Objective To investigate the possibility of creation of tissue engineered heart valve leaflets in vitro . Methods Aorta were obtained from 9 hybrid young pigs. The endothelial cell, fibroblast and smooth muscle cells were isolated and cultured to get enough cell. The expanded fibroblast, smooth muscle cell,and endothelial cells were seeded on the polymers sequentially. The cell polymer constructs were sent for scanning electron microscopy(SEM) examination after cultured for 7, 14, and 28 days. Histological examination were performed after the cell polymer constructs cultured for 28 days. Results SEM showed that the number of cells on the polymers increased as the culture time prolonged, with the formation of matrix. After 28 days, there were a great number of cells and large amount of matrix on the scaffolds. The confluent cell had covered a large area of the polymers. Hematoxylin and eosin(HE) stain showed large amount of cells attached to the polymers. Conclusion With the viability of the cultured cellular scaffolds,it is possible to create tissue engineered heart valve leaflets in vitro.
Objective To develop a reliable method for primary culture of normal human peritoneal mesothelial cells. Methods Human peritoneal mesothelial cells were dissociated by a mixture of pancreatin and ethylene diamine tetraacetic acid with a magnetic puddler. Inverted phase contrast microscope was used to observe the morphological structures of cells, approximate process of growth. Calretinin was used to identify the mesothelial cells. Results On the 4th d of culture, mesothelial cells adhered to the culture dish. After day 14, mesothelial cells confluenced gradually and grew well like the slabstone. Calretinin was positively expressed by mesothelial cells after 5 d of cultivation. The mesothelial cell population of subculture was less than that of the primary culture. Conclusion A reliable method for primary culture of normal human peritoneal mesothelial cells has been successfully developed, by which sufficient amount of highly purified normal human peritoneal mesothelial cells can be obtained.
【Abstract】Objective To study the influence of transplantation of cultured parathyroid cells on the survival of the allografts in rats. Methods Parathyroid cells digested with collagenase and trypsin were cultured and transplanted under the left renal capsule. The survival time of the allografts was recorded and the allografts were examined by transmission electron microscopy.Results In fresh parathyroid cells group, the mean survival time was (9.25±3.45) days. While in cultured parathyroid cells group, the survival time was (46.25±7.44) days (P＜0.01). During the 50 days of observation, serum calcium and PTH remained normal in 6 of 8 rats. There were intact parathyroid cells in the allografts which had abundant rough endoplasmic reticula,mitochondria and secretory granules. Conclusion Transplantation of cultured parathyroid cells in rats can prolong the survival time of allografts and is a potent way to cure hypoparathyroidism.
ObjectiveTo study the effects of recombinant human growth hormone (rhGH) on proliferation of human rectal cancer cell in vitro. MethodsThe experiment was divided into control group,rhGH group,Oxaliplatin (LOHP) group and rhGH+LOHP group. The double proliferation time of cells,cell inhibition rate,cell cycle, proliferation index (PI) and DNA inhibition rate of human rectal cancer line,HR8348,were studied by cell culture, MTT assay and flow cytometry on different concentration of rhGH. ResultsIn vitro the markedly accelerated effects of rhGH on multiplication of HR8348 cell line were not found: there was no statistical significance as compared rhGH group with control group or compared rhGH+LOHP group and LOHP group （Pgt;0.05）. The double proliferation time of cells was markedly lengthened, cell inhibition rate and the cells arrested in G0-G1 phase were obviously increased, meanwhile, the cells in S phase （P＜0.05） and G2-M phase and PI were markedly decreased and DNA inhibition rate was obviously risen as compared rhGH+LOHP group with control group or rhGH+LOHP group and rhGH group （P＜0.01）.ConclusionIn vitro rhGH does not accelerate the multiplication of human rectal cancer cells.
Objective To observe the morphology and growing status of mesenchymal stem cells(MSCs) of human bone marrow in vitro, in order to confirm that MSCs of human bone marrow are ideal seed cells and provide basic theory for further MSCs research. Methods The methods of density gradient centrifugation with lymphocyte separation medium for human and adherent filtration were used to isolate and purify MSCs of human bone marrow. We observed the cellular growth status and morphology of the primary MSCs and the surface antigens of second passage MSCs were tested. Results The primary culture cells fused into monolayer after 14-16 d. The passage cells kept the same morphological characteristics of primary culture cells. Ultrastructure of the second passage MSCs showed that the shape of nuclei was irregular, there were multiple nucleoli in some of the nuclei, and morphological differentiation of intracytoplasm organelles was immature. The growth curve of the first, fifth and tenth passage cells showed a logarithmic growth at day 3, a peak growth at day 5, and no clones occurred after tenth passage. Cloning efficiency of first passage, fifth passage and tenth passage was respectively 25.83%±2.93%, 14.67%±1.63% and 4.67%±0.52%. Test of MSCs phenotypic characteristics showed a high homogeneity among the cells and surface antigen profiles were positive for CD29, CD44 and negative for CD34, CD45. Conclusion The methods of density gradient centrifugation with lymphocyte separation medium for human and adherent filtration are simple, economic and efficient to isolate and purify MSCs from human bone marrow. With a high proliferating ability in vitro, MSCs from human bone marrow are ideal seed cells for tissue engineers.
Abstract： Objective To investigate and improve the method of isolation, induction and culture, amplification in vitro of human endothelial progenitor cells (EPCs) in human bone marrow, thus establish a foundation for EPCs to participate in basic research and clinical application. Methods WHuman bone marrow mononuclear cells (hBMMNCs) were isolated by density gradient centrifugation and cultured in the 6plateds coating human fibronections(HFN group), coating gelatinum (coating gelatinum group) and coating nothing (coating nothing group) respectively. After culturing for 4-7d endothelial cell basal medium-2(EBM-2) cell colonyforming units (CFUs) appeared, then select EPCsliked CFUs for cultivation which was named the pick method, CD34+ KDR+ and CD133+ KDR+ double positive cells were detected in flow cytometry, and CD133, CD34, CD31, vWF and KDR expression were detected with cell immunochemical test. Results hBMMNCs were isolated from human bone marrow more effectively with OptiprepTM cell separating medium, and induced and obtained more EPCs, and cultured and amplificated in vitro. Flow cytometer showed CD133+ KDR+ double positive cells reaching up to 70.4%±5.4%, CD34+ KDR+ double positive cells reaching up to 69.1%±8.7%. EPCs grew vigorously in coating HFN group and coating gelatinum group, both HFN and gelatinum promote EPCs adherence and growth, but there were no statistically difference in two groups (Pgt;0.05).Surface mark of adherent cells cultured 7d such as CD133, CD31, vWF and KDR showed positive, and cells cultured 14d such as CD34 showed positive. Conclusion The method of picking CFUs can obtain more EPCs from human bone marrow with success and can amplificate EPCs in vitro, thus introducing another simple and effective method to purify EPCs, further widening range and increasing method to purify EPCs.
Objective To establish a simple and efficient method to isolate and culture the umbilical vein vascular endothelial cells in canine. Methods Twelve umbilical cords [(13.0 ± 1.5) cm in length] were taken from 12 newborn pups of Beagles. And then the vascular endothelial cells were isolated from these umbilical cords digested by 1% collagenase type I for 5, 7, and 10 minutes respectively (4 umbilical cords in each group). After cultured, the vascular endothelial cells were identified by morphology, immunofluorescence, and flow cytometry. And the growth curvature of umbilical vein vascular endothelial cells was detected by MTT assay. Results Few vascular endothelial cells were collected at 5 and 10 minutes after digestion; many vascular endothelial cells were seen at 7 minutes, and became cobblestone with culture time, with a large nucleus; after passage, cell morphology had no obvious change. Fluorescence microscope results showed that positive von Willebrand factor (vWF) and CD31 cells were observed in most of cells. The flow cytometry test displayed that the positive cell rates of vWF and CD31 were 99.0% ± 0.7% and 98.0% ± 1.2%, respectively. The above results indicated that cultured cells were vascular endothelial cells. MTT assay showed that vascular endothelial cells proliferation increased significantly with culture time. Conclusion Enzyme digestion is a convenient method to isolate vascular endothelial cells from canine umbilical vein, and a large number of cells and high purity of cells can be obtained by the method.
ObjectiveTo establish an efficient method of isolating and culturing high activity and high purity of Schwann cells, and to identify the cells at the levels of transcription and translation. MethodsThe sciatic nerves harvested from a 4-week-old Sprague Dawley rat were digested in the collagenase I for 15 minutes after dissecting, and then the explants were planted in culture flask directly. The cells were cultured and passaged in vitro, the growth state and morphological changes of the cells were observed under inverted phase contrast microscope. MTT assay was used to test the proliferation of cells and the cells growth curve was drawn. RT-PCR and immunohistochemistry staining were used to detect S100 and glial fibrillary acidic protein (GFAP) at the levels of transcription and translation, respectively. The purity of cells was caculated under microscope. ResultsAfter the digestion of collagenase I, fibroblast-like cells appeared around explants within 24 hours, with slender cell body and weak refraction. After tissues were transferred to another culture flask, a large number of dipolar or tripolar cells were seen after 48 hours, with slender ecphyma, plump cell body, and b refraction, and the cells formed colonies within 72 hours. The cells were covered with the bottom of culture flask within 48-72 hours after passaging at a ratio of 1∶2, and spiral colonies appeared. Cells showed vigorous growth and full cytoplasm after many passages. MTT assay results showed that the cells at passage 3 entered the logarithmic growth phase on the 3rd day, reached the plateau phase on the 7th day with cell proliferation, and the growth curve was “S” shape. RT-PCR results showed that the cells expressed S100 gene and GFAP gene, and immunohistochemistry staining showed that most of the cells were positively stained, indicating that the majority of cells expressing S100 protein and GFAP protein. The purity of Schwann cells was 98.37% ± 0.30%. ConclusionHigh activity and high purity of Schwann cells can be acquired rapidly by single-enzyme digestion and explant-culture method.
Objective Dexamethasone is one of the basic agents which could induce osteogenic differentiation of mesenchymal stem cells. To investigate the optimal concentration of dexamethasone in osteogenic differentiation of adiposederivedstem cells (ADSCs) so as to provide the theoretical basis for further bone tissue engineering researches. Methods FiveNew Zealand rabbits (2-3 kg) of clean grade, aged 3 months and male or female, were obtained. ADSCs were isolated from the subcutaneous adipose tissue of inguinal region, and cultured with collagenase digestion, then were detected and identified by CD44, CD106 immunofluorescence staining and adi pogenic differentiation. ADSCs at passage 3 were used and the cell density was adjusted to 1 × 105 cells/mL, then the cells were treated with common cultural medium (group A) and osteogenic induced medium containing 0 (group B), 1 × 10-9 (group C), 1 × 10-8 (group D), 1 × 10-7 (group E), 1 × 10-6 (group F), and 1 × 10-5 mol/ L (group G) dexamethasone, respectively. The cell prol iferation and the mRNA expressions of osteocalcin (OC) and core binding factor α1 (Cbfα1) were detected by MTT and RT-PCR, respectively. The activity of alkal ine phosphatase (ALP) was measured, and the percentage of mineral area was calculated. The mineral nodules were also detected by al izarin red staining. Results ADSCs mostly presented fusiform and polygon shape with positive expression of CD44 and negative expression of CD106. The result of oil red O staining was positive after ADSCs treated with adipogenic induced medium. The result of MTT revealed that the absorbance (A) value decl ined with the ascending of the concentration of dexamethasone, and there was significant difference in A value between groups D and E at 5 and 7 days after osteogenic induction (P lt; 0.05). The mRNA expressions of OC and Cbfα1 reached the peak in groups E and D at 7 days after osteogenic induction, respectively. The activity of ALP and the percentage of mineral area had the maximum value in group D at 14 days, then decl ined gradually. There was no significant difference in the mRNA expressions of OC and Cbfα1, the activity of ALP, and the percentage ofmineral area between groups D and E (P gt; 0.05), but significant differences were found between groups D and E and other groups (P lt; 0.05). After 14 days, the cells of group G died, and the result of al izarin red staining was positive in groups B, C, D, E, and F. Conclusion When the concentration of dexamethasone in osteogenic medium is 1 × 10-8 mol/L, it could not only reduce the inhibitive effect on cells prol iferation, but also induce osteogenic differentiation of ADSCs more efficiently.