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 investigate the method of cultivation and the feature of differentiation of spinal cordderived stem cells in vitro.Methods The neural stemcells from spinal cord of 15 days fetal rats were harvested and cultivated in aserumfree limited medium. The stem cells were induced to differentiate and theresults were identified by cellular immunohistochemistry. Results Lots of stem cells were obtained from the spinal cord of fetal rats and the sphere of stemcells was formed about 10 days. Neural stem cells can give rise to mature neurons and astrocytes.Conclusion Epidermal growth factor/basic fibroblast growth factor serum-free limited medium can promote the proliferation activity ofthe stem cells. Spinal cord-derived stem cells can differentiate into glial cells and neurons.
Objective To locate sinoatrial node (SAN) in suckl ing pigs, to develop a rel iable method for isolation, purification and cultivation of SAN cells and to observe the compatibil ity of SAN cells and Col I fiber scaffold. Methods Five newborn purebred ChangBaiShan suckl ing pigs (male and female), aged less than 1-day-old and weighing 0.45-0.55 kg, wereused. Multi-channels electrophysiological recorder was appl ied to detect the original site of atrial waves. Primary SAN cells harvested from that area were cultured by the conventional culture method and the purification culture method including differential velocity adherent technique and 5-BrdU treatment, respectively. Atrial myocytes isolated from the left atrium underwent purified culture. Cell morphology, time of cell attachment, time of unicellular pulsation, and pulsation frequency were observed using inverted microscope. The purified cultured SAN cells (5 × 105 cells/mL) were co-cultured with prewetted Col I fiber scaffold for 5 days, and then the cells were observed by HE staining and scanning electron microscope (SEM). Results The atrial waves occurred firstly at the area of SAN. The purified cultured SAN cells were spindle, triangular, and irregular in morphology, and the spindle cells comprised the greatest proportion. Atrial myocytes were not spindle-shaped, but primarily triangular and irregular. The proportion of spindle cells in the conventional cultured SAN cells was decreased from 73.0% ± 2.9% in the purified cultured SAN cells, to 44.7% ± 2.3% (P lt; 0.01), and the proportion of irregular cells increased from 7.0% ± 1.7% in the purified cultrued SAN cells to 36.1% ± 2.6% (P lt; 0.01) . The proportion of the triangular cells in the purified and the conventional cultured SAN cells was 20.0% ± 2.1% and 19.2% ± 2.5%, respectively (P gt; 0.05). At 5 days after co-culture, HE staining displayed lots of SAN cells in Col I fiber scaffold, and SEM demonstrated conglobate adherence of the cells to the surface and lateral pore wall of scaffold, mutual connections of the cell processes, or attachment of cells to lateral pore wall of scaffold through pseudopodia. Conclusion With accurate SAN location, the purification culture method containing differential velocity adherent technique and 5-BrdU treatment can increase the proportion of spindle cells and is a rel iable method for the purification and cultivation of SAN cells. The SAN cells and Col I fiber scaffold have a good cellular compatibil ity.
Objective To observe the effects of culture medium of amniotic cells on NO and NOS in retinal tissues of rabbits in vitro in order to provide a protective method for antioxidation in retina transplantation. Methods Thirty adult healthy rabbits (30 right eyes) were divided into 3 groups. Group I: fresh retinal tissue; group II: routine culture medium; group III: culture medium of amniotic cells. The retinal tissues in group II and III were cultured in the corresponding culture medium for 1 week. The content of NO and NOS in retinal tissues in the 3 groups were determined. Results Compared with group I, the content of NO and NOS of group II increased obviously (t=3.821, 3.854; P＜0.001). There was no statistical difference of content of NO and NOS between group I and III (t=1.657, 1.745; P＞0.05). Conclusion Culture medium of amniotic cells may remove free radicals and enhance the ability of antioxidation. (Chin J Ocul Fundus Dis，2004，20：366-368)
Objective To investigate the changes of cellular configuration and polarized characteristic of visual pigment during the development of rod cells of neonatal calf in vitro. Methods Retinal cells of neonatal calf were dissociated and cultivated for 10, 20, 30 and 40 days in vitro were used for immunocytochemical analysis. Retinal rod cells were identified by rho4D2 antibody. The configuration of positive cells and rhodopsin molecular distribution were analysed at different cultivated time. The immuno-reactivity of positive cells was measured by image analysis system. Results The rho4D2 immuno-reactive cells included small round cells without protuberance and the cells with protuberance at the peak. At the 10th day after cultivation, the visual pigment immunoreaction was suffusive in the whole cell membrane and apical process; while at the 30th and 40th day, it gathered at the membrane of apical process or one pole of the cells. The results of quantitative analysis showed that the immunoreactive intensity of positive cells at the 20th day after cultivation was ber than that at the 10th day; while there was no significant difference among the immunoreactive intensity at the 20th, 30th, and 40th day. Conclusions Rod cells at the 30th and 40th day after cultivation have the polarized characterization and visual pigment molecular distribution with high level of expressive ability of protein, which are mature neurons. (Chin J Ocul Fundus Dis, 2005, 21: 394-396)
Objective To establish a rapid in vitro culture method of human choroidal endothelial cells (HCEC) and the cellular Characteristics to provide an in vitro model for researches of choroiretinal diseases which involved the HCEC. Methods The human choroidal tissues were digested in two steps by trypsin and collagenase, and the HCEC were obtained and cultured after the digested cell suspension was sorted and purified with magnetic beads of CD31 Dynabeads. The characteristics of HCMEC were observed by the morphologic observation method, transmission electron microscopy, and immunohistochemical staining with FⅧ factor, CD31, and CD34. Results The cultured HCEC were polygonal and oval, and after amalgamation, the cells had slabstone-like appearance. After the subculture, the configuration of HCEC remained the same, and represented cobblestone appearance with less magnetic beads attached on the cellular surface after HCEC converged into a single layer. The Weibel-Palade body which is the characteristic marker of endothelial cells was found. The staining of FⅧ fatcor, CD31, CD34 were positive. Conclusion HCEC can be cultured in vitro successfully with our method, which is easy to get sufficient number of highly purified HCEC. (Chin J Ocul Fundus Dis, 2007, 23: 126-129)
Objective To discuss the applycation possibility of themicroscopic stripping technique used in the primary culture of human embryonicesophagus squamous epithelial cells, and of the methodds for the isolation, depuration and subculture of the esophagus epithelial cells in vitro. Methods The squamous epithelial cells wereobtained from the esophagus mucous membrane of the 20-week abortion fetus through the microscopic stripping technique, and were digested with trypsin. Then, the morphological, immunohistochemical observation and the growth curve of the isolated cells were studied. Results The isolated cells were spherical in the cell suspension and spherical-like or polygon-like after attachment to the culture flask.The squamous epithelial specialized cytokeratin staining was bly positive. And the morphological studies by the transmission electron microscopy indicated that the cultured cells were squamous epithelial cells. The squamous epithelial cells reached the peak level 3-4 days after the transfer of the culture. The absorbanceat 3 and 4 days was significantly higher than that at 1,2,5 and 6 days (P＜0.05). Conclusion A large mumber of squamous epithelial cells can be available with the microscopic stripping technique and the digestion method. Thecultured squamous epithelial cells can be proliferated quickly, and fit for the tissue engineering study.
Objective To investigate the protective effect and mechanism of erythropoietin (EPO) on injury of human retinal pigment epithelial (hRPE) cell induced by hydrogen peroxide (H2O2). Methods Take subcultured hFRPE cells as study target. They were treated with 800 mu;mol/L of H2O2 for 3 hours to establish the cell injury model. The cultured cells were divided into three groups：control group, simply injury group and therapeutic group which again divided into 10 IU/ml, 20 IU/ml, 40 IU/ml,60 IU/ml subgroups according to the concentration of recombinant human erythropoietin(rhEPO). NF-kappa;B was measured by immunohistochemistry. The content of Malondialdehyde(MDA) which was the product of cellular lipid peroxidation and the releasing rate of lactate dehydrogenase（LDH）were estimated by chromatometry. Results H2O2 could elevate the level of MDA and the releasing rate of LDH, compared simply injury group with control group, the differences were significant.(tLDH＝29.746,tMDA＝20.426,Plt;0.05); Compared all of therapeutics groups with simply injury group, the releasing rate of MAD and LDH were decreased obviously, the differences were significant.（LDH t10IU＝5.770，t20IU＝12.774，t40IU＝19.818，t60IU＝24.833，Plt;0.05；MDA t10IU＝5.345，t20IU＝10.278，t40IU＝18.571，t60IU＝20.247，Plt;0.05）; The correlative analysis results of each therapeutic subgroup were: ①the concentration of rhEPO had negative correlation with the relation rate of LDH and the content of MDA(r=-0.976，P=0.024； r=-0.968，P=0.032) ; ②the concentration of rhEPO had positive correlation with the nuclear translative rate of NF-kappa;B(r=0.998，P=0.002); ③the nuclear translative rate of NF-kappa;B had negative correlation with the content of MDA(r=－0.954，P=0.046). Conclusion EPO can protect hFRPE cells from the injury of H2O2, the mechanism may be related to the activation of NF-kappa;B.
Schwann cells (SC) play an important role in nerve regeneration. The cultures of both human and rabbit SC (gt;99%) were obtained, and were separately derived from the sciatic nerve of the human fetus and the rabbit respectively by "the method of reexplantation". In addition, the cryostore and resuscitation of SC were carried out, and the resuscitated cells could retain their growth properties.
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 .