Protective Effects of Trimetazidine on Bone Marrow Mesenchymal Stem Cells Viability in an ex vivo Model of Hypoxia and in vivo Model of Locally Myocardial Ischemia

Bone marrow mesenchymal stem cells (MSCs) have shown potential for cardiac repair following myocardial injury,but this approach is limited by their poor viability after transplantation.The present study was to investigate whether trimetazidine (TMZ) could improve survival of MSCs in an ex vitro model of hypoxia,as well as survival,differentiation,and subsequent activities of transplanted MSCs in rat hearts with acute myocardial infarction (AMI).MSCs at passage 3 were examined for their viability and apoptosis under a transmission electron microscope,and by using flow cytometry following culture in serumfree medium and exposure to hypoxia (5% CO2,95% N2) for 12 h with or without TMZ.Thirty Wistar rats were divided into 3 groups (n=10 each group),including groupⅠ(AMI control),groupⅡ (MSCs transplantation alone),and group Ⅲ (TMZ+MSCs).Rat MSCs (4×107) were injected into peri-infarct myocardium (MSCs group and TMZ+MSCs group) 30 min after coronary artery ligation.The rats in TMZ+MSCs group were additionally fed on TMZ (2.08 mg?kg-1?day-1) from day 3 before AMI to day 28 after AMI.Cardiac structure and function were assessed by echocardiography at 28th day after transplantation.Blood samples were collected before the start of TMZ therapy (baseline),and 24 and 48 h after AMI,and inflammatory cytokines (CRP,TNF-α) were measured.Then the sur-vival and differentiation of transplanted cells in vivo were detected by immunofluorescent staining.The cellular apoptosis in the peri-infarct region was detected by using TUNEL assay.Furthermore,apoptosis-related proteins (Bcl-2,Bax) within the post-infarcted myocardium were detected by using Western blotting.In hypoxic culture,the TMZ-treated MSCs displayed a two-fold decrease in apoptosis under serumfree medium and hypoxia environment.In vivo,cardiac infarct size was significantly reduced,and cardiac function significantly improved in MSCs and TMZ+MSCs groups as compared with those in the AMI control group.Combined treatment of TMZ with MSCs implantation demonstrated further decreased MSCs apoptosis,further increased MSCs viability,further decreased infarct size,and further improved cardiac function as compared with MSCs alone.The baseline levels of inflammatory cyto-kines (CRP,TNF-α) had no significant difference among the groups.In contrast,all parameters at 24 h were lower in TMZ+MSCs group than those in MSCs group.Furthermore,Western blotting indicated that the expression of antiapoptotic protein Bcl-2 was upregulated,while the proapoptotic protein Bax was down-regulated in the TMZ+MSCs group,compared with that in the MSCs group.It is suggested that implantation of MSCs combined with TMZ treatment is superior to MSCs monotherapy for MSCs viability and cardiac function recovery.

Aspirin inhibits cell viability and mTOR downstream signaling in gastroenteropancreatic and bronchopulmonary neuroendocrine tumor cells

AIM:To investigate the effect of aspirin on neuroendocrine tumor(NET)cell growth and signaling in vitro.METHODS:Human pancreatic BON1,bronchopulmonary NCI-H727 and midgut GOT1 neuroendocrine tumor cells were treated with different concentrations of aspirin(from 0.001 to 5 mmol/L),and the resulting effects on metabolic activity/cell proliferation were measured using cell proliferation assays and SYBR-DNAlabeling after 72,144 and 216 h of incubation.The effects of aspirin on the expression and phosphorylation of several critical proteins that are involved in the most common intracellular growth factor signaling pathways(especially Akt protein kinase B)and mammalian target of rapamycin(mTOR)were determined by Western blot analyses.Propidium iodide staining and flow cytometry were used to evaluate changes in cell cycle distribution and apoptosis.Statistical analysis was performed using a 2-tailed Student’s t-test to evaluate the proliferation assays and cell cycle analyses.The results are expressed as the mean±SD of 3 or 4 independently performed experiments.Statistical significance was set at P<0.05.RESULTS:Treatment with aspirin suppressed the viability/proliferation of BON1,NCI-H727 and GOT1 cells in a time-and dose-dependent manner.Significant effects were observed at starting doses of 0.5-1 mmol/L and peaked at 5 mmol/L.For instance,after treatment with 1 mmol/L aspirin for 144 h,the viability of pancreatic BON1 cells decreased to 66%±13%(P<0.05),the viability of bronchopulmonary NCI-H727 cells decreased to 53%±8%(P<0.01)and the viability of midgut GOT1 cells decreased to 89%±6%(P<0.01).These effects were associated with a decreased entry into the S phase,the induction of the cyclin-dependent kinase inhibitor p21 and reduced expression of cyclindependent kinase 4 and cyclin D3.Aspirin suppressed mTOR downstream signaling,evidenced by the reduced phosphorylation of the mTOR substrates 4E binding protein 1,serine/threonine kinase P70S6K and S6 ribosomal protein and inhibited glycogen synthase kinase3 activity.We observed the(compensatory)activation of tuberous sclerosis 2,the serine/threonine specific protein kinase AKT and extracellular signal-regulated kinases.CONCLUSION:Aspirin demonstrates promising anticancer properties for NETs in vitro.Further preclinical and clinical studies are needed.

Assessment of Benchmark Dose in BEAS-2B Cells by Evaluating the Cell Relative Viability with Particulates in Motorcycle Exhaust via the Air-liquid Interface Exposure

Objective This study aimed to use an air-liquid interface(ALI)exposure system to simulate the inhalation exposure of motorcycle exhaust particulates(MEPs)and then investigate the benchmark dose(BMD)of MEPs by evaluating cell relative viability(CRV)in lung epithelial BEAS-2B cells.Methods The MEPs dose was characterized by measuring the number concentration(NC),surface area concentration(SAC),and mass concentration(MC).BEAS-2B cells were exposed to MEPs at different concentrations via ALI and CRV was determined using Cell Counting Kit(CCK-8)assay.BMD software was applied to calculate BMD and the lower limit of benchmark dose(BMDL)according to Akaike Information Coefficient(AIC),with P-value based on Hill,Linear,Polynomial,and Power model.Results Our results reveal that BMD of NC and SAC were estimated by the best-fitting Hill model,while MC was estimated by Polynomial model.The BMDL for CRV following ALI exposure to MEPs were as follows:364.2#/cm^(3)for NC;0.662×10^(7)nm^(2)/cm^(3)for SAC;and 0.278μg/m^(3)for MC.Conclusion These results indicate that MEPs exposure via ALI system induces a dose-dependent decrease of CRV and provides the potential exposure threshold of MEPs in a lung cell model.

EFFECTS OF SHEAR STRESS ON THE VIABILITY PLANT CELLS

Plant cell lines differred greatly in the ability to withstand shear stresses. Using to-bacco cells and licorice cells as model plant cells, we studied the effects of shear stresses on the vi-ability of plant cells. Our experiments were carried out on a high shear rate Couette rheometerproviding homogeneous and constant shear stresses of laminar flow. The viability was determinedby TTC（2,3,5-Triphenyl tetrazolium chloride）. The results were as follows. （1）The viability（V）dropped exponentially with time（t）, namely V=Exp（-kt）, （k】o is a constant）. This meant thetenability of statistical homogeneity. （2）The value of k was a function of plant cells’ mechanicalproperties and the shear stress acting on the plant cells. The shear rate corresponding to k=o wasthe critical shear rate that the plant cells could withstand. It can be easily determinded by extrapo-lation. For 7-day-old tobacco cells, it was 1090 s-1 and for 9-day-old licorice cells, it was6566 s-1.（3）The plant cell suspensions were pseudoplastic fluids fitting τ=Kγn. For the tobacco cell sus-pension tested, n=O. 73, and for the licorice cellsuspension tested n=0. 7. Thus the critical shearstress for the tobacco cells was 25 dynes/cm2 and for the licorice cells it was 80 dynes/cm2. （4）One of their reasons for licorice cells to have greater tolerance to shear stresses than tobacaccocells may be the geometric features of the cells and the sizes of the cells. The licorice cells wererod-shaped, but the tobacco cells were spherical and larger than the licorice cells.

A review on cell damage,viability,and functionality during 3D bioprinting

Three-dimensional(3D)bioprinting fabricates 3D functional tissues/organs by accurately depositing the bioink composed of the biological materials and living cells.Even though 3D bioprinting techniques have experienced significant advancement over the past decades,it remains challenging for 3D bioprinting to artificially fabricate functional tissues/organs with high post-printing cell viability and functionality since cells endure various types of stress during the bioprinting process.Generally,cell viability which is affected by several factors including the stress and the environmental factors,such as pH and temperature,is mainly determined by the magnitude and duration of the stress imposed on the cells with poorer cell viability under a higher stress and a longer duration condition.The maintenance of high cell viability especially for those vulnerable cells,such as stem cells which are more sensitive to multiple stresses,is a key initial step to ensure the functionality of the artificial tissues/organs.In addition,maintaining the pluripotency of the cells such as proliferation and differentiation abilities is also essential for the 3D-bioprinted tissues/organs to be similar to native tissues/organs.This review discusses various pathways triggering cell damage and the major factors affecting cell viability during different bioprinting processes,summarizes the studies on cell viabilities and functionalities in different bioprinting processes,and presents several potential approaches to protect cells from injuries to ensure high cell viability and functionality.

P53 protein expression and cell viability in irradiated peripheral blood mononuclear cells as bioindicators of radiosensitivity

Cellular radiosensitivity is directly correlated with the mechanism of DNA repair, in which p53 protein plays a major role. In this context, this study correlated cell death with p53 expression in lymphocytes irradiated in vitro with different doses of gammaradiation. For this, peripheral blood samples were collected from 10 healthy subjects. Each sample was divided in aliquots and, separately, irradiated with doses of 0,5;2 and 4 Gy. After this, peripheral blood mononuclear cells (PBMCs) were isolated and cultivated during 72 hours in 5% CO2 at 37oC without mitogen stimulation. The expression of p53 protein was evaluated by flow cytometry. In parallel, cell viability was determined by trypan blue staining. Statistical analysis was performed us-ing analysis of variance (ANOVA), differences were considered as statistically significant when p < 0.05. The results showed an increase of p53 expression with the absorbed dose, which was proportional to cell death, suggesting that p53 can be used as bioindicator of individual radiosensitivity.

Femtosecond Optical Trapping of Cells:Efficiency and Viability

The femtosecond optical trapping capability and the effect of femtosecond laser pulses on cell viability were studied.The maximum lateral velocity at which the particles just failed to be trapped,together with the measured average trapping power,were used to calculate the lateral trapping force(Q-value) .The viability of the cells after femtosecond laser trapping was ascertained by vital staining.Measurement of the Q-values shows that femtosecond optical tweezers are just as effective as continuous wave optical tweezers.The experiments demonstrate that there is a critical limit for exposure time at each corresponding laser power of femtosecond optical tweezers,and femtosecond laser tweezers are safe for optical trapping at low power with short exposure time.

Effect of recombinant human platelet-derived growth factor B on cat corneal endothelial cell viability mediated by adeno-associated virus

AIM: To transduce recombinant human platelet-derived growth factor B (PDGF-B) gene adeno-associated virus(AAV) to in vitro cultured cat corneal endothelial cell (CEC) and observe the effect of the expressed PDGF-BB protein on the viability of cat CEC. METHODS: Cat cornea endothelium was torn under microscope and rapidly cultivated in DMEM to form single layer CEC and the passage 2 endothelial cells were used in this study. The recombinant human PDGF-B gene AAV was constructed and transduced into cat CEC directly. Three groups were as following: blank control group, AAV control group and recombinant AAV group. At 24 hours, 48 hours, and 5 days after transduction, total RNA was extracted from the CEC by Trizol and the expression of PDGF-B gene was detected by fluorescence quantitative polymerase chain reaction. Viability of the transduced CEC was detected at 48 hours after transduction by MTT assay. Cell morphology was observed under inverted phase contrast microscope. RESULTS: With the torn endothelium culture technique, we rapidly got single layer cat CEC. At 24 hours, 48 hours and 5 days after transduction, fluorescence quantitative polymerase chain reaction showed there was no significant difference of the expressed PDGF-B gene mRNA between blank control group and AAV control group (P>0.05). In contrast, there were significant differences between two control groups and recombinant AAV group (P<0.05). MTT assay showed that in recombinant AAV group, the expressed PDGF-BB protein could promote the viability of cat CEC. Morphology observation showed at 48 hours after transduction, cells in CEC-AAV-PDGF-B group proliferated into bigger scales in regular triangle to hexagon shape with distinct boundary, while the number of cells was significantly less in the two control groups. CONCLUSION: The recombinant AAV-PDGF-B expresses biological active PDGF-BB protein in cat CEC, which promotes the viability and proliferation of cells.

Effects of Cryoprotective Agents on the Bovine Articular Chondrocyte Viability

Cryopreservation is the process of choice for long term preservation of cells and tissues. In this study, the effects of cryoprotective agents, dimethyl sulfoxide(DMSO), glycerol and 1,2 propanediol on the bovine articular chondrocyte viability were examined experimentally. The CPA was added at the concentrations of 0 6, 0 9, 1 2 and 1 5 mol/L and at 4 ℃ and 37 ℃ and removed at 37 ℃ in one step. CPA stepwise addition and removal at 0 6 and 1 2 mol/L and at 37 ℃ was also tested as an alternative protocol. Cell volume excursion during DMSO addition and removal was estimated and correlated well with cell survival rates. Solution makeup affects cell survival rate and a stepwise protocol can improve the cell survival rates significantly.

Effect of Cold Plasma on Cell Viability and Collagen Synthesis in Cultured Murine Fibroblasts

An argon atmospheric pressure plasma jet was employed to treat L929 murine fibroblasts cultured in vitro.Experimental results showed that,compared with the control cells,the treatment of fibroblasts with 15 s of plasma led to a significant increase of cell viability and collagen synthesis,while the treatment of 25 s plasma resulted in a remarkable decrease.Exploration of related mechanisms suggested that cold plasma could up-regulate Cyclin D1 gene expression and down-regulate p27 gene expression at a low dose,while it could down-regulate Cyclin D1 expression and up-regulate p27 expression at a higher dose,thus altering the cell cycle progression,and then affecting cell viability and collagen synthesis of fibroblasts.

Protein adsorption, cell viability and corrosion properties of Ti6Al4V alloy treated by plasma oxidation and anodic oxidation

The hardness,wettability,and electrochemical properties of Ti6Al4V alloy surfaces treated with anodic oxidation and plasma oxidation as well as the viabilities of the different cell lines on the obtained surfaces were investigated.The anodic oxidation was performed for 10 min under 100 V potential,and it resulted in a 0.95μm thick nanoporous anatase-TiO2 structure.On the other hand,plasma oxidation was carried out at 650℃ for 1 h and resulted in a dense rutile-TiO2 structure with a thickness of 1.2μm.While a hardness of HV0.025823 and roughness of^220 nm were obtained by plasma oxidation,those obtained by anodic oxidation were HV0.025512 and^130 nm,respectively.The anodic oxidation process created a more hydrophilic surface with a contact angle of 87.2°.Both oxidation processes produced similar properties in terms of corrosion behavior and showed better resistance than the as-received state in a certain range of potential.Moreover,the surface treatments led to no significant change in the protein adsorption levels,which indicates that the difference in viability between the osteoblast and fibroblast cells was not due to the difference in surface protein adsorption.Given all the factors,the surfaces obtained by anodic oxidation treatment revealed higher cell viability than those obtained by plasma oxidation(p=0.05).

Changes in Growth,Photosynthetic Pigments,Cell Viability,Lipid Peroxidation and Antioxidant Defense System in Two Varieties of Chickpea(Cicer arietinum L.)Subjected to Salinity Stress

Salinity is one of the most severe abiotic stresses for crop production.The present study investigates the salinityinduced modulation in growth indicators,morphology and movement of stomata,photosynthetic pigments,activity of carbonic anhydrase as well as nitrate reductase,and antioxidant systems in two varieties of chickpea(Pusa-BG5023,and Pusa-BGD72).On 20^(th) day of sowing,plants were treated with varying levels of NaCl(0,50,100,150 and 200 mM)followed by sampling on 45 days of sowing.Recorded observations on both the varieties reveal that salt stress leads to a significant decline in growth,dry biomass,leaf area,photosynthetic pigments,protein content,stomatal behavior,cell viability,activity of nitrate reductase and carbonic anhydrase with the rise in the concentration of salt.However,quantitatively these changes were less in Pusa-BG5023 as compared to Pusa-BGD72.Furthermore,salinity-induced oxidative stress enhanced malondialdehyde content,superoxide radicals,foliar proline content,and the enzymatic activities of superoxide dismutase,catalase,and peroxidase.The variety Pusa-BGD72 was found more sensitive than Pusa-BG5023 to salt stress.Out of different graded concentrations(50,100,150 and 200 mM)of sodium chloride,50 mM was least toxic,and 200 mM was most damaging.The differential behavior of these two varieties measured in terms of stomatal behavior,cell viability,photosynthetic pigments,and antioxidant defense system can be used as prospective indicators for selection of chickpea plants for salt tolerance and sensitivity.

Inhibition of viability of human retinal microvascular endothelial cells by vialinin A under high glucose condition

AIM:To investigate the effects of vialinin A on viability of human retinal endothelial cells(HRECs)under high glucose condition and its potential mechanism.METHODS:The HRECs were divided into four groups:normal glucose control group(NG,5 mmol/L D-glucose),high glucose group(HG,30 mmol/L D-glucose),HG+1μmol/L vialinin A group,and HG+5μmol/L vialinin A group.The cell viabilities were measured with cell counting kit-8(CCK-8)assay for proliferation,with scratch assay for migration,and tube formation,for evaluation of the impact of vialinin A on cellular behaviour.Real-time PCR and Western blotting were used to determine the expression level of vascular endothelial growth factor(VEGF).RESULTS:The proliferative capacity and migration of HRECs was reduced by 5μmol/L vialinin A in high glucose environment(both P<0.05).Vialinin A also inhibited highglucose-induced tube formation of HRECs.The expression level of VEGF and PI3K in HRECs was also significantly decreased by vialinin A(P<0.05).CONCLUSION:Vialinin A inhibits the cell viability of HRECs.It may serve as a potential target for anti-angiogenic therapy.

Ovarian Follicle Disaggregation to Assess Granulosa Cell Viability

Background: Mammalian ovaries contain follicles containing an oocyte enclosed by layers of granulosa cells (GC). Follicle growth and oocyte maturation are largely dependent on GC numbers and viability, but there is no established, reliable method for assessing the number of viable GC within an isolated follicle. Methods: Centrifugation conditions and the Trypan Blue (TB) Exclusion assay were optimised for low cell densities compatible with the numbers of GC in follicles. Mouse ovarian follicles were disaggregated to produce a single cell suspension of GC which were examined by TB (n = 4), but also by crystal violet assay in a 96-well plate format after 24 h in vitro (n = 3). GC viability in vitro was characterised further by using enzyme-linked immunoassays to quantify GC production of anti-Mullerian hormone (AMH) and estrogen. Results: The centrifugation and low cell density TB protocol could accurately measure the viability of 78 GC in 10 &mu;L, with an intra-assay coefficient of variation (CoV) 22%, and inter-assay CoV 7%. The best follicle disaggregation method (30 min 37°C exposure to 2 mg/mL collagenase prior to 30 min exposure to 0.025% hyaluronidase) yielded (656 &plusmn;87) GC per antral follicle of which 82% &plusmn;5% were viable. Culturing 312 - 20,000 GC per well for 24 hours and assessing viability by crystal violet assay generated a linear correlation between OD value and viable GC number (R2 = 0.98) and estrogen concentration per well (R2 = 0.92). 20,000 GC per well produced 143 &plusmn;16 pg/mL estrogen during 24 hours in vitro, but no detectable AMH. Conclusion: This is the first report describing the isolation of viable, estrogen-producing GC from murine follicles, and their subsequent culture. These procedures are transferrable to other species including humans and can be applied to screening the reproductive toxicity of pharmaceutical agents.

Cell viability in the cadmium-stressed cell suspension cultures of tobacco is regulated by extracellular ATP,possibly by a reactive oxygen species-associated mechanism

Cadmium(Cd)is one of the most widespread and toxic heavy metals to plants.Extracellular ATP(exATP)is thought to be an extracellular effector in regulating the physiological responses of plant cells to environmental stresses.However,the function of exATP in Cd-stressed plant cells is much unknown.The present work showed that treating tobacco(Nicotiana tabacum L.cv.Bright Yellow-2)cell-suspension cultures with exogenous CdCl2 reduced the cell viability,exATP level,and Mg content.However,the production of reactive oxygen species(ROS),Cd content,and electrolyte leakage of the cells were enhanced by exogenous CdCl2.When the Cd-induced accumulation of ROS was decreased by the supplement with DMTU(dimethylthiourea,a scavenger of ROS),the Cd-induced increases of the electrolyte leakage and Cd content were alleviated,and the Cd-induced reductions of cell viability were partly rescued,suggesting that Cd-induced reduction of cell viability could be related to the ROS accumulation.Under the condition of Cd stress,when the reduction of exATP level was partly rescued by exogenous ATP(20μM),the increases of ROS production,electrolyte leakage,and Cd content were attenuated,and the reduction of cell viability was also alleviated.These observations indicate that exATP can regulate the cell viability in the Cd–stressed plant cells possibly by an ROS-associated mechanism.

Poly(Butylene Adipate-Co-Terephthalate)and Poly(ε-Caprolactone)and Their Bionanocomposites with Cellulose Nanocrystals:Thermo-Mechanical Properties and Cell Viability Study

Although nanocomposites have recently attracted special interest in the tissue engineering area,due to their potential to reinforce scaffolds for hard tissues applications,a number of variables must be set prior to any clinical application.This manuscript addresses the evaluation of thermo-mechanical properties and of cell proliferation of cellulose nanocrystals(CNC),poly(butylene adipate-co-terephthalate)(PBAT),poly(ε-caprolactone)(PCL)films and their bionanocomposites with 2 wt% of CNC obtained by casting technique.Cellulose nanocrystals extracted from Balsa wood by acid hydrolysis were used as a reinforcing phase in PBAT and PCL matrix films.The films and pure CNC at different concentrations were cultured with osteoblasts MG-63 and the cell proliferation was assessed by AlamarBlue?assay.The thermal-mechanical properties of the films were evaluated by dynamic-mechanical thermal analysis(DMTA).It was found by DMTA that the CNC acted as reinforcing agent.The addition of CNCs in the PBAT and PCL matrices induced higher storage moduli due to the reinforcement effects of CNCs.The cell viability results showed that neat CNC favored osteoblast proliferation and both PBAT and PCL films incorporated with CNC were biocompatible and supported cell proliferation along time.The nature of the polymeric matrix or the presence of CNC practically did not affect the cell proliferation,confirming they have no in vitro toxicity.Such features make cellulose nanocrystals a suitable candidate for the reinforcement of biodegradable scaffolds for tissue engineering and biomedical applications.

Higher Cell Viability and Enhanced Sample Quality Following Laser-Assisted Liposuction versus Mechanical Liposuction

Background: Despite the popularity of autologous fat transfer applications, high resorption rates, and consequential volume loss, have been reported. Viable adipocyte content has been defined as a key determinant of fat transfer longevity. Moreover, traces of blood, free oil fat and fibrotic tissue accelerate adipocyte degradation. Objective: To compare the effectiveness of a 1470 nm, radial emitting laser-assisted liposection device to a mechanical liposection device in maintaining adipocyte viability in fat tissue harvests. Methods: Bilateral subcutaneous adipose tissue samples were harvested from ten female patients. Fat was harvested from one side using the LipoLife laser-assisted liposuction device and from the other side with a Byron mechanical aspirator. Samples were visually analyzed and blood:fat ratios and cell viability were determined. Results: Laser-harvested samples separated into two distinct phases, with a negligible blood phase at the bottom (1.1%) and a significant adipose phase at the top (98.9%), containing small, uniform-sized cells, of which 95.7% ± 2.7% proved viable. Mechanically harvested samples separated into blood (18%), adipose (60%) and lipid (22%) phases. The adipose phase contained significant amounts of connective tissue, large adipose tissue fragments, large oil droplets and a mean 79.7% ± 18.3% viable adipocytes. Conclusions: Laser liposuctioning was superior to mechanical liposuctioning, providing both higher cell viability and enhanced sample quality. The 1470 nm diode laser bears the potential of improving long-term clinical outcomes of fat transfer procedures. Improved purity of the harvested sample and heightened preadipocyte content are projected to provide for extended graft longevity.

THE EFFECTS OF REFREEZING ON THE CELL'S VIABILITY OF HUMAN PULMONARY ARTERY

The application of human homograft has been popular in cardiovascular surgeryand shown perfect effects.But the difficulty to get graft greatly limited its clinical ap-plication.The thawed homograft can not be used in some cases since it is not clearwhether it can be re-cryopreserved without damage.We conducted the present studyto know the changes in viability of the homograft after refreezing in six human pul-

CCL2 Enhances the Viability of Human Chorionic Trophoblast Cell Line HTR-8/SVneo Cells by Inhibiting Interleukin-24 Expression

Background:To investigate the regulatory effect of interleukin-24(IL-24)on cell viability of human chorionic trophoblast cell line(HTR-8/SVneo cells).Methods:Immunohistochemical staining was used to detect the expression of IL-24 and its receptors IL-20R1,IL-20R2,and IL-22R1 in villus tissue at early normal pregnancy.The effect of thymic stromal lymphopoietin(TSLP)and chemokine CCL2 on the expression of IL-24 in human chorionic trophoblast cell line HTR-8/SVneo cells was analyzed by In-cell Western.In addition,the effect of recombinant human IL-24(rhIL-24)and CCL2 on the viability of HTR-8/SVneo cells was analyzed by MTT assay.Results:IL-24 and its receptors showed a strong positive staining in trophoblasts at early normal pregnancy.Compared with control group,expression of IL-24 in HTR-8/SVneo cells was significantly inhibited after in vitro stimulation of recombinant human CCL2 protein(rhCCL2)(P<0.001).The viability of HTR-8/SVneo cells was significantly decreased after treatment with rhIL-24(P<0.001).In contrast,anti-IL-24 neutralizing antibody significantly enhanced the viability of HTR-8/SVneo cells(P<0.01).In addition,rhCCL2(100μg/L);enhanced the viability of HTR-8/SVneo cells(P<0.01)in vitro,but this effect was inhibited by treatment with rhIL-24.Conclusions:CCL2 enhances the viability of human trophoblast cell line HTR-8/SVneo cells in vitro by inhibiting the secretion of IL-24,which may be beneficial to blastocyst implantation and placental development.