Explant Method Research Articles

5‐Fluorouracil Induces Vascular Calcification in Rat Aortic Smooth Muscle Cells

Background 5-Fluorouracil (5-FU) is a commonly prescribed chemotherapy used to treat malignancies of the gastrointestinal tract, breast, and head/neck. Despite its high clinical efficacy in treating cancer, it is associated with late occurring cardiotoxicity, specifically increases in arterial stiffness along with a heightened risk for developing heart failure and cardiovascular disease. Because increases in arterial stiffness and vessel calcification can occur concurrently and are associated with cardiovascular disease outcomes, we determined if 5-FU treated aortic smooth muscle cells adopted a calcified phenotype. We hypothesized 1) aortic smooth muscle cells resected from 5-FU treated rats would have a faster calcification onset when challenged with inorganic phosphate media and 2) a single in vitro dose of 5-FU in healthy cells would result in a calcified phenotype. Methods Primary aortic smooth muscle cell culture (ASMC) was performed via resection of rat thoracic aorta from 6-month-old Sprague Dawley rats (n= 8) that were treated with a clinically relevant continuous infusion of 5-FU (50mg/kg and 270mg/kg) or saline equivalent volume. After treatment and resection, ASMC were cultured using explant methods until cells were identifiable via smooth muscle actin staining. Cells derived from each group were cultured with normal media (NM) or media including a pathological concentration (3.2 mmol) of inorganic phosphate media (PI) for 5, 8, or 10 days to determine differences in calcification onset between conditions. In a second experiment, healthy rat aortic smooth muscle cells were incubated in NM or media containing 7x10-3 5-FU (NM + 5-FU) to determine if a single dose, in vitro, directly results in calcification. Calcification was quantified using an Alizarin Red staining assay and colorimetric calcium assay. Data are expressed as Alizarin Red Stain concentration (mM) and total calcium (mg/dL). Results In experiment 1, 5-FU+PI presented with significantly higher concentration of Alizarin Red at days 8 (.043 vs .031; p=.048) and 10 (.068 vs .017mM; p<0.0001) compared to 5-FU+NM whereas no differences were found between CON+PI and CON+NM at any timepoint (all p>0.05). These data were confirmed via Calcium assay in both 5-FU (.346 vs.235 mg/dl; p =0.0039) and CON (.293 vs .269 mg/dl; p=0.69) at the 8-day timepoint. Experiment 2 indicated that healthy rat aortic smooth muscle cells treated directly with 5-FU for 72 h expressed significantly greater calcium absorbance compared to CON (5-FU = .3347 mg/dl, CON = .2362 mg/dl; p=0.02). Conclusion After treatment with 5-FU, ASMCs from rat thoracic aorta explants are prone to a calcification phenotype compared to saline control treated cells. This predisposition with treatment with 5-FU is an ongoing investigation into the possible molecular mechanisms involved in oxidative stress signaling.

Isolation and Cryopreservation of Placental Cells: Search for Optimal Biotechniques in Experimental and Regenerative Medicine

High efficacy of placental cells application necessitates their investigation. Preclinical studies require an improvement of the methods for obtaining, standardizing and storage of placental cells of experimental animals. Cells were isolated from rats and mice placentas by means of different enzymatic methods and the one of explants. Cells were cryopreserved with DMSO in DMEM using two-stage freezing. The number, morphological, cultural, metabolic features of cells were studied after isolation and storage. The maximum number of viable cells from the placentas of mice and rats was found to be obtained using the explant method or trypsin with ETDA. Cell cultures from mice and rats placentas after the third passage had stable morphofunctional characteristics. The viability of warmed rat placental cells according to dye exclusion was (92.3 ± 1.6)%, according to the adhesive test this was (81.3 ± 5.8)%. For mice placental cells, these values were (86.7 ± 3.7)% and (79.2 ± 8.1)%, correspondingly. The research results enabled the determining of effective biotechniques for obtaining the cryopreserved placental cells of rats and mice to perform preclinical studies of their biological effect in models of allo- and autotransplantations.

TEGDMA (Triethylene Glycol Dimethacrylate) Induces Both Caspase-Dependent and Caspase-Independent Apoptotic Pathways in Pulp Cells.

Monomers leached from resin-based composites (RBCs) may reach intrapulpal concentrations of the millimolar (mM) range, which could contribute to inflammation. The aim of this investigation was to assess the cytotoxicity of triethylene glycol dimethacrylate (TEGDMA) monomers on pulp cells as well as to identify molecular mechanisms leading to apoptosis. Pulp cells were harvested from molars extracted for orthodontic reasons and cultured through an explant method. To assess cytotoxicity, cells underwent a 5-day exposure to 0.75, 1.5, and 3 mM TEGDMA and were subject to cell counting and WST-1 staining. Based on the findings, cells were subsequently exposed to 0.1, 0.2, 0.75, 1.5, and 3 mM TEGDMA for 24 h to uncover the details of apoptosis. Changes in the production or cleavage of the apoptosis-specific proteins caspase-8, caspase-9, caspase-3, caspase-12, and Apoptosis-Inducing Factor (AIF) were measured by Western blot. The 5-day study showed concentration- and time-dependent cytotoxicity. Significant cell death was detected after 24 h with TEGDMA concentrations of 1.5 and 3 mM. One-day exposure to TEGDMA led to the activation of caspase-8, -9, -3, and -12 and an increased AIF production. Results suggest that relevant concentrations of TEGDMA monomers, leached from RBCs, induce apoptosis in pulp cells through both caspase-dependent as well as caspase-independent mechanisms. Endoplasmic reticulum stress and the activation of caspase-independent apoptotic pathways may be further mechanisms by which monomers induce apoptosis in pulp cells.

Preparation of primary glial tumor cell lines

Objective. The aim of this work was to obtain the primary cell lines of brain malignant tumors using the explant method.&#x0D; Materials and methods. Thirteen patients of both sexes, aged 22 to 66, were recruited. The tumor material of the patients was fragmented and placed in flasks with complete nutrient medium for glial tumor cells. Subsequently, the material was photographed at various stages of cultivation, the cell morphology was determined, and the rate of monolayer formation at the zero and first passages was assessed.&#x0D; Results. As a result, thirteen primary human cell lines of glial tumors were obtained: six glioblastoma lines, two glioblastoma lines with anaplastic astrocytoma, one anaplastic oligodendroglioma line, one diffuse astrocytoma line, one oligoastrocytoma line and one diffuse protoplasmic astrocytoma line, one anaplastic astrocytoma line. In the culture of diffuse astrocytoma, there were observed the cells forming a network at the bottom of the flask. In the culture of anaplastic astrocytoma at a confluence of 3050 %, fibroblast-like cells were presented, and at a confluence of 100 %, a monolayer was formed with cells intimately adjacent to each other. In the culture of oligoastrocytoma, both fibroblast-like cells and islets of closely intertwined fusiform cells were observed. The same was typical for the cells of diffuse protoplasmic astrocytoma. Anaplastic oligodendroglioma during the first week of cultivation was represented mainly by round cells with a contrast agent, which subsequently attached and actively proliferated. At a confluence of 3080 %, fibroblast-like cells were observed, and at 100 %, spindle-shaped cells closely adjacent to each other. In cultures of glioblastomas, no specific character of cell growth was revealed: spindle-shaped, fibroblast-like cells and cells with long processes forming a network were encountered. Glioblastoma cultures against the background of anaplastic astrocytoma were represented by a network of cells with long processes.&#x0D; At the zero passage, the rate of formation of a 100 % confluence monolayer ranged from 22 to 85 days. At the first passage, the cells reached a full monolayer within 4 to 25 days. At the zero passage, the longest time among all the samples to form the monolayer with a 100 % confluence needed glioblastoma lines on average 59 days. The shortest time to reach a 100 % confluence was required for cells of diffuse astrocytoma, anaplastic oligodendroglioma and glioblastoma against the background of anaplastic astrocytoma 2224 days.&#x0D; Conclusions. In our work, it was shown that the explant method ensures the production of viable cells of glial tumors and the possibility of their further cultivation.

Compare features of human dental pulp cells cultured from pulp tissues with and without cryopreservation.

Teeth extracted are usually disposed as bio-waste whereas they could serve as an autologous tissue for culturing multipotent dental pulp cells which have application potential in regenerative medicine. This study aimed to examine the feasibility of cryopreserving dental pulp tissue at teeth extraction for later culturing of cells. The pulp tissue from each of a total of 10 teeth was cut into small fragments which were then divided into two portions. One portion was directly used for culturing pulp cells using the explant method. The other portion was cryopreserved with 10% DMSO in liquid nitrogen for at least one month and then thawed for culturing pulp cells. Vital cells were obtained from all the 10 pulp fragment suspensions which went through cryopreservation. The cell outgrowth from the explants of cryopreserved pulp fragments was two days later than that of corresponding fresh pulp tissue. Otherwise, no difference was observed in proliferation, expression of stem cell markers and differentiation into adipose cells and osteoblasts between the two groups of cells cultured from the fresh or the cryopreserved pulp fragments. Cryopreserving fragmented dental pulp tissue provides a feasible option for saving pulp tissues as autologous cell sources for possible later application.

Мікроскопічне дослідження біосумісності зразків з вмістом мультипотентних мезенхімальних стромальних клітин жирової тканини в умовах експерименту

The purpose of the study was to investigate the biocompatibility of samples containing multipotent mesenchymal stromal cells of adipose tissue to replace bone defects. Material and methods. The study was conducted at Bukovina State Medical University, Chernivtsi, Ukraine. Adipose tissue samples were obtained from the neck of 60 experimental animals (white Wistar rats). We selected 4 samples for the toxicological experiment, which allowed to establish the direct influence of factors in the contact of implantation material at the cellular level. Sample № 1 - Multipotent mesenchymal stromal cells of adipose tissue, which underwent osteogenic differentiation; № 2 - Multipotent mesenchymal stromal cells of adipose tissue with osteogenic differentiation with the addition of platelet-enriched blood plasma; № 3 - “Kolapan” with applied tissue culture of Multipotent mesenchymal stromal cells of adipose tissue cells, which underwent osteogenic differentiation; № 4 - "Kolapan" + Multipotent mesenchymal stromal cells of adipose tissue + platelet-enriched plasma. Multipotent mesenchymal stromal cells of adipose tissue were obtained by grinding adipose tissue of rats in 0.1% collagenase 1A [14]. The study of biocompatibility by cell culture in vitro was performed in accordance with the Working Instruction № 04/2013-VL. The cultures were investigated by the explantation method in a plasma clot in Karelian vials. In order to standardize the nature of growth, their zones were classified into compact, reticular and migrating cells of growing fibroblastic tissues. To assess the probability of the obtained results of the study we used a variation-statistical method of analysis using Microsoft Excel. Statistical calculation of the results of clinical and laboratory studies was carried out according to conventional methods. Results and discussion. Microscopic examination of the surface of samples with culture of fibroblasts showed their satisfactory adhesion on the tooth surface after 5 days of cultivation. In the study of sample № 1 (Multipotent mesenchymal stromal cells of adipose tissue, with osteogenic differentiation), it was noted that the structure of the cells acquired a rounded and oval shape, which indicated their destruction and damage. On the 5th day of observation, cells with numerical intussusception and processes were observed during visual examination of sample № 3 (“Kolapan”, with applied culture of Multipotent mesenchymal stromal cells of adipose tissue). In the study of samples № 4 (Multipotent mesenchymal stromal cells of adipose tissue + platelet-enriched plasma + "Kolapan") on the 5th day of research, signs of growth were manifested by migration of fibroblastic elements that had a spindle-shaped and polygonal shape, with the formation of the primary zone due to strands. On the 7th day of cultivation in experimental samples № 2, № 3, № 4 there was the formation of three growth zones: compact - from cells of polygonal and spindle-shaped form; reticulate - from strands and bundles of cells that were located reticulate and areas of single migrating elements of spindle-shaped. External characteristics and cell growth surface did not differ from control samples. On the 10th day of cultivation in the experimental samples, as well as in the control, the areas of compact and reticular growth zone and the zone of migrating fibroblasts were increased. At the same time, tissue-like growth of cells was observed. Visualization of compact and stack-like zones of the studied experimental samples revealed signs of the beginning of degenerative changes, which was characterized in the form of rounding of the shape and vacuolation of cells. This trend was most pronounced in samples № 2 and № 4. Conclusion. Thus, tissue equivalents of bone tissue based on Multipotent mesenchymal stromal cells of adipose tissue can be candidates for use in regenerative medicine, and studies of their application in experimental animals will provide an opportunity to expand the understanding of the characteristics of Multipotent mesenchymal stromal cells of adipose tissue in order to optimize their further clinical application and implement new approaches in different areas of dentistry

Establishment and Characterization of Eye Muscle Cell Line from Snow Trout, Schizothorax richardsonii (Gray, 1832), a Vulnerable Coldwater Fish, for In Vitro Studies

Schizothorax richardsonii, commonly called snow trout is a vulnerable coldwater fish distributed in India and adjoining countries, is an important resource as food, ornamental and game fishery. A cell line, designated as SREM-1, has been established from the eye muscle of S. richardsonii using explant method and cultured in Leibovitz’s L-15 medium. The growth studies at 20-32°C temperatures and 5-20% of fetal bovine serum (FBS) concentrations revealed that SREM-1 cells exhibited optimal growth at 28°C in L-15 medium containing 20% FBS. Cell type was confirmed primarily as epithelial in nature by immuno-phenotyping assay and was sub-cultured for &gt;55 times since its development. The origin of the cell line was confirmed by sequencing of cytochrome oxidase c subunit I and 16S rRNA genes. SREM-1 cell line is free from mycoplasma contamination and has been cryopreserved at different passage levels with revival efficiency of 70-80% after 6 months. The SREM-1 cell line was successfully transfected and also employed for evaluating in vitro cytotoxicity against a heavy metal, mercuric chloride. The newly established cell line of this vulnerable species would be useful as a model for aquatic toxicological and transfection studies.

Infusion of Human Mesenchymal Stem Cells Improves Regenerative Niche in Thioacetamide-Injured Mouse Liver.

This study investigated whether xenotransplantation of human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) reduces thioacetamide (TAA)-induced mouse liver fibrosis and the underlying molecular mechanism. Recipient NOD/SCID mice were injected intraperitoneally with TAA twice weekly for 6weeks before initial administration of WJ-MSCs. Expression of regenerative and pro-fibrogenic markers in mouse fibrotic livers were monitored post cytotherapy. A hepatic stallate cell line HSC-T6 and isolated WJ-MSCs were used for in vitro adhesion, migration and mechanistic studies. WJ-MSCs were isolated from human umbilical cords by an explant method and characterized by flow cytometry. A single infusion of WJ-MSCs to TAA-treated mice significantly reduced collagen deposition and ameliorated liver fibrosis after 2-week therapy. In addition to enhanced expression of hepatic regenerative factor, hepatocyte growth factor, and PCNA proliferative marker, WJ-MSC therapy significantly blunted pro-fibrogenic signals, including Smad2, RhoA, ERK. Intriguingly, reduction of plasma fibronectin (pFN) in fibrotic livers was noted in MSC-treated mice. In vitro studies further demonstrated that suspending MSCs triggered pFN degradation, soluble pFN conversely retarded adhesion of suspending MSCs onto type I collagen-coated surface, whereas pFN coating enhanced WJ-MSC migration across mimicked wound bed. Moreover, pretreatment with soluble pFN and conditioned medium from MSCs with pFN strikingly attenuated the response of HSC-T6 cells to TGF-β1-stimulation in Smad2 phosphorylation and RhoA upregulation. These findings suggest that cytotherapy using WJ-MSCs may modulate hepatic pFN deposition for a better regenerative niche in the fibrotic livers and may constitute a useful anti-fibrogenic intervention in chronic liver diseases.

Peculiarities of the metabolic activity of mesenchimal stem cells under oxidative stress

Aim. Oxidative stress is considered to be one of the major damaging factors that limits the therapeutic potential of mesenchymal stem cells (MSCs). The purpose of our work was to study the metabolic activity of Wharton jelly-derived MSC of different donor origin under oxidative stress conditions induced by hydrogen peroxide. Methods. MSC were obtained by the explant method and cultured according to standard methods. Oxidative stress was caused by treating cells with different concentrations of hydrogen peroxide. The metabolic activity of MSCs was evaluated using the MTT test. Results. Analysis of the MTT test showed a biphasic dependence of the MSC response to the concentration of H2O2. Concentrations of hydrogen peroxide from 6.25 to 50 μM increased the level of metabolic activity of MSCs, and concentrations from 50 to 440 μM inhibited metabolic activity. The maximum stimulating effect was observed at concentrations of 12.5 μM and 25 μM depending on the donor. Conclusions. The response of cells to oxidative stress corresponded to the hormetic dependence, and the points of critical concentration and maximum stimulation were individual for each donor. Processes such as preconditioning MSCs with hydrogen peroxide to increase their survival rate during transplantation also require personalization of the approach depending on the points of maximum stimulation.&#x0D; Keywords: mesenchymal stem cells, hydrogen peroxide, oxidative stress.

THE CHARACTERISTICS OF RAT EMBRYONIC FIBROBLASTS (REFS) AND RAT BONE MARROW DERIVED MESENCHYMAL STEM CELLS (RAT-BMMSCS) FROM THE WISTAR RAT: A COMPARATIVE STUDY

Objective: Rat embryonic fibroblasts (REFs) and rat bone marrow-derived mesenchymal stem cells (rat-BMMSCs) an be used as in vitro models for a variety of studies, including for degenerative diseases such as arterial ischemia, tissue engineering and development of induced pluripotent stem cells (iPSCs). Therefore, the further developments of the use of these two cells of great importance.&#x0D; Methods: The experiments were performed with Wistar rat, those with 15-17 d gestation (aged 32 w) as a REFs source and those aged 12 w as a BMMSCs source. Dulbecco's modified eagle medium (DMEM) was used for both cell cultures but with different media supplements. Proliferation ability was determined for both by calculating population doubling time (PDT). Characterization was performed by differentiation testing into osteocyte, chondrocyte and adipocyte cells by staining with Alizarin Red, Alcian Blue and Oil Red O and by an investigation of specific antigen characteristics using flow cytometry with positive CD90 and CD29 and negative CD34 markers.&#x0D; Results: Morphologically, the REFs and rat-BMMSCs had the same fibroblasts like shape. PDT was higher for the REFs than the BMMSCs (p&lt;0.05), and both could differentiate into osteocytes, chondrocytes and adipocyte. The characteristics of the positive markers (CD29 and CD90) were higher in rat-BMMSCs than in REFs.&#x0D; Conclusion: In this study demonstrated that the explant method for REFs isolation and flushing method for rat-BMMSC isolation are both effective. It also showed that rat-BMMSC grow faster than REFs, and that both cells have the same differentiation ability as rat-BMMSCs but with different specific surface antigen characteristics.

Allogeneic umbilical cord-derived mesenchymal stem cells for treating critical-sized bone defects: a translational study.

The current 'gold-standard' treatment of critical-sized bone defects (CSBDs) is autografts; however, they have drawbacks including lack of massive bone source donor site morbidity, incomplete remodeling, and the risk of infection. One potential treatment for treating CSBDs is bone marrow-derived mesenchymal stem cells (BM-MSCs). Previously, there were no studies regarding the use of human umbilical cord-mesenchymal stem cells (hUC-MSCs) for treating BDs. We aim to investigate the use of allogeneic hUC-MSCs for treating CSBDs. We included subjects who were diagnosed with non-union fracture with CSBDs who agreed to undergo hUC-MSCs implantation. All patients were given allogeneic hUC-MSCs. All MSCs were obtained and cultured using the multiple-harvest explant method. Subjects were evaluated functionally using the Lower Extremity Functional Scale (LEFS) and radiologically by volume defect reduction. A total of seven (3 male, 4 female) subjects were recruited for this study. The subjects age ranged from 14 to 62years. All seven subjects had increased LEFS during the end of the follow-up period, indicating improved functional ability. The follow-up period ranged from 12 to 36months. One subject had wound dehiscence and infection, and two subjects developed partial union. Umbilical cord mesenchymal stem cells are a potential new treatment for CSBDs. Additional studies with larger samples and control groups are required to further investigate the safety and efficacy of umbilical cord-derived mesenchymal stem cells for treating CSBDs.

Comparison of umbilical cord tissue-derived mesenchymal stromal cells isolated from cryopreserved material and extracted by explantation and digestion methods utilizing a split manufacturing model

Background aimsUmbilical cord (UC) tissue is recognized as an advantageous source of mesenchymal stromal cells (MSCs), whose therapeutic properties are being actively evaluated in pre-clinical and clinical trials. In recognition of its potential value, storage of UC tissue or cells from UC tissue in newborn stem cell banks is now commonplace; however, strategies for isolating UC-derived MSCs (UCMSCs) from UC tissue have not been standardized. The majority of newborn stem cell banks take one of two approaches to cord tissue processing and cryopreservation: enzymatic digestion of the fresh tissue with cryopreservation of the subsequent cell suspension or cryopreservation of the tissue as a composite whole with later, post-thaw isolation of cells by explantation. Evaluation of UCMSCs derived by these two principal preparation and cryopreservation strategies is important to understanding whether the methods currently employed by newborn stem cell banks retain the desirable clinical attributes of UC cells. MethodsUCMSCs were isolated from 10 UC tissue samples by both explantation and enzymatic digestion methods to allow for comparison of cells from the same donor. Cell isolates from both methods were compared pre- and post-cryopreservation as well as after serial passaging. Cell viability, morphology, growth kinetics, immunophenotype, cytokine secretion and differentiation capacity were evaluated. ResultsUCMSCs could be derived from fresh UC tissue by both explantation and digestion methods and from thawed UC tissue by explantation. Initial cell populations isolated by digestion were heterogeneous and took longer to enrich for UCMSCs in culture than populations obtained by explantation. However, once isolated and enriched, UCMSCs obtained by either method showed no significant difference in viability, morphology, rate of proliferation, surface marker expression, levels of cytokine secretion or differentiation capacity. ConclusionsDerivation of UCMSCs by explantation after thawing UC cryopreserved as a composite tissue may be favorable in terms of initial purity and number of cells achievable by a specific passage. However, we observed no evidence of functional difference between UCMSCs derived by explanation or digestion, suggesting that cells isolated from cryopreserved material obtained by either method maintain their therapeutic properties.

Establishment and characterization of fantail goldfish fin (FtGF) cell line from goldfish, Carassius auratus for in vitro propagation of Cyprinid herpes virus-2 (CyHV-2).

BackgroundHerpesviral hematopoietic necrosis disease, caused by cyprinid herpesvirus-2 (CyHV-2), is responsible for massive mortalities in the aquaculture of goldfish, Carassius auratus. Permissive cell lines for the isolation and propagation of CyHV-2 have been established from various goldfish tissues by sacrificing the fish. Here, we report the development of a cell line, FtGF (Fantail Goldfish Fin), from caudal fin of goldfish using non-lethal sampling. We also describe a simple protocol for successful establishment and characterization of a permissive cell line through explant method and continuous propagation of CyHV-2 with high viral titer using this cell line.MethodsCaudal fin tissue samples were collected from goldfish without killing the fish. Cell culture of goldfish caudal fin cells was carried out using Leibovitz’s L-15 (L-15) medium containing 20% FBS and 1X concentration of antibiotic antimycotic solution, incubated at 28 °C. Cells were characterized and origin of the cells was confirmed by sequencing fragments of the 16S rRNA and COI genes. CyHV-2 was grown in the FtGF cells and passaged continuously 20 times. The infectivity of the CyHV-2 isolated using FtGF cells was confirmed by experimental infection of naïve goldfish.ResultsThe cell line has been passaged up to 56 times in L-15 with 10% FBS. Karyotyping of FtGF cells at 30th, 40th and 56th passage indicated that modal chromosome number was 2n = 104. Species authentication of FtGF was performed by sequencing of the 16S rRNA and COI genes. The cell line was used for continuous propagation of CyHV-2 over 20 passages with high viral titer of 107.8±0.26 TCID50/mL. Following inoculation of CyHV-2 positive tissue homogenate, FtGF cells showed cytopathic effect by 2nd day post-inoculation (dpi) and complete destruction of cells was observed by the 10th dpi. An experimental infection of naïve goldfish using supernatant from infected FtGF cells caused 100% mortality and CyHV-2 infection in the challenged fish was confirmed by the amplification of DNA polymerase gene, histopathology and transmission electron microscopy. These findings provide confirmation that the FtGF cell line is highly permissive to the propagation of CyHV-2.

Development of primary cell culture from spleen of giant gourami Osphronemus goramy for propagation of giant gourami iridovirus (GGIV).

The severe mortality of fish due to the infection of megalocytivirus caused significant economic losses. Since 2011, megalocytivirus (giant gourami iridovirus (GGIV)) has become the main pathogen in giant gourami (Osphronemus goramy), particularly in West Java, Central Java and Bali. This study aimed to develop primary cell culture from spleen as the target organ for propagating megalocytivirus in vitro, which was developed by explant method with enzymatic dissociation. Optimization was carried out at incubation temperature, medium and serum concentrations. The origin of the primary cell, cell susceptibility and GGIV pathogenicity were observed. The results showed that the primary cell (GP cells) can grow well in 10% foetal bovine serum L-15 medium at 27°C, which was sufficient for cell growth. PCR and BLAST analyses showed the primary cell was originated from giant gourami. In infected GP cells, cell enlargement and cell rounding were observed. Virus propagated in GP cells was highly virulent when injecting giant gourami in an artificial infection experiment. Intraperitoneal injection of diluted virus supernatant showed 100% mortality in 7-11 days post-injection and 97% mortality in 21days post-cohabitation, with abnormalities observed in spleen and kidney. In conclusion, GP cell was successfully subcultured for more than 30 passages and susceptible to GGIV.

Cyclic stretch promotes the ossification of ligamentum flavum by modulating the Indian hedgehog signaling pathway.

The Indian hedgehog (IHH) signaling pathway is an important pathway for bone growth and development. The aim of the present study was to examine the role of the IHH signaling pathway in the development of the ossification of ligamentum flavum (OLF) at the cellular and tissue levels. The expression levels and localization of the osteogenic genes Runt-related transcription factor 2 (RUNX2), Osterix, alkaline phosphatase (ALP), osteocalcin (OCN) and IHH were evaluated in OLF tissues by reverse transcription-quantitative PCR (RT-qPCR) and immunohistochemistry. Non-ossified ligamentum flavum (LF) sections were used as control samples. The tissue explant method was used to obtain cultured LF cells. In addition, OLF cells were subjected to cyclic stretch application for 0, 6, 12 or 24 h. The expression levels of osteogenic genes, and the IHH signaling pathway genes IHH, Smoothened (SMO), GLI family zinc finger 1 (GLI1), GLI2 and GLI3 were evaluated with RT-qPCR and western blotting. Osteogenic differentiation was further evaluated by assessing ALP activity and staining. Moreover, the effect of cyclopamine (Cpn), an IHH signaling inhibitor, on osteogenic differentiation was examined. The RT-qPCR and immunohistochemical results indicated that the mRNA and protein expression levels of RUNX2, Osterix, ALP, OCN and IHH were significantly higher in the OLF group compared with the LF group. Furthermore, application of cyclic stretch to OLF cells resulted in greater ALP activity, and significant increases in mRNA and protein expression levels of RUNX2, Osterix, ALP and OCN in a time-d00ependent manner. Cyclic stretch application also led to significant increases in IHH signaling pathway genes, including IHH, SMO, GLI1 and GLI2, while no significant effect was found on GLI3 expression level. In addition, it was found that Cpn significantly reversed the effect of cyclic stretch on the ALP activity, and the expression levels of RUNX2, Osterix, ALP, OCN, GLI1 and GLI2. Collectively, the present results suggested that the IHH signaling pathway may mediate the effect of cyclic stretch on the OLF cells.

Comparative separation methods and biological characteristics of human placental and umbilical cord mesenchymal stem cells in serum-free culture conditions

BackgroundMesenchymal stem cells (MSCs) are considered to be an effective tool for regenerative medicine with promising applications for clinical therapy. However, incongruent data has been reported partially owing to their functional heterogeneity. To provide sufficient and suitable clinical seed cells derived from the placenta for MSC therapy, we compared the various current isolation methods, as well as the biological characteristics, of different human placenta mesenchymal stem cells (hPMSCs).MethodsWe selected placentas from 35 informed donors and exploited three commonly used methods. MSCs were isolated from different parts of placental tissue including umbilical cord (UC), amniotic membrane (AM), chorionic membrane (CM), chorionic villi (CV), and deciduae (DC). The appropriate isolation methods for each type of hPMSCs were first assessed. The resulting five MSC types from the same individuals were identified based on their surface marker expression, proliferation capacity, transcriptome, differentiation, multipotency and karyotype.ResultsAll three methods successfully isolated the five hPMSC types from placental tissues. However, the UC-MSCs were most effectively separated via the tissue explant method, while the enzymatic digestion method was found to be more suitable for separating CV-MSCs, owing to its higher output efficiency compared to the other methods. Alternatively, the perfusion method was complicated and exhibited the lowest efficiency for cell isolation and uniformity. Furthermore, we determined that UC-MSCs and CV-MSCs express a higher level of paracrine cytokines and display much stronger proliferative capacity as well as superior extraction efficiency. Finally, karyotype analysis revealed that DC-MSCs are derived from the mother, while the other cell types are derived from the fetus. Moreover, the different hPMSCs exhibited unique gene expression profiles, which may prove advantageous in treatment of a broad range of diseases.ConclusionshPMSCs from different sources are similar yet also unique. Our results describe the biological characteristics of five hPMSCs and provide insights to aide in the selection process of candidates for MSCs treatment. Overall, UC- and CV-MSCs appear to be ideal sources of primary MSCs for clinical treatment and future research.

Quarantine Time Validation for UC-MSC Isolation

Background & Aim Umbilical cord tissue is a potential source of Mesenchymal Stem Cells (MSCs). These cells have been widely used especially in pre-clinical and clinical applications resulting its various effectiveness. Banking process of umbilical cord involves transportation from the hospital where the cord is received to the banking facility to be processed further. Prior to processing, pre-sterility test must be conducted and while waiting for the result, the umbilical cord is quarantined. This quarantine process has to be carried out carefully to maintain cell's recovery and viability. Suitable range of time should be chosen to deliver high quality cells. In this study, we aim to determine the maximum time for quarantined umbilical cord to be processed. Methods, Results & Conclusion One umbilical cord (UC2018030001) was used in this study. Medium transportation is used to carry the umbilical cord to banking facility. Prior to processing, pre sterility was done using spread plate method by spreading the medium transport onto TSA agar. After the results were out and passed, the tissues were put into quarantine medium and were processed at day 0, 7, 14, and 21 for MSCs isolation. The tissue was processed using explant method and sub-cultured until 3rd passage. Graphic shown below are the cell doubling level from different passages at various days of quarantine. The doubling level remain constant with maximum deviation at 0.512. Results showed that tissues which was quarantined until day 21 still produce cells normally. The cell number are slowly increasing because the higher the passage, the more pure the cells are so they can propagate easily. This shows normal growth of normal cells. Their doubling level are also on track with no significant difference. In brief, umbilical cord tissue can be quarantined until 21 days and still produce cells normally.

Reproducible Derivation and Expansion of Corneal Mesenchymal Stromal Cells for Therapeutic Applications.

PurposeA reproducible protocol for the production of corneal mesenchymal stem/stromal cells (cMSCs) is necessary for potential clinical applications. We aimed to describe successful generation and expansion of cMSCs using an explant method.MethodsCorneoscleral rims of human cadaveric eyes were divided into four pieces and used as explants to allow outgrowth of cMSCs (passage 0, or P0). The cells were subcultured at a 1:10 ratio until passage 5 (P5). The characteristics as well as therapeutic effects of expanded cMSCs were evaluated both in vitro, using a scratch assay, and in vivo using epithelial debridement and chemical injury mouse models.ResultsAll explants demonstrated outgrowth of cells by 7 days. Although the initial outgrowth included mixed mesenchymal and epithelial cells, by P1 only cMSCs remained. By subculturing each flask at a ratio of 1:10, the potential yield from each cornea was approximately 12 to 16 × 1010 P5 cells. P5 cMSCs demonstrated the cell surface markers of MSCs. The secretome of P5 cMSCs induced faster closure of wounds in an in vitro scratch assay. Subconjunctival injection of P5 cMSCs in mouse models of mechanical corneal epithelial debridement or ethanol injury led to significantly faster wound healing and decreased inflammation, relative to control.ConclusionscMSCs can be reproducibly derived from human cadaveric corneas using an explant method and expanded with preservation of characteristics and corneal wound healing effects.Translational RelevanceThe results of our study showed that cMSCs produced using this scheme can be potentially used for clinical applications.

Effects of Phytohormone Compatibility on Callus Growth of Rosa rugosa tunb

To obtain superior quality callus and to regenerate root system of Rosa rugosa tunb and improve culture efficiency, this experiment used a multi-factor orthogonal test method to establish a sterile system with young leaves of R. rugosa tunb as experimental materialsand different concentrations of hormones in each stage. Explore the effects of plant callus growth. The results showed that the optimal disinfection method of explants was 70% alcohol 30s+0.1% HgCl2 8min. At this time, the browning rate was 46.70%, and the infection rate was 10.00%, which was 14.40% and 72.67% lower than that before disinfection. [Primary medium]: MS+1.5 mg/L 6-BA+3.0 mg/L NAA+3% sucrose+0.9% agar, the induction rate at this time is 75.00%, an increase of 62.50% compared with no added hormone. [Proliferation medium] MS+2.0mg/L 6-BA+0.2mg/L NAA+3% sucrose+0.9% agar, the callus diameter reached 2.49cm after proliferation, the growth was strong, and the callus was highly available. [Rooting medium] MS+0.4mg/L IBA+0.5g/L AC+3% sucrose+ 0.9% agar, the rooting rate is 75.00%, the root system is long and the growth is robust.

Overexpression of microRNA-136-3p Alleviates Myocardial Injury in Coronary Artery Disease via the Rho A/ROCK Signaling Pathway

Coronary artery disease (CAD) is a cardiovascular disease that poses a fatal threat to human health, and the identification of potential biomarkers may help to delineate its pathophysiological mechanisms. Accumulating evidence has implicated microRNAs (miRNAs) in the pathogenesis and development of cardiovascular diseases. The present study aims to identify the expression of miRNA-136-3p (miR-136-3p) in CAD and further investigate its functional relevance in myocardial injury both in vitro and in vivo. Initially, CAD models were induced in rats by high-fat diet and intraperitoneal injection of pituitrin. Next, the effect of overexpressed miR-136-3p on cardiac function and pathological damage in myocardial tissue, cardiomyocyte apoptosis, oxidative stress and inflammatory response were assessed in CAD rats. Rat cardiac microvascular endothelial cells (CMECs) were isolated and cultured by the tissue explant method, and the CMEC injury model was induced by homocysteine (HCY). The function of miR-136-3p in vitro was further evaluated. miR-136-3p was poorly expressed in the myocardial tissue of CAD rats and CMEC injury models. In vivo assays indicated that overexpressed miR-136-3p could improve cardiac function and alleviate pathological damage in myocardial tissue, accompanied by reduced oxidative stress and inflammatory response. Moreover,in vitro assays suggested that overexpression of miR-136-3p enhanced proliferation and migration while inhibiting apoptosis of HCY-stressed CMECs. Notably, we revealed that EIF5A2 was a target gene of miR-136-3p, and miR-136-3p inhibited EIF5A2 expression and activation of the Rho A/ROCK signaling pathway. In conclusion, the overexpression of miR-136-3p could potentially impede myocardial injury in vitro and in vivo in CAD through the blockade of the Rho A/ROCK signaling pathway, highlighting a potential miR-136-3p functional relevance in the treatment of CAD.