Logistic regression analysis showed that the type of basal medium (MEM: OR= 11.717, 95% CI: 3.274-41.934, P < 0.001) and FBS concentrations (5% FBS: OR= 24.625, 95% CI: 3.027-200.299, P= 0.003; 10% FBS: OR= 13.449, 95% CI: 1.618-111.813, P= 0.016) were factors influencing fetal wound healing. Under the culture condition of 5% FBS+ MEM, the dermis and epidermis healed well without epidermal thickening. The cutaneous stem cell labeling technique showed that the papillary dermis and basal layer of the epidermis were the two major regions with active cell proliferation in the wound of fetal mouse skin. Moreover, AP-1 was expressed abundantly in these two regions as well. Conclusions The culture condition of 5% FBS+ MEM is considered to be optimal for in vitro wound healing in fetal mice. The papillary dermis and basal layer of the epidermis are two major regions with active cell proliferation during wound healing in fetal mouse skin, and play important roles in wound healing process. Key words: Wound healing; Cicatrix; Transcription factor AP-1; Scarless wound healing; Fetal mice