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 establish a better method of isolating andculturing ofneural stem cells(NSCs) in neonatal rat brain. Methods Tissue of brain was isolated from neonatal rats. Different medium and culture concentration were used toculture NSCs of neonatal rat. The culture concentration used were 1×10 4, 1×105, 1×106and 1×107/ml respectively. Ingredient of medium was classified into group 1 to 8 respectively according to whether to add 2% B27, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) as well as the difference in culture concentration. The cells were induced to differentiate asto be confirmed as NSCs, and then were checked by phase contrast microscopy and identified by immunocytochemistry. Results The cells isolated and cultured gathered into neurospheres. The cells were capable of proliferating and maintaining longterm survival in vitro. The cells could be differentiated into neurons and glia.It was to the benefit of the survival of NSCs to add 5% fetal bovine serum(FBS)into the medium at the beginning of the culturing. When 10% FBS was added intothe medium, the neurospheres differentiated quickly. When concentration 1×106/ ml was used, the growth rate of the cells was the highest of all the concentrations. Reasonably higher cell concentration promoted the proliferation of NSCs. It was necessary to add 2% B27, EGF, and bFGF into the medium. The cells had the best growth when 2% B27, 20 ng/ml bFGF and 20 ng/ml EGF were added into the culture medium. EGF and bFGF had cooperative effect. Conclusion A better method of isolating and culturing of NSCs in neonatal rat brain is established and the foundation for future research is laid.
Abstract An experiment was carried out to investigate the possibility of the establishment of an osteoblasts bank which could supply osteoblasts in repairing bone defect. Osteoblasts were isolated from thetibial periosteum of eight New-Zealand rabbits and cultured in votro. A bone defect, 1.5cm in length was made in both radii of each of the 8 rabbits. The cultivated osteoblasts, gelfoam as a carrier were randomly implanted into the defects of the radii of rabbits. Accordingly, the contralateral radial defects wereimplanted with gelfoam absorbed with the Hanks solution as control. The healing of bone defects was evaluated by roentgenographic examination at 2, 4, 8 and 12 weeks after operation, respectively. It was shown that the implanted cells had osteogenetic capability and could be possible to promote healing of the bone defects. It was suggested that further study needed to be carried out in this field.
【Abstract】ObjectiveTo establish an efficient, effective hepatocyte isolation technique in order to increase cell production and decrease the prime cost. Methods The inferior vena cava below diaphragm was dissected and ligatured, and the inferior vena cava below liver was separated. Subsequently, the liver was perfused with EGTA through the portal vein while the inferior vena cava below liver was opened, and then the liver was harvested. The liver tissue was cut into 1 mm×1 mm×1 mm and digested at 37 ℃ water bath with Ⅳ collagenase for 30-40 minutes, then the hepatocytes were purified and cultured in CO2 incubator. The production and function of hepatocytes were assessed. ResultsThe isolated hepatocytes using this technique were more than 95% among the all isolated cells. No statistic difference was found in cell production and cell function comparing with traditional technique. But this technique was simplified and more economically. ConclusionThis modified hepatocyte isolation technique is efficient and effective. It can ensure the amount of production and purity of hepatocytes.
Objective To establish an effective way to cryopreserveprecartilaginous stem cells(PSCs) of neonate rat. Methods PSCs ［fibroblast growth factor-3(FGFR-3) positive cells］ were isolated and purified by magnetic cell sorting method. PSCs were cultured and amplified to the third generation. PSCs were preserved in liquid nitrogen. The biological properties of cryopreserved PSCs were investigated by reverse transcriptase polymerase chain reaction(RT-PCR), immunohistochemistry, and immunofluorescence. Results Immunohistochemical and immunofluorescent analysis showed widespread expression of FGFR-3 in cryopreserved PSCs. FGFR-3 could be dectected by RT-PCR in cryopreserved PSCs.Cryopreserved PSCs kept high cell viability, and phenotypic and proliferation characteristics of PSCs in vivo.Conclusion Cryopreservation of PSCs can supply adequate qualified cells for repairing the defects of epiphyseal growth plate by tissue engineering technique.
Objective To investigate the effect of acid, basic fibroblast growth factor (aFGF, bFGF) and epidermal growth factor (EGF), andtheir combination on the proliferation of rabbit anterior cruciate ligament (ACL) and medial collateral ligament (MCL) in vitro. Methods Thecells of ACL and MCL were isolated and subcultured from the knee joints of tenweek-old New Zealand white rabbits. The cells were seeded into 96-well corning cluster plates. Three growth factors of different concentration alone or in combination were added into the culture medium respectively, which were 0, 1, 5, 10, 50 and 100 ng/ml for aFGF, bFGF and 0, 1.56, 3.13, 6.25, 12.5, 25 and 50 ng/ml for EGF. The proliferation of the fibroblasts was measured for 48 h with XTT method. Results All of the three growth factors alone promoted the cell proliferation of ACL and MCL fibroblasts. The concentration of aFGF hada significant effect on the proliferation of both ACL and MCL fibroblasts. The concentration of 1 ng/ml bFGF and 5 ng/ml EGF was most effective in promoting the proliferation of ACL, and both bFGF and EGF had a significant effect on MCL. 5ng/ml aFGF with 50 ng/ml EGF had effect on ACL. 1 ng/ml aFGF with 3.13 ng/ml EGF had effect on MCL. Conclusion The three growth factors may promote the cell proliferation of ACL and MCL. These findings suggest that topical application of aFGF, either alone or in combination with EGF may have the potential to promote the proliferation of rabbit ACL and MCL,and aFGF of low concentration in combination with EGF is more effective than single growth factor.
ObjectiveTo explore the distribution and features of the optic cup stem cells in embryonic rat at tailbud stage.MethodsThe distribution of optic cup stem cells in optic cup tissue in 12.5-embryonic-day-old rats was observed by immunohistochemistry. The separated cells from optic cup were cultured with serum-free media, and immunofluorescence technique was used to detect the ability of hyperplasia of stem cells and expression of CHX10 antigen and specific antigens of mature retinal cells before and after differentiation.ResultsThe optic cup stem cells in embryonic rat at tailbud stage were mainly located at inner, outer, and marginal layer of optic cup. No expression of specifically marked protein of mature retinal cells was detected. The cells separated from optic cup had the ability of single-cell clone, positive expression of CHX10 and expression of several specific antigens of mature retinal cells after the inducement, including Thy1.1, glial fibrillary acidic protein (GFAP), protein kinase C (PKC) α, and rhodopsin.ConclusionOptic cup of 12.5-embryonic-day-old rats composes of undifferentiated cells, and the stem cells are mainly located in optic cup inner and marginal. High ability of hyperplasia of the optic cup stem cells cultured in vitro is found. The cells, which are retinal stem cells, can express several specifically marked proteins of mature retinal cells after inducement and differentiation.(Chin J Ocul Fundus Dis, 2005,21:159-162)
Objective To explore an effective method to culture and purify canine bladder transitional epithelial cells.Methods Bladder tissue was obtained from healthy puppy under sterile conditions. Bladder mucosa was removed from the remaining tissue with fine scissor and minced into small pieces, and then were dissociated into single cell suspensions with 0.125% trypsin. The bladder epithelial cells were cultured in defined keratinocyte serum free medium. The cells were passaged and purified by 0.05% trypsin and 0.02% EDTA. Morphological characterization were studied under inverted phase contrast microscope and transmission electron microscope. Expression of cell specific marker protein was assessed by immunohistochemistry. Results Canine bladder transitional epithelial cells could be efficiently cultivated and expanded in serum-free medium without fibroblast contamination. The cells could be passaged 4-6 times without a distinguished decrease in cell proliferation. The cells were characterized by well-developed micro filament and desmosome junction under transmission electron microscope. Immunohistochemical staining with broadly reacting anticytokeratin antibodies (AE1/AE3) confirmed the epithelial phenotype of the cells.Different generations of cells showed diploid cells. Conclusion A large number of bladder transitional epithelial cells can be obtained from small bladder tissue with our digestion method. The cultured bladder epithelial cells can be proliferated to sufficient quantities for further reconstructive purposes.
Human fibroblasts and human epidermal keratinocytes were used for culture. Chitosan solution were added in the culture solution(DMEM). After 72 hours, the fibroblasts showed rapid growth in the control culture without Chitosan, But the numbers of human fibroblasts from growth was decreased as the concentration of Chitosan was increasing. On the contrary the human epidermal keratinocytes growed more rapidly in the culture with Chitosan than in the culture without Chitosan. The results showed that Chitosan inhibited the growwth of human fibroblast and stimulated the growth of human epidermal keratinocyte .
Objective To investigate the effect of hypoxia on the exp ression and function of integrin receptor αvβ3 of bovine retinal vascular endotheliocytes. Methods Bovine retinal vascular endotheliocy tes in the culture dishes coated by vitronectin was put into the normal and hypoxemic condition, respectively. Enzyme linked immunosorbent assay and cell adhesion analysis were used to detect the expression and function of integrin receptor αvβ3 in bovine retinal vascular endotheliocytes, respectively. Results Under the condition of hypoxia, the expression of αvβ3 increased gradually, and reached the peak at the 48th hour. The expression of αvβ3 at the 60th and 72nd hour in hypoxia group was higher than that in the normal group. Bovine retinal vascular endotheliocytes absorbed more Vn of extra-cellular matrixes (ECM) after cultured under hypoxemic condition for 24 hours.Conclusion Hypoxia may up-regulate the expression of αvβ3, which promote the adsorbability of endotheliocytes.(Chin J Ocul Fundus Dis，2004，20：360-363)