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Objective To explore the isolation, culture and identification of mouse amniotic fluid-derived mesenchymal stem cell (AF-MSC). Methods The uteruses of pregnant mice were obtained under sterile conditions. The amniotic fluid was collected, filtered and centrifuged, and the precipitated cell mass was cultured and passaged. The morphology of AF-MSC was observed and the proliferation characteristics of AF-MSC were analyzed. The surface markers of AF-MSC were identified by flow cytometry. The osteogenic, chondrogenic and adipogenic differentiation capability of AF-MSC and cell vitality after cryopreservation and resuscitation were evaluated. Results The mouse AF-MSC was seen in typical spindle shape, and vortex structure could be observed when the cell confluency exceeded 80%. No evident latency was noted in the passage and culture of mouse AF-MSC. After 2-3 d culture, AF-MSC proliferated in the logarithmic growth stage with the fastest growth rate, which was slowed down and entered into the plateau period. AF-MSC expressed stem cell antigen (Sca)-1, CD29 and CD44 rather than CD34 and CD45. After the osteogenic differentiation of mouse AF-MSC, the mineralized crystals were stained in dark red spots by Alizarin red S staining. After chondrogenic differentiation, the secreted acid mucopolysaccharide was stained in light blue by Alcian blue. After adipogenic differentiation, cytoplasmic lipid droplets were stained in red by oil red O staining. After cryopreservation and resuscitation, the survival rate of AF-MSC exceeded 95%, and the growth status was excellent. The proliferation ability at 6 d was significantly better than that before cryopreservation (P < 0.05), and the proliferation ability at other time points did not significantly differ from that before cryopreservation (all P > 0.05). Conclusions Mouse AF-MSC may be successfully isolated with convenient procedure and the low cost. In addition, the isolated AF-MSC may be purified along with the increasing times of passage. Cryopreservation does not affect the proliferation ability of AF-MSC.
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Objective To explore a practical and feasible method for isolation,culture and identification of mouse bone marrow endothelial progenitor cells(EPCs).Methods Bone marrow-derived mononuclear cells isolated by density gradient centrifugation were cultured in endothelial cell growth medium-2 MV medium.Growth and morphological changes of the cells were observed under inverted microscopy.Cell proliferation was observed by cell counting kit-8 assay.Surface markers of the EPCs were detected by flow cytometry.Angiogenic tube formation was determined by Matrigel tube formation assay.Fluores cein isothiocyanat e-ulex europaeus agglutinin-1 (FITC-UEA-1) binding and Dil-Ac-LDL uptake capabilities were observed by fluorescent microscopy.Results In the early stage,the cells were round and spindle-shaped after induced culture for 4 days.After 7 days,the cells grew in colony arrangement and gradually increased in number.After 14 days,the cells were differently shaped,such as short shuttle and triangle.After 21 days,the typical "paving stone" appearance of the cells was observed.The cells were positive for endothelial markers in flow cytometry:CD34 + (84.3％),vascular endothelial growth factor receptor 2 + (74.1％),but CD45 + (4.04％).The cells were capable of forming capillary-like tubes,up-taking Dil-Ac-LDL and binding FITC-UEA-1 in Matrigels.Conclusions A reliable method for isolation,culture and identification of mouse bone marrow EPCs may be improved on the basis of previous experiences.Since the EPCs obtained by this method may be capable of good proliferation,large in number,and stable in biological characteristics,they can serve as ideal seed cells for related subsequent studies.
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Objeetive To improve the method for the isolation and purification of rat hepatic stellate(HSC) cells and to provide a stable cell source for the research on liver-related diseases.Methods Rat liver was digested in situ by a two-step infusion assay under a strict control of the infusion temperature,flow rate and time with a combined utilization of Pronase E and Collagenase Ⅳ.And then,the HSC cells were separated by Percoll density gradient centrifugation.The cell growth curve and survival rate were measured by CCK-8 and trypan blue staining,respectively.The HSC cells were identified by flow cytometry and immunofluorescence cytochemistry.Results With the improved methods,there were (2.1 ± 0.2) × 107 HSC cells isolated from one rat and the survival rate was (96.2 ± 0.8) ％.The percentage of HSC cells with a spontaneous fluorescent characteristic from the isolated cells was 96.3％.The immunofluorescence cytochemistry was used to detect the expressions of the surface antigens α-SMA and Desmin in the isolated HSC cells.Conclusion By strict control of infusion temperature,flow rate and perfusion time as well as the combined application of Pronase E and Collagenase Ⅳ,there is an increased harvest of HSC cells with improved cell viability and purity,which is helpful for further research on HSC cells.
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·AIM:To establish a simple and efficient method for the primary culture of rabbit corneal limbus stem cells. ·METHODS:Obtained the limbal tissues from rabbits, used tissue block and enzyme digestion method to culture the corneal limbus stem cells in vitro. The growth characteristics of the cultured cells in vitro were observed under inverted microscope. By means of HE, the morphology and construction features of cells were observed.And immunohistochemical method was used to identify the cultured cells. ·RESULTS:Rabbit corneal limbus stem cells could be fast and simply cultured by using tissue block and enzyme digestion method. The dynamic observation under microscope showed that rabbit corneal limbus stem cells grew well with a higher proliferative capacity. In HE staining, the morphology and structure of cells were normal.AE5 and P63 cellular immune identification were positive. · CONCLUSION: Tissue block and enzyme digestion method could be a simple and efficient mode for the primary culture of rabbit corneal limbus stem cells.
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Obej ctive Knockout mice are widely used in the studies of joint diseases .This article investigated the effects of joint processing methods , collagenase types ,and collagenase digestion time on the number of primary fibroblast -like synoviocytes (FLSs) obtained from mice. Methods The hind legs of 6 of the 12 male mice were cut open from the hip joints , but not those of the other 6.FLSs were isolated using the type-Ⅳcollagenase digestion method and purified by differential digestion .Cell morphology was observed under the inverted microscope .The type, viability, and purity of the cells were determined by flow cytometry . Rse ults Significantly fewer FLSs were obtained from the mice with the hind legs cut open ( 19 133 ±115 ) than from those without (24 933 ± 503) (P<0.05).The numbers of FLSs collected from the cell suspension at 1, 2, 3, 4, 5,6 , and 7 hours after digestion were 700 ±300 , 600 ±100 , 15 200 ±900 , 5100 ±800 ,2700 ±300 , 900 ±200, and 300 ±100, respectively, the highest at 3 hours. There were statistically significant differences in the total number of FLSs obtained by type-Ⅳ and type-Ⅱ collagenase digestions (24900 ±500v s 18 100 ±400, P<0.05). Conclusion For in virt o culture of primary mouse FLSs, it is recommended that the hip joints be not cut open, and type-Ⅳcollagenase be used with cell sus-pension at 2-6hours after digestion .
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Objective To conduct extensive quality control tests on Madin-Darby Canine Kidney ( MDCK) cells used for the production of influenza vaccine .Methods Tests for characteristics , extraneous agents, endogenous agents and tumorigenicity were performed on MDCK cells according to Chinese Pharma -copeia Book III .Cell lysate and DNA of MDCK cells were tested for oncogenicity in the light of new interna -tional requirements .Results The MDCK cells extracted from canis were adherent cells with an epithelial morphology, whose average number of chromosome was 80±1.No bacteria, fungi and mycoplasma contami-nation were detected . The detection for extraneous and endogenous virus showed that there was no nonspecific virus causing cytopathic effect , hemadsorption , hemagglutination or animal death .Tests for re-verse transcriptase , bovine viruses and canine viruses were all negative .Each nude mouse was injected with 107 viable cells to observe their tumorigenicity .Twelve weeks after cell injection , no node was found at the injection site and in large organs by gross anatomy .There was no significant difference between test group and negative control group .The test for tumorigenicity of viable cells was negative .Cell lysate and cellular DNA collected from equivalent amount of cells were respectively injected into nude mice , and no node forma-tion was found.There was no significant difference between the test cells and negative controls in pathology indicating that the tested MDCK cells were non-oncogenic .Conclusion It showed the possibility of using MDCK cells for the production of influenza vaccine .
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Background To optimize the culture method of human retinal microvascular endothelial cells is very important for the study of retinal angiogenesis disease.Human retinal microvascular endothelial cells have been successfully cultured in previous studies,but further improvement of the culture method to harvest higher yields and purity cells is still needed.Objective This study was to design a modified method to isolate and purify human retinal microvascular endothelial cells much easily and quickly,and to compare the expression of specific markers of vascular endothelial cells,factor Ⅷ and CD31/CD34 in the cells.Methods The use of human donor eyeballs was approved by the Ethic Commission of Zhongshan Ophthalmic Center of Sun Yat-sen University.The retina tissue from healthy donor was isolated and digested by the two-step digestion method with 2％ trypsin and 0.133％ collagenase Ⅳ.Human retinal microvascular endothelial cells were collected and plated in 60 mm dishes coated by 0.1％ fibronectin and cultured in endothelial cell-specialized medium supplemented with 10％ fetal bovine serum,0.3 mg/L β-endothelial cell growth factor (ECGF) and 100 ng/L sodium heparin.During the culturing,the growth situation of the cells was monitored by morphological observation,and immunohistochemical staining was performed to probe vascular endothelial cell-specific membrane protein CD31,CD34 and factor Ⅷ for identification of the cell purity.Results Human retinal microvascular endothelial cells were isolated successfully from the retina by the twostep digestion method.The primary cultured cells adhered to well 72 hours later and achieved confluence with the typical cobblestone appearance 9 to 10 days after cultured.The cells exhibited the blue nuclei and reddish cytoplasm by regular haematoxylin and eosin stain and showed a strong positive response for CD31,CD34 and factor Ⅷ by immunohistochemistry.The positive dye of CD31 and CD34 was lower than Ⅷ factor in both endothelial cells.Conclusions Modified culture method of human retinal microvascular endothelial cells can improve cell culture result and purify target cells.
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Recently, study on cencer stem cells has been a focus of study. Cancer stem cell is a small population of cencer cells possessing the properties of stem cells: self-renewal, differentiation and proliferation. To date, the existence of cancer stem cells has been proven in acute and chronic myeloid leukemia, breast cancer, brain tumors, liver cancer and colon cancer, etc.. In this article we reviews the current progress on cancer stem cells, including the defination, existing evidence, research methods, and challenges in clinical application.
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Objective To study the application of short tandem repeat (STR) profiling in quality control of human cell lines used for biological production. Methods The methods detecting 9 and 16 STR loci to identify human cell lines by PCR-capiilary electrophoresis were established respectively. Human cell lines, which were derived from many corporations and including diploid cell strains used for virus-vaccine production and 293 cell lines used for gene therapy products, were analyzed and compared by these two methods. Results The STR profiling methods used for authentication of human cell lines were established. Most of human diploid cell strains(20/21 ) used for virus-vaccine production from 13 corporations were iden-tiffed as the intended cells and no cross-contamination was found. However, one MRC-5 cells was identified as a false cell line and one MRC-5 had 3 alleles in D13S317 locus. For 12 strains of 293 cell lines, there were significant differences in STR profiling from different manufactures, which was likely be explained that the sources and gene modifications of these 293 cell lines are not well known and their genes are unstable during passage. Conclusion The STR profiling method has the advantages of high sensitivity and specifici-ty, and can be used for authentication of each of human cell lines for biological production.
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Objective To isolate and identify the photoreceptor precursor cells isolated from newborn mouse retina. Methods Fourteen newborn littermate mice were randomly divided into seven groups (P1-P7,2 each). The retinal cells were isolated from the newborn mice on postnatal 1-7d respectively,and then cultured in vitro. The cellular growth state was observed under inverted phase contrast microscope. After in vitro expansion,5-bromo-2-deoxy-uridine (BrdU),neuroepithelial stem protein (Nestin),neural retina leucine zipper (Nrl) and Opsin in cultured cells were detected by immunohistochemistry and immunofluorescence techniques. Results Most of retinal cells isolated from the seven groups (P1-P7) of newborn mice proliferated and adhered to the plate in the particular medium. Most of the growing cells were BrdU-positive cells. A part of the cells could express neural stem cell specific antigen-Nestin,the positive expression rate of Nestin in the seven groups (P1-P7) were 31.0%,31.6%,32.3%,30.2%,31.2%,30.9% and 29.5%,respectively,with no significant difference. Some growing cells expressed Nrl,and the expression of Nrl was increased significantly on the third day after birth,and then increased in small range,the positive expression rate of Nrl in the seven groups (P1-P7) were 20.6%,35.2%,65.5%,68.6%,71.6%,73.0% and 73.3%,respectively. The positive expression of Opsin was found only in a few cells of P5-P7,while the cells in groups P1-P4 did not express Opsin. Conclusions The photoreceptor precursor cells are presented in neonatal mouse retina. They have the ability of proliferation and may differentiate into retinal photoreceptor cells,having a potentiality of expressing Opsin. The photoreceptor precursor cells are increased significantly on the third day after birth,and then differentiate into mature retinal photoreceptor cells gradually.
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Objective:To investiate biological properties of Mesenchymal stem cell(BMSC) from rat bone marrow in vitro,and their effects in inducing immune tolerance.Methods:BMSCs were obtained from rat bone marrow by density gradient centrifugation and selected by cell attachment.The morphology of BMSCs was observed by electron microscope.The expression of surface molecules of CD34 and CD44 was analyzed by flow cytometry.The cells were injected ex vivo intravenously after in vitro culture and then CD4+ CD25+ Treg ratio were determined.The proliferation of thymic cells was estimated by 3H-TdR incorporation method.Results:BMSCs were fibroblast-like cells.The expression ratio of CD44 in BMSCs was 98%,but expression of CD34 was negative.After administration of BMSCs,the ratio of CD4+CD25+Treg(2.5%?0.69%) from peripheral blood of rats was higher than in normal group(0.8%?0.14%) and PBS control group(1.0%?0.23%),P
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This study is to set up a method for separating,culturing and identifying neural stem cells from cortex in rat embryo,observing their characteristics of multipotentiality and differentiation in vitro. By using serum-free cell culturing and clone technology,the stem cells were isolated,cultured and passaged from cortex in 14-day old rat embryo. The expression of Nestin antigen of brain cells were investigated by immunocytochemistry and H-E staining. It proved that cells separated from cortex in rat embryo possessed the potential to form clones and express nestin special antigen,the differentiated cells could express special antigen of neurons,astrocytes,and oligodendrocytes,they were stem cells of central nervous system.
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Recently,study on cencer stem cells has been a focus of study.Cancer stem cell is a small population of cencer cells possessing the properties of stem cells:self-renewal,differentiation and proliferation.To date,the existence of cancer stem cells has been proven in acute and chronic myeloid leukemia,breast cancer,brain tumors,liver cancer and colon cancer,etc..In this article we reviews the current progress on cancer stem cells,including the defination,existing evidence,research methods, and challenges in clinical application.