First cloned swamp buffalo produced from adult ear fibroblast cell

The world’s first cloned swamp buffalo (Bubalus bubalis) derived from adult ear skin fibroblast has been reported. Donor fibroblast cells were produced from biopsies taken from adult male ear skin and in vitro matured oocytes obtained from a slaughterhouse were used as cytoplasts. A total of 39 blastocysts and 19 morulae fresh embryos were transferred into 12 recipient buffaloes. Progesterone assays indicated establishment of pregnancy in 10 of the 12 buffaloes (83.3%) after 45 days, with six animals still pregnant at 3 months. One recipient maintained pregnancy to term and naturally delivered a 40 kg male calf after 326 days of gestation. DNA analysis showed that the cloned calf was genetically identical to the donor cells. Genotype analyses, using 12 buffalo microsatellite markers, confirmed that the cloned calf was derived from the donor cell lines. In conclusion, the present study reports, for the first time, the establishment of pregnancy and birth of the first cloned Thai swamp buffalo derived from adult ear skin fibroblast cells.     

A comparative study on efficiency of adult fibroblasts and amniotic fluid-derived stem cells as donor cells for production of hand-made cloned buffalo (Bubalus bubalis) embryos

The efficiency of two cell types, namely adult fibroblasts, and amniotic fluid stem (AFS) cells as nuclear donor cells for somatic cell nuclear transfer by hand-made cloning in buffalo (Bubalus bubalis) was compared. The in vitro expanded buffalo adult fibroblast cells showed a typical “S” shape growth curve with a doubling time of 40.8 h and stained positive for vimentin. The in vitro cultured undifferentiated AFS cells showed a doubling time of 33.2 h and stained positive for alkaline phosphatase, these cells were also found positive for undifferentiated embryonic stem cell markers like OCT-4, NANOG and SOX-2, which accentuate their pluripotent property. Further, when AFS cells were exposed to corresponding induction conditions, these cells differentiated into osteogenic, adipogenic and chondrogenic lineages which was confirmed through alizaran, oil red O and alcian blue staining, respectively. Cultured adult fibroblasts and AFS cells of passages 10–15 and 8–12, respectively, were used as nuclear donors. A total of 94 embryos were reconstructed using adult fibroblast as donor cells with cleavage and blastocyst production rate of 62.8 ± 1.8 and 19.1 ± 1.5, respectively. An overall cleavage and blastocyst formation rate of 71.1 ± 1.2 and 29.9 ± 2.2 was obtained when 97 embryos were reconstructed using AFS cells as donor cells. There were no significant differences (P > 0.05) in reconstructed efficiency between the cloned embryos derived from two donor cells, whereas the results showed that there were significant differences (P < 0.05) in cleavage and blastocyst rates between the cloned embryos derived from two donor cell groups. Average total cell numbers for blastocyst generated using AFS cells (172.4 ± 5.8) was significantly (P < 0.05) higher than from adult fibroblasts (148.2 ± 6.1). This study suggests that the in vitro developmental potential of the cloned embryos derived from AFS cells were higher than that of the cloned embryos derived from adult fibroblasts in buffalo hand-made cloning.     

Efficient genome editing in cultured cells and embryos of Debao pig and swamp buffalo using the CRISPR/Cas9 system

Myostatin (MSTN), a protein encoded by growth differentiation factor 8 (GDF8), is primarily expressed in skeletal muscle and negatively regulates the development and regeneration of muscle. Accordingly, myostatin-deficient animals exhibit a double-muscling phenotype. The CRISPR/Cas9 system has proven to be an efficient genome-editing tool and has been applied to gene modification in cells from many model organisms such as Drosophila melanogaster, zebrafish, mouse, rat, sheep, and human. Here, we edited the GDF8 gene in fibroblasts and embryos of Debao pig and swamp buffalo using the CRISPR/Cas9 system. The CRISPR/Cas9-mediated mutation efficiency in fibroblasts was as high as 87.5% in pig and 78.9% in buffalo. We then obtained single-cell clones with mutations at the specific sites of the GDF8 gene by screening with G418 in fibroblasts of pig and buffalo. In addition, the frequencies of Cas9/gRNA-mediated mutations were at 36 and 25% in the intracytoplasmic sperm injection embryos of pig and in vitro fertilization embryos of buffalo, respectively. Our work demonstrates that the Cas9/gRNA system is a highly efficient and fast tool for genome editing in cultured cells and embryos of Debao pig and swamp buffalo. These results can be helpful for the establishment of a new animal strain that can generate more meat.     

Establishment and biological characterization of fibroblast cell line from the Langshan chicken

Objective: We needed to establish an embryonic fibroblast cell line from the Langshan chicken (LSCEF61) to preserve their important genetic resources at the cellular level. Material and methods: The cell line was established from 9‐day‐old embryos by direct explant culture and cryopreservation techniques. Cell morphology, dynamic proliferation and any contamination present were tested, and the karyotype and levels of isoenzymes of lactic dehydrogenase and malic dehydrogenase were analysed. Four types of fluorescent protein exogenous genes for pEGFP‐C₁, pEGFP‐N₃, pEYFP‐N₁ and pDsRed1‐N₁ were transfected into the cells. Results: Showed that the cells were healthy and were of spindle shaped structure, without change in morphology. Cell growth curves were of typical S‐shape. Assays for microbial contamination were negative. The LSCEF61 line showed no cross‐contamination when assessed by isoenzyme analysis. Chromosome number (2n) = 78 on more than 90% of occasions. The four types of fluorescent protein extro‐genes appeared to be expressed effectively with high transfection efficiency between 15.6% and 38.6%. Conclusion: The cell line met each of the quality control standards required for the American Type Culture Collection. It had not only preserved the genetic resources of the important Langshan chicken at the cellular level, but also provided valuable material for genomic, post‐genomic and somatic cell cloning research and other applications.     

The role of MC1R gene in buffalo coat color

Melanocortin-1 receptor (MC1R) plays a major role in pigmentation in many species. To investigate if the MC1R gene is associated with coat color in water buffalo, the coding region of MC1R gene of 216 buffalo samples was sequenced, which included 49 black river buffalo (Murrah and Nili-Ravi), 136 swamp buffalo (Dehong, Diandongnan, Dechang, Guizhou, and Xilin) with white and gray body, and 31 hybrid offspring of river buffalo Nili-Ravi (or Murrah) and swamp buffalo. Among the three variation sites found, SNP684 was synonymous, while SNP310 and SNP384 were nonsynonymous, leading to p.S104G and p.I128M changes, respectively. Only Individuals carrying homozygote EBR/EBR were black. The genotype and phenotype analysis of the hybrid offspring of black river buffalo and gray swamp buffalo further revealed that the river buffalo type allele EBR or the allele carrying the amino acid p.104S was important for the full function of MC1R. The in silico functional analysis showed that the amino acid substitutions p.G104S and p.M128I had significant impact on the function of MC1R. Above results indicate that the allele EBR or the allele carrying the amino acid p.104S was associated with the black coat color in buffalo.     

Independent maternal origin of Chinese swamp buffalo (Bubalus bubalis)

To obtain more knowledge on the origin and genetic diversity of the swamp buffalo (Bubalus bubalis) in China, the complete mitochondrial D-loop sequences of 119 samples representing seven native types were compared. Two mitochondrial DNA (mtDNA) lineages (lineages A and B) were determined for the Chinese swamp buffalo. Examination of the diversity patterns suggest that lineage A has undergone a population expansion event. Divergence of lineages A and B was estimated at 18,000 years ago. Combined analyses of mtDNA sequences from Chinese, Indian, Brazilian/Italian and Southeast Asian/Australian buffalo samples showed independent domestication events in the swamp buffalo from China and the river buffalo from the India subcontinent. The spread of swamp and river buffalo from China and India respectively to mainland Southeast Asia suggests that Southeast Asia is a hybrid zone for buffalo. Our data support the hypothesis of the evolution of domesticated swamp and river buffalo from ancestral swamp-like animals. These ancestral animals were extensively distributed across mainland Asia and most likely are represented today by the wild Asian buffalo (Bubalus arnee).     

Genetic diversity of mitochondrial cytochrome b gene in swamp buffalo

The swamp buffalo (Bubalus carabanensis) is mainly bred for meat, transport and rice cultivation in China and Southeast Asian countries. In the current study, we investigated the genetic diversity, maternal origin and phylogenetic relationship of swamp buffalo by analyzing 1,786 mitochondrial cytochrome b (cytb) sequences from China, Vietnam, Laos, Thailand, India and Bangladesh. Our results indicated that the swamp buffalo can be divided into two major lineages (SA and SB with the sublineages) and three rare lineages (SC, SD and SE), which showed strong geographic differentiation. The SA1 lineage represented a major domestication event, which involved population expansion. Regions III and V showed higher haplotype diversity than the other regions, indicating that the regions of Southwest China and IndoChina are potential domestication centers for the swamp buffalo. In addition, the swamp buffalo showed a closer phylogenetic relationship to tamaraw. In conclusion, our findings revealed a high level of genetic diversity and the phylogenetic pattern of the swamp buffalo.     

Establishment, characterization and immortalization of a fibroblast cell line from the Chinese red belly toad Bombina maxima skin

The skin of the amphibian Bombina maxima is rich in biologically active proteins and peptides, most of which have mammalian analogues. The physiological functions of most of the mammalian analogues are still unknown. Thus, Bombina maxima skin may be a promising model to reveal the physiological role of these proteins and peptides because of their large capacity for secretion. To investigate the physiological role of these proteins and peptides in vitro, a fibroblast cell line was successfully established from Bombina maxima tadpole skin. The cell line grew to form a monolayer with cells of a uniform shape and abundant rough endoplasmic reticulum, which are typical characteristics of fibroblasts. Further identification at a molecular level revealed that they strongly expressed the fibroblast marker protein vimentin. The chromosome number of these cells is 2n = 28, and most of them were diploid. Growth property analysis showed that they grew well for 14 passages. However, cells showed decreased proliferative ability after passage 15. Thus, we tried to immortalize the cells through the overexpression of SV40 T antigen. After selecting by G418, cells stably expressed SV40 large T antigen and showed enhanced proliferative ability and increased telomerase activity. Signal transduction analysis revealed functional p42 mitogen-activated protein (MAP) kinase in immortalized Bombina maxima dermal fibroblasts. Primary fibroblast cells and the immortalized fibroblast cells from Bombina maxima cultured in the present study can be used to investigate the physiological role of Bombina maxima skin-secreted proteins and peptides. In addition, the methods for primary cell culturing and cell immortalization will be useful for culturing and immortalizing cells from other types of amphibians.     

Collagen modulates cell shape and cytoskeleton of embryonic corneal and fibroma fibroblasts: Distribution of actin, α-actinin,and myosin

In the embryo, fibroblasts migrating through extracellular matrices (ECM) are generally elongate in shape, exhibiting a leading pseudopodium with filopodial extensions, and a trailing cell process. Little is known about the mechanism of movement of embryonic cells in ECM, for studies of fibroblast locomotion in the past have been largely confined to observations of flattened cells grown on planar substrata. We confirm here that embryonic avian corneal fibroblasts migrating within hydrated collagen gels in vitro have the bipolar morphology of fibroblasts in vivo, and we show for the first time that highly flattened gerbil fibroma fibroblasts, grown as cell lines on planar substrata, can also respond to hydrated collagen gels by becoming elongate in shape. We demonstrate that the collagen-mediated change in cell shape is accompanied by dramatic rearrangement of the actin, α-actinin, and myosin components of the cytoskeleton. By immunofluorescence, the stress fibers of the flattened corneal fibroblasts grown on glass are seen to stain with antiactin, anti-α-actinin, and antimyosin, as has been reported for fibroma and other fibroblasts grown on glass. Stress fibers, adhesion plaques, and ruffles do not develop when the corneal or fibroma fibroblast is grown in ECM; these features seem to be a response to strong attachment of the cell underside to a planar substratum. When the fibroblasts are grown in ECM, antimyosin staining is distributed diffusely through the cytoplasm. Antiactin and anti-α-actinin stain the microfilamentous cell cortex strongly. We suggest that locomotion of the fibroblast in ECM is accompanied by adhesion of the cell to the collagen fibrils and may involve an interaction of the myosin-rich cytosol with the actin-rich filamentous cell cortex. Interestingly, the numerous filopodia that characterize the tips of motile pseudopodia of cells in ECM are very rich in actin and α-actinin, but seem to lack myosin; if filopodia use myosin to move, the interaction must be at a distance. Soluble collagen does not convert flattened fibroblasts on planar substrata to bipolar cells. Thus, the effect of collagen on the fibroblast cytoskeleton seems to depend on the presence of collagen fibrils in a gel surrounding the cell.     

Melanoma cell-derived factors stimulate hyaluronan synthesis in dermal fibroblasts by upregulating HAS2 through PDGFR-PI3K-AKT and p38 signaling

In many cancers hyaluronan content is increased, either by tumor cells or the surrounding stromal cells and this increased hyaluronan content correlates with unfavorable clinical prognosis. In the present work, we studied the effects of melanoma cell (aggressive melanoma cell line C8161)-derived factors on fibroblast hyaluronan synthesis, intracellular signaling, MMP expression and invasion. Treatment of the fibroblast cultures with melanoma cell conditioned medium (CM) caused accumulation of hyaluronan in the culture medium and formation of thick pericellular hyaluronan coat and hyaluronan cables. The expression of Has2 was increased approximately 20-fold by the C8161 melanoma cell CM, while Has1 and Has3 were increased twofold. Knock-down of Has2 expression with siRNA showed that Has2 was responsible for the increased hyaluronan synthesis induced by the melanoma cell CM. To find out the signaling routes, which led to Has2 upregulation, the phosphorylation profiles of 46 kinases were screened with phosphokinase array kit. Melanoma cell CM treatment strongly induced a rapid phosphorylation of p38, JNK, AKT, CREB, HSP27, STAT3 and cJUN. Treatment of the fibroblasts with specific inhibitors of PI3K, AKT and p38 reduced the melanoma cell CM-induced hyaluronan secretion, while the inhibitor of PDGFR totally blocked it. In addition, siRNA for PDGFRα/β inhibited Has2 upregulation in melanoma cell CM-treated fibroblasts. In parallel with the increased hyaluronan synthesis the melanoma cell CM-treated fibroblasts showed spindle shape, numerous long cell protrusions, enhanced MMP expression and increased invasion into collagen-Cultrex matrix. siRNA blocking of Has2 or PDGFRα/β expression reversed the stimulatory effect of melanoma cell CM on fibroblast invasion. PDGF secreted by melanoma cells thus mediated fibroblasts activation, with HAS2 upregulation as a major factor in the fibroblast response. This effect on stromal matrix is suggested to favor tumor growth.     

Rapid sexing of water buffalo (Bubalus bubalis) embryos using loop-mediated isothermal amplification

Loop-mediated isothermal amplification (LAMP) is a novel DNA amplification method that amplifies a target sequence specifically under isothermal conditions. The objective of this study was to identify a Y chromosome-specific sequence in water buffalo and to establish an efficient procedure for embryo sexing by LAMP. The homologues of a Y chromosome-specific sequence, bovine repeat Y-associated.2, in swamp and river buffalo were cloned, and designated swamp buffalo repeat Y-associated.2 and river buffalo repeat Y-associated.2, respectively. Sexing by LAMP was performed using primers for swamp buffalo repeat Y-associated.2. A 12S rRNA was also amplified by LAMP as a control reaction in both male and female. The minimal amount of the template DNA required for LAMP appeared to be 0.1-10 pg. The sensitivity was further examined using swamp buffalo fibroblasts as templates. When fibroblasts were lysed with NaOH, the minimal cell number required for detection of both male-specific and male-female common DNA appeared to be two cells, whereas correct determination of sex could not be achieved using fibroblasts lysed by heat denaturation. Embryo sexing was also performed using blastomeres from interspecies nuclear transfer embryos. The sex determined by LAMP for blastomeres corresponded with the sex of nuclear donor cells in analyses using four or five blastomeres as templates. The LAMP reaction required only about 45 min, and the total time for embryo sexing, including DNA extraction, was about 1 h. In conclusion, the present procedure without thermal cycling and electrophoresis was reliable and applicable for water buffalo embryos.     

Cancer-Associated Fibroblasts Promote Proliferation of Endometrial Cancer Cells

Endometrial cancer is the most commonly diagnosed gynecologic malignancy worldwide; yet the tumor microenvironment, especially the fibroblast cells surrounding the cancer cells, is poorly understood. We established four primary cultures of fibroblasts from human endometrial cancer tissues (cancer-associated fibroblasts, CAFs) using antibody-conjugated magnetic bead isolation. These relatively homogenous fibroblast cultures expressed fibroblast markers (CD90, vimentin and alpha-smooth muscle actin) and hormonal (estrogen and progesterone) receptors. Conditioned media collected from CAFs induced a dose-dependent proliferation of both primary cultures and cell lines of endometrial cancer in vitro (175%) when compared to non-treated cells, in contrast to those from normal endometrial fibroblast cell line (51%) (P<0.0001). These effects were not observed in fibroblast culture derived from benign endometrial hyperplasia tissues, indicating the specificity of CAFs in affecting endometrial cancer cell proliferation. To determine the mechanism underlying the differential fibroblast effects, we compared the activation of PI3K/Akt and MAPK/Erk pathways in endometrial cancer cells following treatment with normal fibroblasts- and CAFs-conditioned media. Western blot analysis showed that the expression of both phosphorylated forms of Akt and Erk were significantly down-regulated in normal fibroblasts-treated cells, but were up-regulated/maintained in CAFs-treated cells. Treatment with specific inhibitors {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 and U0126 reversed the CAFs-mediated cell proliferation (P<0.0001), suggesting for a role of these pathways in modulating endometrial cancer cell proliferation. Rapamycin, which targets a downstream molecule in PI3K pathway (mTOR), also suppressed CAFs-induced cell proliferation by inducing apoptosis. Cytokine profiling analysis revealed that CAFs secrete higher levels of macrophage chemoattractant protein (MCP)-1, interleukin (IL)-6, IL-8, RANTES and vascular endothelial growth factor (VEGF) than normal fibroblasts. Our data suggests that in contrast to normal fibroblasts, CAFs may exhibit a pro-tumorigenic effect in the progression of endometrial cancer, and PI3K/Akt and MAPK/Erk signaling may represent critical regulators in how endometrial cancer cells respond to their microenvironment.     

Genetic diversity of mitochondrial cytochrome b gene in Chinese native buffalo

The origins of the domestic water buffalo remain contentious. To better understand the origins of Chinese water buffalo, we sequenced the complete mitochondrial cytochrome b (MT-CYB) gene from 270 individuals representing 13 Chinese domestic swamp buffalo populations. We found genetic evidence of introgression of river buffalo into Chinese swamp buffalo herds. Swamp buffalo haplotypes can be divided into two highly divergent lineages (A and B), suggesting that Chinese native swamp buffalo have two maternal origins. We found that the A→G transition in the buffalo MT-CYB gene stop codon resulted in buffalo haplotypes being terminated by one of two stop codons: AGA or AGG. AGA is common to river buffalo and lineage A of swamp buffalo, while AGG is specific to lineage B of swamp buffalo. Lineage A appears to have been domesticated in China. Further genetic evidence is required to clarify the origins of lineage B.     

Establishment and genetic characteristics analysis of in vitro culture a fibroblast cell line derived from Wuzhishan miniature pig

Establishment of fibroblast cell lines of endangered pig breeds and research on the gene functions based on the cells made a significant contribution to the conservation and utilization of genetic resources. The Wuzhishan miniature pig ear marginal tissue fibroblast cell line (WPF22) from 22 samples, stocking 87 cryogenically-preserved vials, was successfully established by using primary explants technique and cell cryopreservation techniques. WPF22 cells were adherent, with a population doubling time of 30.2 h. Chromosome karyotyping and G-banding analysis showed that >90.2% of cells were diploid (2n = 38) prior to the 4th generation. Neither microbial contamination nor cross-contamination was detected by isoenzyme analyses. Cell viability was 97.8% before cryopreservation and 94.9% after recovery. To determine cell permeability, intracellular path and stability of exogenous proteins during the transduction, six fluorescent protein genes were transferred into fibroblasts by lipofectamine-mediated method. The transfection efficiency of six fluorescent protein genes fluctuated between 8.1% and 42.6%. ECFP and DsRed were mostly shown in cytoplasmic in dots around the nucleus, and EYFP and EGFP had a slightly stronger expression in the nucleus than in the cytoplasm, but without expression in some vacuoles. Every index of the WPF22 cell line meets all the standard quality controls of American type Culture Collection (ATCC). This research thus does not only preserve important genetic resources of Wuzhishan miniature pig at the cell level, but also serve as a valuable resource for genome, postgenome and somacloning research.     

Induction of pluripotency in mammalian fibroblasts by cell fusion with mouse embryonic stem cells

BackgroundCell fusion is a phenomenon that is observed in various tissues in vivo, resulting in acquisition of physiological functions such as liver regeneration. Fused cells such as hybridomas have also been produced artificially in vitro. Furthermore, it has been reported that cellular reprogramming can be induced by cell fusion with stem cells.MethodsFused cells between mammalian fibroblasts and mouse embryonic stem cells were produced by electrofusion methods. The phenotypes of each cell lines were analyzed after purifying the fused cells.ResultsColonies which are morphologically similar to mouse embryonic stem cells were observed in fused cells of rabbit, bovine, and zebra fibroblasts. RT-PCR analysis revealed that specific pluripotent marker genes that were never expressed in each mammalian fibroblast were strongly induced in the fused cells, which indicated that fusion with mouse embryonic stem cells can trigger reprogramming and acquisition of pluripotency in various mammalian somatic cells.ConclusionsOur results can help elucidate the mechanism of pluripotency maintenance and the establishment of highly reprogrammed pluripotent stem cells in various mammalian species.     

Genomic differentiation between swamp and river buffalo using a cattle high-density single nucleotide polymorphisms panel

Buffalo (Bubalus bubalis) is an important livestock species in many tropical and subtropical regions. In recent decades, the interest in buffalo’s milk have expanded and intensive buffalo farms start to emerge. However, breeding programs and population genetics information for this species is scarce or inexistent. The present study aims to test the suitability of the commercial high-density single nucleotide polymorphisms (SNP) genotyping panel, the Illumina BovineHD BeadChip, to estimate population genetics parameters, pedigree control and identification of common variants in major production candidate genes. From a total of 777 962 SNPs included in the panel, 20 479 were polymorphic in water buffalo at a call rate of 86% and an average expected heterozygosity (H_E) of 0.306. From these, 357 were mapped within or around the flanking regions of several major candidate genes. A principal components analysis identified three different clusters, each representing pure swamp buffalo type, pure river buffalo type and admixed river buffalo. The hybrids between swamp and river buffalo were clearly identified as an intermediary cluster. The suitability of these SNPs data set for parentage and identity testing demonstrated that the combination of just 30 to 50 SNPs were enough to attain high probabilities of parentage exclusion (0.9999) in both types and identity (2.3×10⁻⁵ and 2.0×10⁻⁷) for river and swamp buffalo, respectively. Our analysis confirms the suitability of the BovineHD BeadChip to assess population structure, hybridization and identity of the water buffalo populations.     

In vitro cytotoxic potential of Solanum nigrum against human cancer cell lines

Plants have natural products which use to possess antiproliferative potential against many cancers. In the present study, six isolated fractions (ethyl acetate, petroleum ether, chloroform, n-butanol, ethanol and aqueous) from Solanum nigrum were evaluated for their cytotoxic effect on different cell lines. Hepatic carcinoma cell line (HepG2), cervical cancer cell line (HeLa) and baby hamster kidney (BHK) used as normal non-cancerous cells were evaluated for cytotoxicity against isolated fractions. Cell viability assay was performed to evaluate the cytotoxicity of all fractions on different cell lines followed by the lactate dehydrogenase and vascular endothelial growth factor assays of most active fraction among all screened for cytotoxic analysis. HPLC analysis of most active fractions against cytotoxicity was performed to check the biological activity of compounds. Results displayed the potent cytotoxic activity of ethyl acetate fraction of S. nigrum against HepG2 cells with IC₅₀ value of 7.89 μg/ml. Other fractions exhibited potent anticancer activity against HepG2 cells followed by HeLa cells. Fractions in our study showed no cytotoxicity in BHK cells. Cytotoxic activity observed in our current study exposed high antiproliferative potential and activity of ethyl acetate fraction against HepG2 cells. The results demonstrated that S. nigrum fractions exhibited anticancer activity against hepatic and cervical cancer cell lines with non-toxic effect in normal cells. These results reveal significant potential of S. nigrum for the therapeutic of cancers across the globe in future.     

Establishment and characterization of two fetal fibroblast cell lines from the yak

The objective of this work was to not only establish two fetal fibroblast cell lines from yak lung and ear tissue using a primary explant technique and cell cryogenic preservation technology but also check for their quality and biological characteristics. The cells showed typical morphologic characteristics of fibrous and long spindle appearance. Outgrowth of fibroblast-like cells from the lung and ear explants was around 2 and 3 d, and reaching 90% confluence level was in the ninth day and the thirteenth day, respectively. Biological analysis showed that the average viability of the lung fibroblast cells (ear fibroblast cells) was 97.5% (95.0%) before freezing and 91.0% (89.5%) after thawing. Analysis of the growth of the fifth passage culture revealed an ??S??-shaped growth curve with the population doubling times of 30 h for lung fibroblast cell line and 35 h for ear fibroblast cell line. Karyotyping indicated the chromosome number of yak was 2n?=?60, comprising 29 pairs of autosomes and one pair of sex chromosomes (XY). All somatic chromosomes were telocentric autosomes except that the two sex chromosomes were submetacentric. Assays for bacteria, fungi, and mycoplasmas were negative. Immunocytochemical staining showed that the cells were positive for the expression of vimentin and negative for the expression of cytokeratin. In conclusion, two yak fetal fibroblast cell lines (YFLF and YFEF) from lung and ear explants are successfully established in culture. It will not only preserve the genetic resources of yaks at the cellular level but also provide valuable materials for somatic cell cloning and transgenic research.     

Bioactive Polymeric Nanoparticles for Periodontal Therapy

Aimsto design calcium and zinc-loaded bioactive and cytocompatible nanoparticles for the treatment of periodontal disease.MethodsPolymP-nActive nanoparticles were zinc or calcium loaded. Biomimetic calcium phosphate precipitation on polymeric particles was assessed after 7 days immersion in simulated body fluid, by scanning electron microscopy attached to an energy dispersive analysis system. Amorphous mineral deposition was probed by X-ray diffraction. Cell viability analysis was performed using oral mucosa fibroblasts by: 1) quantifying the liberated deoxyribonucleic acid from dead cells, 2) detecting the amount of lactate dehydrogenase enzyme released by cells with damaged membranes, and 3) by examining the cytoplasmic esterase function and cell membranes integrity with a fluorescence-based method using the Live/Dead commercial kit. Data were analyzed by Kruskal-Wallis and Mann-Whitney tests.ResultsPrecipitation of calcium and phosphate on the nanoparticles surfaces was observed in calcium-loaded nanoparticles. Non-loaded nanoparticles were found to be non-toxic in all the assays, calcium and zinc-loaded particles presented a dose dependent but very low cytotoxic effect.ConclusionsThe ability of calcium-loaded nanoparticles to promote precipitation of calcium phosphate deposits, together with their observed non-toxicity may offer new strategies for periodontal disease treatment.