Primary Culture Medium Research Articles

A novel medium for long-term primary culture of hemocytes of Metapenaeus ensis

The development of a suitable shrimp cell medium is essential for achieving a long-term cell culture and finite cell line of shrimps routinely. In this study, we have successfully developed an optimal shrimp cell medium that can be used for long-term in vitro culture and continuous subculture of the hemolymph cells (or hemocytes) of greasyback shrimp Metapenaeus ensis, designated as MeH cells, by shrimp serum-based and supplements-based optimization of the basic and growth medium. In this article, we have focused on the details for the preparation of the optimal shrimp cell medium by diluting and mixing of various stock solutions as well as the methods for isolation and primary culture of MeH cells.•A novel shrimp cell growth medium is developed for long-term shrimp hemocytes culture.•The preparation method of shrimp cell growth medium is successfully established.•Obvious cell activity and proliferation potential of isolated shrimp cells can be maintained beyond 30 days.

Adenosine promoted angiogenesis mediated by the release of small extracellular vesicles from human endothelial progenitor cells.

Endothelial progenitor cells (EPCs) are stem cells mainly derived from bone marrow; from where they migrate to repair and regenerate damaged tissues. eEPCs have been classified into two sub-populations, early (eEPC) and late EPCs (lEPC), depending on maturation stages in vitro. In addition, eEPC release endocrine mediators, including small extracellular vesicles (sEVs), which in turn may enhance the eEPC-mediated wound healing properties. Nevertheless, adenosine contributes to angiogenesis by recruiting eEPC at the injury site. However, whether ARs may enhance the secretome of eEPC, including sEVs, is unknown. Therefore, we aimed to investigate whether AR activation increase the release of sEVs in eEPC, which in turn has paracrine effects on recipient endothelial cells. Results shown that 5'-N-ethylcarboxamidoadenosine (NECA), a non-selective agonist, increase both the protein levels of the vascular endothelial growth factor (VEGF), and the number of sEVs released to the conditioned medium (CM) in primary culture of eEPC. Importantly, CM and EVs harvested from NECA-stimulated eEPC promote in vitro angiogenesis, without changes in cell proliferation, in recipient ECV-304 endothelial cells. This constitutes the first evidence showing that adenosine enhances sEVs release from eEPC, which has pro-angiogenic capacity on recipient endothelial cells.

Analysis of Aggrecan Glycanation by Western Blot in Cell Culture.

Several experimental protocols are available to study the synthesis and secretion of proteoglycans in health and diseases, but there are few methods to analyse the intracellular processing of these macromolecules. We report a western blot analysis on medium and cell layer of primary chondrocyte culture to determine the glycanation status of aggrecan. Using a specific antibody against the aggrecan core protein and digesting an aliquot of sample with chondroitinase ABC, it is possible to analyse the whole aggrecan macromolecule and the core protein in order to evaluate defects in aggrecan glycanation.

Neuroprotective action of α-Klotho against LPS-activated glia conditioned medium in primary neuronal culture

The α-Klotho is an anti-aging protein that, when overexpressed, extends the life span in humans and mice. It has an anti-inflammatory and protective action on renal cells by inhibiting NF-κB activation and production of inflammatory cytokines in response to TNF-α. Furthermore, studies have shown the neuroprotective effect of α-Klotho against neuroinflammation on different conditions, such as aging, animal models of neurodegenerative diseases, and ischemic brain injury. This work aimed to evaluate the effects of α-Klotho protein on primary glial cell culture against the proinflammatory challenge with LPS and how this could interfere with neuronal health. Cortical mixed glial cells and purified astrocytes were pretreated with α- α-Klotho and stimulated with LPS followed by TNFα, IL-1β, IL-6, IFN-γ levels, and NF-κB activity analysis. Conditioned medium from cortical mixed glia culture treated with LPS (glia conditioned medium (GCM) was used to induce neuronal death of primary cortical neuronal culture and evaluate if GCM-KL (medium from glia culture pretreated α-Klotho followed by LPS stimulation) or GCM + LPS in the presence of KL can reverse the effect. LPS treatment in glial cells induced an increase in proinflammatory mediators such as TNF-α, IL-1β, IL-6, and IFN-γ, and activation of astrocyte NF-κB. GCM treated-cortical neuronal culture induced a concentration-dependent neuronal death. Pretreatment with α-Klotho decreased TNF-α and IL-6 production, reverted NF-κB activation, and decreased neuronal death induced by GCM. In addition, KL incubation together with GCM + LPS completely reverts the neuronal toxicity induced by low concentration of GCM-LPS. These data suggest an anti-inflammatory and neuroprotective effect of α-Klotho protein in the CNS. This work demonstrated the therapeutic potential of α-Klotho in pathological processes which involves a neuroinflammatory component.

Inhibition of Cervical Cancer Cell Migration by Human Wharton’s Jelly Stem Cells

Background: One of the most prevalent malignancies to strike women, both in Iran and globally, is cervical cancer. Metastasis, which is a significant cause of mortality, is one of the most significant pathological processes of this cancer. Therefore, preventing the migration of cancer cells may be a useful therapeutic approach. The aim of this work was to investigate the impact of conditioned media and human Wharton's jelly stem cells (hWJSCs) on the migration and growth of the cervical cancer cell line Hela as well as the in vitro mRNA expression of genes involved in metastasis. Methods: After primary culture, cellular extract and conditioned medium of hWJSCs were prepared. The viability of cervical cancer cells was investigated by MTT assay after treatment with cellular extract and conditioned medium of hWJSCs. Moreover, the anti-migratory effects of cellular extract and conditioned medium of hWJSCs on the cervical cancer cells were evaluated by wound‐healing migration assay. Finally, the mRNA expression of migration-related genes (E-cadherin and Vimentin) was detected by real-time PCR. Results: Our results indicated that the cellular extract and conditioned medium of hWJSCs (with 32% concentration) inhibited the proliferation of 100% and 20% of Hela cancer cells, respectively. In addition, the cellular extract and conditioned medium of hWJSCs significantly decreased morphological alteration and migration of the cancer cells. The cellular extract and conditioned medium of hWJSCs modified the expression of Vimentin and E-cadherin genes to inhibit the cancer cell migration (p<0.05). However, the cellular extract indicated significantly profound inhibitory effects on the cervical cancer cells compared to the conditioned medium. Conclusions: Our study demonstrated that the cellular extract and conditioned medium of hWJSCs inhibit the proliferation and migration of cervical cancer cells through the modification of migration-related gene expression. However, further in vitro and in vivo studies are required for more accurate results.

337 DermaCult™, a Serum- and BPE-free Medium Supporting Long-term Feeder-free Expansion of Primary Human Epidermal Keratinocytes that Retain Their Differentiation Potential at Air Liquid Interface

Primary human epidermal keratinocytes (HEKs) typically undergo growth arrest at approximately 15 - 30 population doublings (PDs) when maintained in traditional feeder-free culture medium formulations. To circumvent this early senescence, we developed DermaCult™, a serum- and BPE-free medium that promotes long-term growth (> 30 PDs) of HEKs while maintaining their air liquid interface (ALI) differentiation potential. To measure cell expansion over the entire lifespan of the culture, HEKs derived from either neonatal or adult skin were thawed and seeded onto uncoated tissue cultureware at a density of 1 - 2.5 x 103 cells/cm2, and passaged when cultures reached approximately 50 - 70% confluence. At various time points, keratinocytes were analyzed by qPCR and flow cytometry to detect expression of basal and differentiated cell markers. In separate cultures, keratinocytes were also differentiated by increasing the calcium concentration in feeder- free monolayer culture or establishing ALI culture. Keratinocytes derived from adult or neonatal skin and cultured in DermaCult™ proliferated between 50 - 70 or >70 PDs before undergoing senescence, respectively (n=4). In monolayer cultures, keratinocytes predominantly maintained a cobblestone morphology, expressed basal cell markers (TP63, KRT14, ITGA6), and were devoid of suprabasal marker KRT10 (n=4). Induction of differentiation resulted in the expression of KRT10, LOR, and formation of ZO-1 tight junctions, with concomitant down-regulation of basal markers TP63 and KRT14. In ALI culture, HEKs generated a stratified epithelium with the appropriate cellular architecture and expression of markers similar to their in vivo counterparts. In summary, we have developed an improved and first BPE-free medium for primary HEKs culture that promotes greater expansion of HEKs and maintenance of their differentiation potential at later passages.

Direct effects of phenolic compounds on the mammary gland: In vivo and ex vivo evidence

We assessed the potential of Pistacia lentiscus (lentisk) phenolic compounds to enhance production of milk composition in lactating goats and caprine primary mammary epithelial cells (MEC). Damascus goats were given a lentisk infusion (LI) or fresh water (FW) to drink, in a crossover design. Milk from LI vs. FW goats was 0.43% richer in fat and 30% in omega 3 fatty acids. Lentisk infusion enhanced antioxidant capacity of plasma and milk by 37% and 30% respectively, and induced transcriptional activation of antioxidant genes. To assess the direct effect of polyphenols on milk quality in terms of composition and antioxidant capacity, we used plasma collected from goats fed hay (HP) or browsed on phenolic compounds-rich pasture (primarily lentisk; PP) as a conditioning medium for primary culture of MEC. PP increased 2-fold cellular triglyceride content and 2.4-fold intracellular casein, and increased ATP production and non-mitochondrial oxygen consumption. Taken together, the results imply that lentisk phenolic compounds affect blood, MEC and milk oxidative status, which increase fat production by the mammary gland.

Comparison of Chromogenic (HiCrome Urinary Tract Infection Agar) Medium with Cysteine Lactose Electrolyte Deficient Agar in a Resource-Limited Setting.

Background and Objectives:Urinary tract infections (UTI) are one of the most frequent infections encountered in hospital settings as well as in community, making urine the most cultured specimens in laboratories across the world. Urine samples occupy most of the time and manpower in the form of resources in the microbiology laboratories. The microbiological performance of HiCrome UTI agar was compared with cysteine lactose electrolyte deficient (CLED) agar for isolation and presumptive identification of bacteria from urine culture with ease of reporting with less human resource and reduction in the cost.Materials and Methods:The study was conducted in a total of 208 collected midstream catch urine samples from patients attending the Department of Microbiology, Khartoum Teaching Hospital Central Laboratories. Urine samples received in the bacteriology laboratory were inoculated on CLED agar and for HiCrome UTI agar simultaneously and incubated overnight. Isolates were identified by the colony's color for HiCrome UTI agar and by standard microbiological techniques for CLED agar.Results:Out of 208 urine samples tested, significant growth was obtained in 94 (45.2%) plates of CLED agar and 84 (40.4%) of HiCrome UTI; moreover, 15 (7.2%) and 28 (13.5%) plates showed mixed with no growth observed in 99 (47.6%) and 96 (46.1%) on CLED agar and HiCrome UTI agar, respectively. The rate of presumptive identification was found significantly higher on HiCrome UTI agar (94%) than CLED agar (84%) (P < 0.05) as the primary urine culture medium. out of 43 (100%) polymicrobial growths 28 (65.1%) were demonstrated distinctly on HiCrome UTI agar and only 15 (34.9%)were obtained fromCLED agar.Conclusions:HiCrome UTI agar was found to be more useful as a primary urine culture medium in both higher rate of isolation and presumptive identification of uropathogens in comparison to conventional media. Its inherent characteristics in demonstrating polymicrobial growth and ease of rapid identification by distinct colony color are unique.

Research on key techniques of tissue culture of Qinghai Rhubarb

Rheum palmatum L. is a perennial herb of the Rheum genus Polygonaceae. It is a traditional Chinese herbal medicine which has extremely high medicinal value. Rhubarb has scarce seeds in subtropical regions, and natural planting conditions have limitations. In recent years, the demand of rhubarb has increased, and the means of sowing and raising seedlings can no longer meet the needs of the market. Traditional sowing and propagation methods have limitations such as long cycle and low reproduction coefficient. Tissue culture technology can shorten the reproduction cycle that increase the reproduction coefficient and maintain the excellent characteristics of the parents, which is an effective way to promote the large-scale and commercial production of fine varieties of rhubarb. The results of the study showed that: (1) Analysis of the characteristics of Qinghai Rhubarb seeds. (2) Seed sterilization treatment. (3) Primary culture medium selection. (4) Callus induction. (5) Callus proliferation culture. (6) Hypocotyl callus bud differentiation.(7)Rooting culture. (8) Qinghai Rhubarb tissue culture seedling transplantation. Using the method of the present invention to establish a Qinghai Rhubarb in vitro culture aseptic propagation system, high-quality Qinghai Rhubarb seeds as explants and different hormone medium ratios, Qinghai Rhubarb hypocotyl induction rate reached 90%, and the proliferation coefficient reached 4.3. The survival rate of transplanting tissue cultured seedlings reaches 95% which can improve the reproduction efficiency of Qinghai Rhubarb, and provide theoretical basis and technical support for subsequent research on transgenic and genetic improvement. It can also provide a basis for medicinal value for the systematic research and commercial production of Qinghai Rhubarb.

Mechanism of lncRNA-SRLR induced invasion and metastasis in U2OS osteosarcoma cells

Objective: To explore the potential mechanism of sorafenib resistance associated long non-coding RNA (lncRNA-SRLR) promoted invasion and metastasis in U2OS osteosarcoma cells. Methods: We transfected U2OS cells with negative control lentivirus (LV-NC) or lncRNA-SRLR overexpressed lentivirus (LV-over/SRLR) particles. LV-NC and LV-over/SRLR stable transfected cells (U20S/NC and U20S/SRLR) were selected by primary cell culture medium containing puromycin. The mRNA expressions of lncRNA-SRLR and procollagen-lysine, procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The effect of lncRNA-SRLR on the invasion of U2OS cells were determined by wound-healing assay and Transwell migration assay. The effect of SRLR on the interleukin-6 (IL-6) secretion of U2OS cells was evaluated by enzyme-linked immunosorbent assay (ELISA) analysis. The subcellular distribution of SRLR in U2OS cells was detected by fluorescence in situ hybridization (FISH) analysis.The expression of PLOD2 in cells was detected by immunofluorescence (IF). The expressions of PLOD2 and focal adhesion kinase (FAK)/signal transducer and activator of transcription 3 (STAT3) signal pathway related proteins in U2OS/NC and U2OS/SRLR cells were detected by western blotting. Results: qRT-PCR assay showed that mRNA expressions of lncRNA-SRLR and PLOD2 in U2OS/SRLR cells were (3 964.97±0.05) and (2.77±0.11), respectively, significantly higher than those in U2OS/NC cells (P<0.001 or P<0.01). The results of wound-healing and Transwell migration assay showed that over-expression of SRLR markedly promoted the invasion ability of U2OS cells (P<0.05). The result of ELISA analysis showed that the IL-6 secretions in U2OS/NC or U2OS/SRLR cells were (125.38±11.22) pg/ml or (119.97±13.43) pg/ml, without statistical significance (P>0.05). The subcellular distribution assay revealed that lncRNA-SRLR is predominately located in the nucleus. The result of IF showed that compared with U2OS/NC cells, the expression of PLOD2 was up-regulated in U2OS/SRLR cells. The result of western blotting showed that over-expression of SRLR significantly increased the expression levels of PLOD2, phosphorylation (p)-FAK and p-STAT3 in U2OS cells (P<0.01). Conclusion: lncRNA-SRLR promotes invasion and metastasis of osteosarcoma by activating PLOD2-FAK/STAT3 signal axis.

Mouse whole embryo culture: Evaluating the requirement for rat serum as culture medium.

BackgroundWhole embryo culture is a valuable research method in mammalian developmental biology and birth defects research, enabling longitudinal studies of explanted organogenesis‐stage rodent embryos. Rat serum is the primary culture medium, and can sustain growth and development over limited periods as in utero. However, the cost, labor, and time to produce culture serum are factors limiting the uptake of the methodology. The goal of replacing or at least reducing rat serum usage in culture would be in accordance with the principles of “replacement, reduction, and refinement” of animals in research (the 3Rs).MethodsWe performed cultures of mouse embryos for 24 hr from embryonic day 8.5 in serum‐free media or in rat serum diluted with defined media, compared with 100% rat serum. Developmental parameters scored after culture included yolk sac circulation, dorsal axial length, somite number, protein content, and completion of cranial neural tube closure.ResultsA literature review revealed use of both serum‐free and diluted rat serum‐based media in whole embryo culture studies, but with almost no formal comparisons of culture success against 100% rat serum. Two serum‐free media were tested, but neither could sustain development as in 100% rat serum. Dilution of rat serum 1:1 with Glasgow Minimum Essential Medium plus defined supplements supported growth and development as well as whole rat serum, whereas other diluent media yielded substandard outcomes.ConclusionRat serum usage cannot be avoided, to achieve high quality mouse embryo cultures, but rat usage can be reduced using medium containing diluted serum.

Comparative Study of Hicrome Agar Medium with Conventional Culture System for the Isolation of Uropathogens

Objective: The present study was done to compare the performance of chromogenic agar medium and conventional culture media for the isolation and presumptive identification of uropathogen.Methodology: A total 300 sample were collected from Rajshahi Medical College Hospital, Bangladesh during January to June, 2008. Urine samples of the suspected UTI cases, showing pus cells &gt;5/HPF on microscopic examination were included for urine culture simultaneously onto 2 conventional media (Blood agar and MacConkey agar) and chromogenic agar medium (HiCrome UTI agar medium). Results: Culture yielded 139 (46.33%) bacterial growth among them, 133 (44.33%) showed single organism and remaining 06 (2.00%) showed mixed growth of two organisms in different combinations. It is evident from the present study that both HiCrome UTI agar and Blood agar (BA) media supported growth of all 145 bacteria, while MacConkey (MAC) agar yielded 133(91.72%) bacterial growths. The rate of presumptive identification of the isolates was found significantly higher (97.24%) on HiCrome UTI agar when compared with the MacConkey agar (80.68%) and Blood agar (27.58%) media. Out of 91 E. coli isolated, 88(96.70%) could be identified differentially on HiCrome UTI agar medium in contrast to 85(93.40%) on MacConkey agar and only 06(06.59%) on Blood agar. Again, all 06 (100%) of the isolate-pairs of mixed growth were identified distinctly on HiCrome UTI agar, whereas both Blood agar and MacConkey agar media could revealed only 01(16.66%) of the polymicrobial growth.Conclusion: HiCrome UTI agar medium has been documented for its very high yielding rate, rapid presumptive identification of both single and polymicrobial growths with greater precision and avoidance of biochemical tests for further identification of uropathogens. Thus it can be recommended as primary urine culture medium to be used by the clinical microbiology laboratories.TAJ 2011; 24(2): 128-135

Chromogenic Agar Medium : A Versatile Tool for the Diagnosis of UTI

Objective: The present study was done on Chromogenic agar media to identify uropathogens more efficiently by its characteristic colony colour for each of the organism.Methodology: A total 300 sample were collected from Rajshahi Medical College Hospital, Bangladesh. Urine samples of the suspected UTI cases, showing pus cells &gt;5/HPF on microscopic examination were included for urine culture simultaneously onto Chromogenic agar media, Blood agar and MacConkey agar media.Results: Culture yielded 139 (46.33%) bacterial growth among them, 133 (44.33%) showed single organism and remaining 06 (2.00%) showed mixed growth of two organisms in different combinations. It is evident from the present study that both Chromogenic agar media and Blood agar (BA) media supported growth of all 145 bacteria, while MacConkey (MAC) agar yielded 133(91.72%) bacterial growths. The rate of presumptive identification of the isolates was found significantly higher (97.24%) on Chromogenic agar media when compared with the MacConkey agar (80.68%) and Blood agar (27.58%) media. Out of 91 E. coli isolated, 88(96.70%) could be identified differentially on Chromogenic agar media in contrast to 85(93.40%) on MacConkey agar and only 06(06.59%) on Blood agar. Again, all 06 (100%) of the isolate-pairs of mixed growth were identified distinctly on Chromogenic agar media, whereas both Blood agar and MacConkey agar media could revealed only 01(16.66%) of the polymicrobial growth.Conclusion: Chromogenic agar media has been documented for its very high yielding rate, rapid presumptive identification of both single and polymicrobial growths with greater precision and avoidance of biochemical tests for further identification of uropathogens. Thus it can be recommended as primary urine culture medium to be used by the clinical microbiology laboratories.TAJ 2012; 25: 64-71

Effect of chemokine CC ligand 2 (CCL2) on α‑synuclein‑induced microglia proliferation and neuronal apoptosis.

The present study aimed to investigate the effect of chemokine CC ligand 2 (CCL2) on α-synuclein-mediated microglia proliferation and neuronal apoptosis. Primary cultured microglia and primary neurons were isolated and cultured in vitro. Microglia were divided into four groups: The cells in the control group were treated with an identical amount of PBS, whereas the cells in the CCL2 group were cultured in medium containing 0.05 ng/µl CCL2; cells in the α-synuclein group were treated with medium containing 0.2 ng/µl α-synuclein; and cells in the CCL2 plus α-synuclein group were cultured in medium containing 0.05 ng/µl CCL2 and 0.2 ng/µl α-synuclein. After incubation for 24 h, the proliferation of glial cells, and the level of α-synuclein in the cells, were measured. The levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and nitric oxide (NO) in the culture medium were also measured. Levels of cleaved caspase-3, Akt and phosphorylated (p)-Akt in neurons treated with primary microglia culture medium in each group were subsequently monitored. The proliferation activity and secretion of TNF-α, IL-1β and NO in the CCL2, α-synuclein and CCL2 plus α-synuclein groups were significantly higher compared with that in the control group (P<0.05), as were the levels of α-synuclein (P<0.01). The levels of neuronal apoptosis and cleaved caspase-3 protein in the CCL2, α-synuclein and CCL2 plus α-synuclein groups were also significantly higher compared with that in the control group (P<0.01). Taken together, these results have demonstrated that CCL2 is able to promote α-synuclein secretion and the apoptosis of neurons induced by α-synuclein, thus inducing proliferation of the microglia and secretion of TNF-α, IL-1β and NO.

Research on influence mechanism of G protein coupled receptor kinase interacting protein 1 on differentiation of bone marrow mesenchymal stem cells into endothelial cells

To investigate the mechanism of G protein coupled receptor kinase interacting protein 1 (GIT1) affecting angiogenesis by comparing the differentiation of bone marrow mesenchymal stem cells (BMSCs) differentiated into endothelial cells between GIT1 wild type mice and GIT1 gene knockout mice. Male and female GIT1 heterozygous mice were paired breeding, and the genotypic identification of newborn mice were detected by PCR. The 2nd generation BMSCs isolated from GIT1 wild type mice or GIT1 gene knockout mice were divided into 4 groups, including wild type control group (group A), wild type experimental group (group A1), GIT1 knockout control group (group B), and GIT1 knockout experimental group (group B1). The cells of groups A1 and B1 were cultured with the endothelial induction medium and the cells of groups A and B with normal cluture medium. The expressions of vascular endothelial growth factor receptor 2 (VEGFR-2), VEGFR-3, and phospho-VEGFR-2 (pVEGFR-2), and pVEGFR-3 proteins were detected by Western blot. The endothelial cell markers [von Willebrand factor (vWF), platelet-endothelial cell adhesion molecule 1 (PECAM-1), and vascular endothelial cadherin (VE-Cadherin)] were detected by flow cytometry. The 2nd generation BMSCs of GIT1 wild type mice were divided into 4 groups according to the different culture media: group Ⅰ, primary cell culture medium; group Ⅱ, cell culture medium containing SAR131675 (VEGFR-3 blocker); group Ⅲ, endothelial induction medium; group Ⅳ, endothelial induction medium containing SAR131675. The endothelial cell markers (vWF, PECAM-1, and VE-Cadherin) in 4 groups were also detected by flow cytometry. Western blot results showed that there was no obviously difference in protein expressions of VEGFR-2 and pVEGFR-2 between groups; and the expressions of VEGFR-3 and pVEGFR-3 proteins in group A1 were obviously higher than those in groups A, B, and B1. The flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group A1 than in groups A, B, and B1 ( P<0.05), and in group B1 than in groups A and B ( P<0.05); but no significant difference was found between groups A and B ( P>0.05). In the VEGFR-3 blocked experiment, the flow cytometry results showed that the expressions of vWF, PECAM-1, and VE-Cadherin were significantly higher in group Ⅲ than in groupsⅠ, Ⅱ, and Ⅳ, and in group Ⅳ than in groups Ⅰ and Ⅱ ( P<0.05); but no significant difference was found between groups Ⅰ and Ⅱ ( P>0.05). GIT1 mediates BMSCs of mice differentiation into endothelial cells via VEGFR-3, thereby affecting the angiogenesis.

Influence of nutrient medium composition on the morphological characteristics of culture of dorsal root ganglion cells of neonatal piglets

Dorsal root ganglion (DRG) is a potential source of neural stem cells because it contains neural crest derived cells that are capable to differentiate into neurons and glial cells. Cell cultures obtained from animals that are close to humans by physiological characteristics can be regarded as an adequate modern model for in vitro studies. In this respect, DRG cell culture obtained from the domestic pig (Sus scrofa domesticus) is a convenient model. The aim of the work was to obtain a primary cell culture of DRG of neonatal piglets and to study its morphological and proliferative properties depending on culture medium composition. The composition of the media prepared on the basis of α-MEM varied depending on the presence of fetal calf serum (FCS) or its modern supplements B-27 and NeuroMax. It is established that morphological differences of primary DRG cell cultures of neonatal pigs depend on the composition of the nutrient medium. When cultured in the presence of 10% FCS, the formation of monolayer which includes satellite glial cells (SGC) and fibroblast-like cells was observed. Small colonies of neurons producing long processes were on the monolayer. When cultured in the presence of NeuroMax and B-27 supplements, the bulk of the cells is not attached, but organized into floating multicellular spheroids (MS). With the passage of culture obtained in the presence of 10% FCS, rapid attachment and proliferation of cells was observed. When MS obtained in the presence of NeuroMax and B-27 were transferred to the medium with 10% FCS, the attachment of MS to the substrate and cell migration were observed. The cells retain the ability to actively proliferate, because the monolayer achieves confluence by 5–7 days of subculture. Regardless of the composition of the primary culture medium, there were 3 morphologically different types of cells in the subcultures: SGC, neuron-like and fibroblast-like cells. The type of cells prevailing in the subculture depends on the composition of the nutrient medium. When MS is transferred from a B-27-containing medium, a significant growth of fibroblast-like cells is observed, whereas when MS is transferred from NeuroMax-containing medium MG and neuron-like cells were abundant.

Inflammatory response in craniopharyngiomas and its clinical significance

Objective To investigate the clinical significance of inflammatory reaction in craniopharyngiomas. Methods Antibody microarray was employed for detection of inflammatory cytokines expression in craniopharyngiomas and other common tumors in sellar region (including the pituitary adenoma, meningioma, pilocytic astrocytoma). The methods of qPCR and ELISA were further used for verifying the expression of inflammatory factors in each group. At the same time, the mouse brain neuron cell line (CATH.a) was treated with supernatant of the primary culture medium of craniopharyngiomas, and the apoptotic state of neurons was explored by flow cytometry. Results The craniopharyngioma was more likely to have an inflammatory response than other sellar tumors. The results of antibody microarray showed that the relative expression levels of monocyte chemotactic protein-1 (MCP-1), interleukin 6 (IL-6) and interleukin 1 (IL-1) in adamantinomatous craniopharyngiomas (ACP) (7±1, 6±1, 8±2) were significantly higher than those in papillary craniopharyngiomas (PCP) (2±1, 2±1, 2±1), pilocytic astrocytomas (2±1, 1±0, 2±1), pituitary adenomas (1±0, 2±1, 2±1) and meningiomas (1±0, 1±0, 1±0) (all P<0.05). The qPCR results further verified that the transcriptional levels of 3 inflammatory factors mentioned above in ACP (180±24 pg/ml, 266±19 pg/ml, 243±31 pg/ml) were significantly higher than those in PCP (68 ±14 pg/ml, 98 ±29 pg/ml, 154±21 pg/ml), pilocytic astrocytomas (60±14 pg/ml, 142±29 pg/ml, 149±39 pg/ml), pituitary adenomas (180±24 pg/ml, 266±19 pg/ml, 134±27 pg/ml) and meningiomas (56±14 pg/ml, 89±19 pg/ml, 121±21 pg/ml) (all P<0.05), while Elisa results showed that three kinds of inflammatory factors in the ACP primary cell culture medium supernatant (168±19 pg/ml, 155±34 pg/ml, 228±31 pg/ml) were significantly higher than those in PCP (66±24 pg/ml, 89±19 pg/ml, 112±21 pg/ml) (all P<0.05). After CATH.a cells were stimulated by ACP medium supernatant, their apoptosis increased significantly, while the promotion of apoptosis by supernatant of PCP medium was not obvious. Immunohistochemistry results showed that the inflammatory response in ACP tissue was characterized by infiltration of macrophage inflammatory cells; and activated microglias were observed in the front area between tumor and the third ventricle floor. Conclusions Inflammatory reaction could be found in the craniopharyngioma and the levels of MCP-1, IL-6 and IL-1 were higher in ACP than PCP. The inflammatory reaction of craniopharyngioma seems toxic to CATH.a cells. Key words: Craniopharyngioma; Inflammation reaction; Prognosis; Endocrine

Generation of embryonic stem-like cells from in vivo-derived porcine blastocysts at a low concentration of basic fibroblast growth factor.

Although basic fibroblast growth factor (bFGF) is an essential factor supporting the maintenance of porcine embryonic stem (ES) cell self-renewal and pluripotency, its high cost has limited previous studies, and the development of a low-cost culture system is required. For these systems, in vivo blastocysts were progressively cultured under various conditions consisting of different culture mediums and/or different feeder cell numbers at a low concentration of bFGF. As the results, the sequential culture of in vivo-derived porcine blastocysts on 5.0×105 mouse embryonic fibroblast (MEF) feeder cells in alpha minimum essential medium-based medium for primary culture, on 2.5×105 MEF feeder cells in Mixture medium for the 1st subpassage, and on 2.5×105 MEF feeder cells in DMEM/Ham's F10-based medium for the post-2nd subpassage could support the establishment and maintenance of porcine ES-like cells at the low concentration of bFGF. The established porcine ES-like cells showed ES cell-specific characteristics such as self-renewal and pluripotency. We confirmed that porcine ES-like cells could be generated from in vivo-derived porcine blastocysts at a low concentration of bFGF.

Involvement of thromboxane A2 in interleukin-31-induced itch-associated response in mice.

Atopic dermatitis is a chronic and severe pruritic skin disease. Interlukin-31 (IL-31) has been recently demonstrated to be one of the key pruritogens in atopic dermatitis. However, the mechanisms underlying IL-31-induced itching remains unclear. In our previous study, we have shown that thromboxane (TX) A2 is involved in itch-associated responses in mice with atopy-like skin diseases. IL-31 was given intradermally into the rostral back of ICR mice and the hind-paw scratching to the injection site were counted. Expression of TX synthase and IL-31 receptors were analyzed using immunohistochemical staining or RT-PCR in mouse skin or primary cultures of mouse keratinocytes. The concentration of TXB2, a metabolite of TXA2, in the skin and the culture medium of primary cultures of mouse keratinocytes was measured using enzyme immunoassay. The concentration of intracellular Ca2+ ions in mouse keratinocytes was measured using the calcium imaging method. An intradermal injection of IL-31 elicited scratching, an itch-related response, in mice. The scratching was inhibited by TP TXA2 receptor antagonist DCHCH. The distribution of TX synthase and IL-31RA receptor was mainly epidermal keratinocytes in the skin. The primary cultures of keratinocytes expressed the mRNAs of TX synthase and IL-31 receptors. IL-31 increased the concentration of TXB2, which was inhibited by TX synthase inhibitor sodium ozagrel and EGTA, in the skin and the culture medium of primary cultures of keratinocytes. IL-31 increased the concentration of intracellular Ca2+ ions in mouse keratinocytes. It is suggested that IL-31 elicits itch-associated responses through TXA2 produced from keratinocytes.

Primary culture of Zhikong scallop Chlamys farreri hemocytes as an in vitro model for studying host-pathogen interactions

Primary cultured cells can be a useful tool in studies on physiology, virology, and toxicology. Hemocytes play an important role in animal rapid response to pathogen invasion. In this study, an appropriate medium for primary culture of hemocytes of the bivalve Chlamys farreri was developed by adding 5% fetal bovine serum and 1% C. farreri serum to Leibovitz L-15 medium. These primary cultured hemocytes were maintained for more than 40 d in vitro and were classified into 3 types: (1) granulocytes containing numerous granules in the cytoplasm, (2) hyalinocytes with no or few granules, (3) a small percentage of macrophage-like cells. Furthermore, the primary cultured hemocytes were observed to be sensitive to bacterial and viral challenges. These hemocytes could phagocytose the bacterium Vibrio anguillarum, and presented cytopathic effects on the extracellular products (ECPs) of V. anguillarum; the mRNA level of QM, which plays an important role in immune response, also significantly increased 12 h after infection. When these hemocytes were challenged with ostreid herpesvirus 1 (OsHV-1), virus particles and empty capsids in the cells infected for 48 h were observed by transmission electron microscopy, and the QM mRNA level increased significantly at 12 h and 24 h following OsHV-1 challenge. This primary culture system is available for C. farreri hemocytes which can be used in the future to study host-pathogen interactions.