Primary Culture of Bovine Mammary Epithelial Cells

[ Objective] To investigate the feasibility of the primary culture of bovine mammary epithelial cells in biochemical incubator. [ Method] In vitro, bovine mammary epithelial cells were isolated and cultured by the tissue explant method in order to investigate the optimal culture conditions. The morphology observation and identification of the cultured cells were performed by inverted microscope observation, Giemsa staining and cytokeratin immunohistochemistry. [ Result] Observed with inverted microscope, most of the bovine mammary epithelial cells were polygonal and displayed typical slabstone-like appearance. As it can be seen from cell staining results, the cell body was big and the nucleus was stained dark blue and was round or oval in shape, with clearly visible nucleoli, generally 2 -4 nucleoli. The tissue-specific expression of cytokeratin 14 and cytokeratin 18 genes in mammary epithelial cells was identified by cytokeratin immunohistochemistry. [ Conclusion] Primary bovine mammary epithelial cells were successfully cultured in biochemical incubator.

Protective Effects of Quercetin on Cadmium-induced Cytotoxicity in Primary Cultures of Rat Proximal Tubular Cells

Objective To investigate the protective effects of quercetin on cadmium-induced cytotoxicity in primary cultures of rat proximal tubular （rPT） cells. Methods Primary cultures of rPT cells undergoing exponential growth were incubated with 1.0 ug/mL quercetin and/or cadmium （2.5, 5.0 umol/L）, in a serum-free medium at 37℃ at different time intervals. Commercial kits were used and flow cytometric analyses were performed on rPT cell cultures to assay apoptosis and oxidative stress. Results Exposure of rPT cells to cadmium acetate （2.5, 5.0 umol/L） induced a decrease in cell viability, caused an increase in apoptotic rate and apoptotic morphological changes. Simultaneously, elevation of intracellular reactive oxygen species, malondialdehyde and calcium levels, depletion of mitochondrial membrane potential and intracellular glutathione, and inhibition of Na＋, K＋ -ATPase, Ca2＋ -ATPase, glutathione peroxidase （GSH-Px）, catalase （CAT）, and superoxide dismutase （SOD） activities were revealed during the cadmium exposure of rPT cells. However, simultaneous supplementation with 1 ug/mL quercetin protected rPT cells against cadmium-induced cytotoxicity through inhibiting apoptosis, attenuating lipid peroxidation, renewing mitochondrial function and elevating the intracellular antioxidants （non-enzymatic and enzymic） levels. Conclusion The present study has suggested that quercetin, as a widely distributed dietary antioxidant, contributes potentially to prevent cadmium-induced cytotoxicity in rPT cells.

Transplantation of primary cultured embryonic mesencephalic neural precursor cells for treating Parkinsonian rats

BACKGROUND： Choosing proper donor cells is one of keys in experimental and clinical studies on cell replacement therapy （CRT） for treating Parkinson disease （PD）. Embryonic mesencephalic precursor cells （MPCs） can stably differentiate into dopaminergic neuron after in vitro proliferated culture. As compared with embryonic stem cell and neural stem cell strains, cell composition of embryonic MPCs after primary culture is also the most close to that of embryonic mesencephalic ventral cell suspension without proliferated culture. Successful experience accumulated in the latter suggests that primary cultured embryonic MPCs might be the most potential donor cells in clinical application with CRT for treating PD so far. OBJECTIVE： To investigate the feasibility of primary cultured embryonic precursor cells cultured primarily as donor cells in CRT for treating PD in rats. DESIGN ： A randomized and controlled trial taking SD rats as experimental animals.SETTING： Department of Neurosurgery, Huashan Hospital Affiliated to Fudan University.MATERIALS： This experiment was carried out at the Institute of Neuroscience, Shanghai Institute for Biological Science, Chinese Academy of Sciences from July 2003 to June 2004. Totally 26 female SD rats, with body mass of 200 to 220 g, were provided by Shanghai Experimental Animal Center of Chinese Academy of Sciences. METHODS ： Stereotaxic injection of 6-hydroxydopamine into the medial forebrain bundle were perfored to develop PD model rat. Among 26 SD rats, 20 rats achieved a more than 5 turns/min in apomorphine induced rotation test, reaching the standard of PD model rats. Immunohistochemical detection was performed on 1 out of 20 model rats after execution, and the other 19 rats were randomly divided into control group （n=5）, sham transplantation group （n=5）and cell grafted group （n=9）. Primary cultured E12 MPC cell suspension （1.2×10^11 L^-1）were used as donor cells. 4μL primary cultured E12 MPC cell suspension prepared freshly was injected into the lesioned corpus striatum of rats in cell grafted group, and 4μL D-Hank＇s solution was injected in sham transplantation group in the same way. There was no injection in control group. Apomorphine-induced rotation rate of PD rats were recorded respectively in cell grafted group and sham transplantation group pre-operation （initial value） and at postoperative 2, 4, 6 and 16 weeks. Apomorphine-induced rotation rate of PD rats was recorded in control group at postoperative 2 months （initial value） and following 2,4,6 and 16 weeks. To determine TH antigen with immunohistological ABC method （DAB developing） at 6 months post-transplantation to investigate the differentiation and survival of donor cells in the host body.MAIN OUTCOME MEASURES： Apomorphine-induced rotation behavior before and after transplantation and the survival and differentiation of implanted cells in the host body at 6 months post-transplantation. RESULTS： Among 19 model rats, one rat died after transplantation respectively in the cell grafted group and sham transplantation group; finally 17 model rats entered the stage of result analysis. Relative apomorphine-induced rotation rate was significantly decreased in the cell grafted group as compared with that before transplantation , with significant difference （P 〈 0.01 .P 〈 0.05）;the mean value of relative apomorphine-induced rotation rate was significantly decreased at postoperative 16 weeks in cell grafted group as compared with that of corresponding relative rotation rate in control group , also with significant difference （P 〈 0.05）.Immunohistological results showed that donor cells could differentiate into large and multi-polar dopaminergic neurons in the host body. CONCLUSION ： Primary cultured embryonic MPCs can be used as the donor cells in CRT for treating PD.

Establishment of primary cultures of craniopharyngioma cells

Craniopharynigoma samples were collected from 36 patients. Out of the 36 samples, 29 achieved successful sub-culturing, with a success rate of 80.6%. Immunohistochemistry staining showed that cytokeratin-7 was positively expressed in the cytomembrane and cytoplasm of craniopharyngioma cells at 6-8 passages, confirming that all cultured cells were squamous epithelial cells. The doubling time of craniopharyngioma cells was 3 days, as confirmed by the 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay. In this study, craniopharyngioma cells cultured in vitro were established; however, establishment of immortalized craniopharyngioma cell lines requires further research.

Bis(7)-Tacrine, a Promising Anti-Alzheimer's Agent,Attenuates Glutamate-Induced Cell Injury in Primary Cultured Cerebrocortical Neurons of Rats

The effects of bis(7) tacrine, a novel dimeric acetylcholinesterase (AChE) inhibitor, on glutamate induced cell injury were investigated in primary cerebral cortical neurons of rats. Exposure of cultured neurons (12 days after plating) to 0.5 mmol/L glutamate for 30 min resulted in significant cell damage. Pretreatment with bis(7) tacrine (0.03 1.0 μmol/L) reduced the glutamate induced neurotoxicity in a concentration dependent manner and the maximal response was seen at 1 μmol/L with approximately 30% protection. A receptor binding assay showed that bis(7) tacrine can completely displace MK 801 binding to rat cortical membrane with an IC 50 of 0.57 μmol/L. These findings suggest that bis(7) tacrine can directly interact with N methyl D aspartate receptor channel complex, which may contribute to the inhibitor's protective effects against glutamate induced excitotoxicity. Thus, it is possible that anti glutamate/anti AChE synergism is responsible for potentially better Alzheimer's therapy of bis(7) tacrine relative to tacrine.

Manganese enhances the expression of the manganese superoxide dismutase in cultured primary chick embryonic myocardial cells

In the present study, the effect of manganese（Mn） on antioxidant status and the expression of the manganese superoxide dismutase（MnSOD） gene in cultured primary myocardial cells collected from the chick embryos was investigated. The hypothesis that Mn supplementation would enhance the expression of MnSOD in cultured primary myocardial cells of chick embryos was tested. Eggs collected from Mn-depleted Arbor Acres laying breeder hens were incubated for 10 days and then myocardial cells were isolated and cultivated for 8 days. The embryonic myocardial cells on day 6 were treated with Mn in the cell culture medium at different time points when the proportion of cells showing spontaneous contraction was over 95% after the 3-day primary culture. A completely randomized design involving a 3 Mn levels（0, 0.5 and 1.0 mmol L^（-1））×3 incubation time points（12, 24 and 48 h） factorial arrangement of treatments（n=6） was used in the current experiment. The results showed that MnSOD activity and m RNA expression level were induced by Mn and increased with incubation time, which supported the hypothesis that Mn would enhance the expression of the MnSOD gene, and thus might protect myocardial cells from oxidative stress during the chick embryonic development.

Primary Culture of Alveolar Epithelial Type Ⅱ Cells and Its Bionomic Study

To establish a better method of primary culture for alveolar epithelial type Ⅱ cells （AEC Ⅱ ） and to study its bionomics, alveolar epithelial type Ⅱ cells were isolated by digestion with trypsin and collagenase, which were then purified by plated into culture flask coated with rat immunoglobulin （i The purified AEC Ⅱ were identified by alkaline phosphatase staining, electron mi- croscopy, immunocytochemical staining of pulmonary surfactant protein A （SPA）. The SPA expression and transfection characteristics were compared with those of A549 cell line. The results showed that AEC Ⅱ could be isolated by digestion with trysin and collagenase and purified by adhesive purification by using IgG, with a yield of about 2-3 × 10^7, and a purity of about 75%-84 %. Cells could be quickly identified with AKP staining. AEC Ⅱ were different from A549 cell line in terms of SPA expression and transfection characteristics. It is concluded that adhesive purification with IgG can improve the purity of AEC Ⅱ, and AKP staining is simple in cell identification. AEC Ⅱ can not be completely replaced by A549 cells in some studies because the differences between them, such as SPA expression.

Effect of the gene silencing of phosphorus transporters on phosphorus absorption across primary cultured duodenal epithelial cell monolayers of chick embryos

The aim of the study was to investigate whether phosphorus(P) transporters, type IIb sodium-dependent phosphate cotransporter(NaP-IIb) and inorganic phosphate transporter 2(PiT2), were directly involved in P absorption across primary cultured duodenal epithelial cell monolayers of chick embryos. The siRNAs against NaP-IIb or PiT2 were designed, synthesized and transfected into primary cultured duodenal epithelial cells of chick embryos. Then, the inhibitory efficiency of siRNAs against NaP-IIb or PiT2 was analyzed, and the most efficacious siRNAs were selected to be used for subsequent P absorption experiments. Briefly, primary cultured duodenal epithelial cells of chick embryos were transfected with either NaP-IIb or PiT2 siRNAs and grown in confluent monolayers on transwell plates. The untransfected or transfected cell monolayers were then incubated in an uptake medium containing 0 or 0.25 mmol L^(–1) of P as KH_(2) PO_(4) to measure the P absorption across duodenal epithelial cell monolayers. The results showed that among the siRNAs designed, si-1372 and si-890 were demonstrated to be the most effective in inhibiting the NaPIIb and PiT2 expressions, respectively. Supplemental P increased(P=0.065) the protein abundance of PiT2 and enhanced(P<0.0001) P absorption in primary cultured duodenal epithelial cell of chick embryos. Furthermore, NaPIIb silencing decreased(P=0.07) P absorption across duodenal epithelial cell monolayers, while PiT2 silencing had no effect(P=0.345). It is concluded that the NaP-IIb, but not PiT2, might be directly involved in the P absorption of chick duodenal epithelial cells.

Primary culture and identification about brain microvascular endothelial cells of rabbits

Objective:To establish a simple and efficient culture method of primary rabbit brain microvascular endothelial cells,provide important carriers and tool cells for the research of related cerebrovascular diseases.Methods:The cerebral cortexes of rabbits were collected aseptic and inoculated after cutting,passing through cell sieve,bovine serum albumin density gradient centrifugation,typeⅡcollagenase digestion,finally inoculated and cultured.The cultured cells were identified by cell morphological observation and angiogenesis experiment.Results:Under the inverted microscope,the cells were short fusiform or polygonal,and grew in clusters and adhere to the wall.After the cells were densely fused,they would be in a typical monolayer flat,“pebbled"mosaic arrangement.Tube formation test had the ability to form tubes structure.Conclusion:This method can successfully separate and cultivate primary rabbit brain microvascular endothelial cells.

Comparison of biological behavior of lacrimal gland adenoid cystic carcinoma with high-grade transformation cells

AIM:To evaluate the differences between human lacrimal gland adenoid cystic carcinoma with high-grade transformation(LACC-HGT)primar y cells cultured by high-grade transformation tissue and non-high-grade transformation(non-HGT)primary cells cultured by non-highgrade transformation tissue in proliferation,metastasis,drug susceptibility,and genes.METHODS:LACC-HGT primary cells were established by tissue block culture,and the 4^(th)to 10^(th)generation primary cells were selected as research objects.The cells were preliminarily identified by immunofluorescent staining.The differences between non-HGT and LACC-HGT primary cells in terms of proliferation,metastasis,and drug susceptibility were compared by cell counting kit-8(CCK-8)assay,wound healing,and drug sensitivity experiments.Differentially expressed genes were screened using mRNA array.Gene expression was analyzed using real-time quantitative polymerase chain reaction(RT-qPCR).RESULTS:LACC-HGT primary cells were successfully cultured by tissue block culture.Immunofluorescence staining results showed that cytokeratin(CK)and CK7 expression levels were positive in LACC-HGT primary cells.CCK-8 results showed that the proliferation ability of LACCHGT cells was significantly higher than that of non-HGT cells.Wound healing experiment showed that the migration ability of LACC-HGT cells was significantly higher than that of non-HGT cells.LACC-HGT cells were also less sensitive to cisplatin and paclitaxel than non-HGT cells.Compared with non-HGT cells,9566 differentially expressed genes were found in LACC-HGT primary cells,of which 5162 were upregulated and 4404 were down-regulated.The expression of N-acetylneuraminate pyruvate lyase(NPL),MARVEL domain containing 3(MARVELD3),syntabulin(SYBU),and allograft inflammatory factor 1(AIF1)was higher in LACCHGT cells than in non-HGT cells,whereas that of periostin(POSTN)was lower.CONCLUSION:LACC-HGT primary cells have faster proliferation,stronger migration ability,and poorer sensitivity to chemotherapy drugs than non-HGT primary cells.The expression of mRNAs in non-HGT and LACC-HGT primary cells are significantly different.These features are speculated to be the reasons why high-grade transformation tissues exhibit higher malignant degree and poorer prognosis than their counterparts.

Comparison of two methods used to culture and purify rat retinal Mller cells

AIM:To study two methods for culturing and purifying Sprague-Dawley(SD)rat retinal Muller cells and determine which one is better.METHODS:The passage culture method of Muller cells was respectively carried out by complete pancreatic enzyme digestion method and repeated incomplete pancreatic enzyme digestion method.After culturing retinal cells for one month through these two methods,fluorescence-activated cell sorter(FACS),RT-PCR,and immunohistochemistry technology were performed to examine the enrichment and purity of Muller glial cells,and carried out two-sample approximate t test using SSPS 13.0 to further compare the Muller cell positive rate in both methods.RESULTS:The statistical results showed that the purity of Muller cells was 83.2%±5.16%in group A,and the purity was 98.5%±1.08%in group B.The two-sample approximate t test analysis demonstrated that the difference between group A and group B was statistically significant(t=-9.178,P【0.005).The results clearly exhibited a difference between the purity of Muller cells cultured by the complete pancreatic enzyme digestion method(group A)and the repeated incomplete pancreatic enzyme digestion method(group B).CONCLUSION:Compared with the complete pancreatic enzyme digestion method,this novel method was more efficient and a higher purity of Muller cells could be obtained using this approach.

Simplified methods to isolate,culture and purify olfactory ensheathing cells

Conventional methods for harvesting, culturing and purifying olfactory ensheathing cells are complicated, time-consuming, and poorly reproducible. Olfactory bulbs were detached from adult Sprague Dawley rats and olfactory ensheathing cells were isolated using shearing, dispersion processes. After the primary cultures reached confluence, the cells were purified using a three-step process. The olfactory ensheathing cells attached and grew rapidly. The purity of the olfactory ensheathing cells increased following the three purification steps, eventually exceeding 95%. These cells could be maintained for an extended period time in culture. This simple, inexpensive, reproducible method of harvesting, culturing and purifying olfactory ensheathing cells shortens the culture cycle and provides sufficient olfactory ensheathing cells of controllable purity.

Which form of collagen is suitable for nerve cell culture?

In this study, we investigated the effects of hydrolyzed and non-hydrolyzed collagen and two-di- mensional and three-dimensional collagen matrices on cell survival, attachment and neurite out- growth of primary cultured nerve cells using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide （MTT） colorimetric assay and inverted microscopy. Hydrolyzed collagen facilitated nerve cell survival and neurite outgrowth, but it had no obvious influences on cell attachment. In contrast, non-hydrolyzed two-dimensional collagen matrix had no obvious effects on neurite outgrowth. These findings suggest that hydrolyzed collagen is an ideal nerve cell culture media.

Adherence of uropathogenic Escherichia coli to human primary epithelial cells of renal pelvis

Human primary epithelial cells of renal pelvis was established to investigate the adherence of uropathogenic Escherichia coli （UPEC） to this cell line, in which the primary cell culture was performed by using cultivation of the normal epithelium of renal pelvis in keratinocyte serum free medium （K-SFM） with epidermal growth factor （EGF） and bovine pituitary extract （BPE）. Both UPEC132 obtained from urine specimen of patients with pyelonephritis and the pilus-free representative strain E. coli K-12p678- 54 were used to study the adherence of these strains on human primary epithelial cells of renal pelvis. The UPEC adherence was performed with observation on the morphological changes of the adhered cells, while the adhesion rates and indices were calculated in different times of experiment. In addition, the virulence genes hly and cnfl of UPEC132 were detected by multiplex PCR assay. In this study, the human primary epithelial cells of renal pelvis was found to exhibit the character of the transitional epithelial cells. Compared with the control group, the adhesion rates and indices began to increase from 15 min of the experiment time and reached its peak in 120 min. The adhesion rate and index of UPEC132 to human primary epithelial cells of renal pelvis were 74.4% and 34.0 respectively. Many microscopic changes in the primary cells adhered with UPEC132 could be detected, such as rounding or irregularity in shape, unevenness in staining and the cytoplasmic and nuclear changes. It suggests that human primary epithelial cells of renal pelvis can be used for the experiment on UPEC adhesion, thus providing a basis for the further study on the pathogenesis of UPEC.

Effects of lysophosphatidic acid on human periodontal ligament stem cells from teeth extracted from dental patients

Despite their potential applications in future regenerative medicine, periodontal ligament stem cells(PDLSCs) are difficult to obtain in large amounts from patients. Therefore, maintaining sternness while expanding the cell numbers for medical use is the key to transitioning PDLSCs from the bench to the clinic. Lysophosphatidic acid(LPA), which is present in the human body and saliva, is a signaling molecule derived from phospholipids. In this study, we examined the effects of LPA on sternness maintenance in human PDLSCs. Several spindle-shaped and fibroblast-like periodontal ligament stem-like cell lines were established from PDLSC isolation. Among these cell lines, the most morphologically appropriate cell line was characterized. The expression levels of OCT4, NANOG(a stem cell marker), and CD90(a mesenchymal stem cell marker) were high. However, CD73(a negative marker of mesenchymal stem cells) expression was not observed. Notably, immunofluorescence analysis identified the expression of STRO-1, CD146(a mesenchymal stem cell marker), and sex determining region Y-box 2 at the protein level. In addition, lipid droplets were stained by Oil red O after the induction of adipogenesis for 21 days, and mineralized nodules were stained by Alizarin Red S after the induction of osteogenesis for 14 days. Alkaline phosphate staining also demonstrated the occurrence of osteogenesis. In summary, we established a human PDLSC line, which could be applied as a cell source for tissue regeneration in dental patients. However, further studies are needed to determine the detailed effects of LPA on PDLSCs.

Three-dimensional collagen-based scaffold model to study the microenvironment and drug-resistance mechanisms of oropharyngeal squamous cell carcinomas

Objective:Squamous cell carcinoma(SCC)represents the most common histotype of all head and neck malignancies and includes oropharyngeal squamous cell carcinoma(OSCC),a tumor associated with different clinical outcomes and linked to human papilloma virus(HPV)status.Translational research has few available in vitro models with which to study the different pathophysiological behavior of OSCCs.The present study proposes a 3-dimensional(3 D)biomimetic collagen-based scaffold to mimic the tumor microenvironment and the crosstalk between the extracellular matrix(ECM)and cancer cells.Methods:We compared the phenotypic and genetic features of HPV-positive and HPV-negative OSCC cell lines cultured on common monolayer supports and on scaffolds.We also explored cancer cell adaptation to the 3 D microenvironment and its impact on the efficacy of drugs tested on cell lines and primary cultures.Results:HPV-positive and HPV-negative cell lines were successfully grown in the 3 D model and displayed different collagen fiber organization.The 3 D cultures induced an increased expression of markers related to epithelial–mesenchymal transition(EMT)and to matrix interactions and showed different migration behavior,as confirmed by zebrafish embryo xenografts.The expression of hypoxia-inducible factor 1α(1α)and glycolysis markers were indicative of the development of a hypoxic microenvironment inside the scaffold area.Furthermore,the 3 D cultures activated drug-resistance signaling pathways in both cell lines and primary cultures.Conclusions:Our results suggest that collagen-based scaffolds could be a suitable model for the reproduction of the pathophysiological features of OSCCs.Moreover,3 D architecture appears capable of inducing drug-resistance processes that can be studied to better our understanding of the different clinical outcomes of HPV-positive and HPV-negative patients with OSCCs.

Neuroanatomy and morphological diversity of brain cells from adult crayfish Cherax quadricarinatus

As in vertebrates, brains play key roles in rhythmic regulation, neuronal maintenance, diff erentiation and function, and control of the release of hormones in arthropods. But the structure and functional domains of the brain are still not very clear in crustaceans. In the present study, we reveal the structural details of the brain in the redclaw crayfish using hematoxylin-eosin staining and microscopic examination, firstly. The brain of crayfish is consist of three main parts, namely, protocerebrum, deutocerebrum, and tritocerebrum, including some tracts and commissures, briefly. Secondly, at least 9 kinds of brain cells were identified on the basis of topology and cell shapes, as well as antibody labeling. We also provide morphological details of most cell types, which were previously un-described. In general, four types of glia and three types of neurosecretory cells were described except cluster 9/11 and cluster 10 cells. Glia were categorized into another three main kinds:(1) surface glia;(2) cortex glia; and(3) neuropile glia in addition to astrocytes identified by GFAP labelling. And neurosecretory cells were categorized into I, Ⅱ and III types based on morphological observation. Finally, cluster 9/11 and 10 cells derived from the brain of crayfish, could be used for primary culture about 7–9 d under the optimized conditions. There results provide a resource for improving the knowledge of the still incompletely defined neuroendocrinology of this species. Using the crayfish as an animal model, we are easy to carry out further research in manipulating their endocrine system, exploring cellular and synaptic mechanisms so much as larval production on a small scale, such as in a cell or tissue.

The expressions of brain-derived neurotrophic factor and nerve growth factor in purified rat choroid plexus epithelial cells in vitro

Objective： To detect the expressions of brain-derived neurotrophic factor （BDNF） and nerve growth factor （NGF） in purified rat choroid plexus epithelial cells in vitro. Methods： Primary and passage choroid plexus epithelial cells were obtained from newborn, one-day Spragne-Dawley rats. The expressions of BDNF and NGF were measured by qRT-PCR and Western blottingting. The secretions of BDNF and NGF were detected by ELISA. Cell supematants of primary cells, purified cells and passage 1 cells were harvested. Results： The expression of BDNF in the purified cells was significantly lower than that in the primary cells （P〈0.05）, and it in the primary cells and the purified cells was significantly higher than that in the passage 1 cells （P〈0.05）. The expression of NGF was significantly higher in the purified cells than in the primary cells and the passage 1 cells （P〈0.05）. It in the passage 1 cells was significantly higher than that in the primary cells （P〈0.05）. Conclusion： The time of CPECs transplantation for central nervous system diseases should be selected based on their secretory function and features,which could lead to better and more effective treatment.

Cytotoxicity of Modified Nonequilibrium Plasma with Chlorhexidine Digluconate on Primary Cultured Human Gingival Fibroblasts

The aim of this study was to investigate the cytotoxicity of modified nonequilibrium plasma with chlorhexidine digluconate（CHX） on human gingival fibroblasts（HGFs）, and to evaluate the biosecurity of modified nonequilibrium plasma with 2% CHX as a new method of root canal treatment. Tissue samples taken from human gingiva were primarily cultured and passaged. Cells from passages 3–7 were used. HGFs were treated by modified nonequilibrium plasma with 2% CHX for 0 min（control group）, 30 s, 1 min, 1.5 min, 3 min, 5 min, and 10 min, respectively, and then they were incubated for 0, 24, and 48 h. After that, cell counting kit-8（CCK-8） assay was applied to analyze the cytotoxicity of modified nonequilibrium plasma with 2% CHX on HGFs. There was no significant difference between the 0 h group treated with the modified nonequilibrium plasma for 1 min and the control group（P〉0.05）. However, there were significant differences between all the other treated groups and the control group（P〈0.05）. When treated for 1.5 min or shorter, the cell viability was obviously increased; while treated for 3 min or longer, it was obviously reduced. Moreover, when successively cultured for 0, 24, and 48 h, cell viability was decreased at first and then increased in the 3-min-treated and 5-min-treated groups. The modified nonequilibrium plasma with 2% CHX was of no influence on cell viability in 1.5 min treatment, and it could be safely used on root canal treatment.