Objective To establish a method for primary culture of iris pigment epithelial cells(IPE). MethodsEnzyme-Assisted microdissection was used to isolate and cultivate the IPE cells.An identification was made with microscopic and immunohistochemical observations.Results IPE were successfully sultured and showed on differences with RPE in primary culture and subculture.ConclusionEnzyme-Assisted microdissection is a reliable and quick method for the isolation of IPE.
Objective To culture astrocytes of human optic nerve and establish the cell lines for further study of healing process after optic nerve trauma. Methods Astrocytes of infantile optic nerve were cultured by tissue inoculation or tissue digestion with 0.25 % trypsin and 0.06% EDTA. The second and fourth passage cells were stained with HE and anti-GFAP, S-100 protein, vimentin, and CD34 antibodies. Results The trypsinized astrocytes of infantile optic nerver eached confluence in 7 days. The cultured cells were in polygonal shape with processes and the cytoplasm was abundant. These cells were positive in GFAP, S-100 protein and vimentin staining, and negative in CD34 staining. Conclusions Astrocytes of human optic nerve can be successfully cultured by trypsinization rather than tissue inoculation. (Chin J Ocul Fundus Dis, 2001,17:144-146)
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.
Objective To prepare the silk fibroin microcarrier loaded with clematis total saponins (CTS) (CTS-silk fibroin microcarrier), and to investigate the effect of microcarrier combined with chondrocytes on promoting rabbit knee articular cartilage defects repair. Methods CTS-silk fibroin microcarrier was prepared by high voltage electrostatic combined with freeze drying method using the mixture of 5% silk fibroin solution, 10 mg/mL CTS solution, and glycerin. The samples were characterized by scanning electron microscope and the cumulative release amount of CTS was detected. Meanwhile, unloaded silk fibroin microcarrier was also prepared. Chondrocytes were isolated from knee cartilage of 4-week-old New Zealand rabbits and cultured. The 3rd generation of chondrocytes were co-cultured with the two microcarriers respectively for 7 days in microgravity environment. During this period, the adhesion of chondrocytes to microcarriers was observed by inverted phase contrast microscope and scanning electron microscope, and the proliferation activity of cells was detected by cell counting kit 8 (CCK-8), and compared with normal cells. Thirty 3-month-old New Zealand rabbits were selected to make bilateral knee cartilage defects models and randomly divided into 3 groups (n=20). Knee cartilage defects in group A were not treated, and in groups B and C were filled with the unloaded silk fibroin microcarrier-chondrocyte complexes and CTS-silk fibroin microcarrier-chondrocyte complexes, respectively. At 12 weeks after operation, the levels of matrix metalloproteinase 9 (MMP-9), MMP-13, and tissue inhibitor of MMP 1 (TIMP-1) in articular fluid were detected by ELISA. The cartilage defects were collected for gross observation and histological observation (HE staining and toluidine blue staining). Western blot was used to detect the expressions of collagen type Ⅱ and proteoglycan. The inflammatory of joint synovium was observed by histological staining and inducible nitric oxide synthase (iNOS) immunohistochemical staining. Results The CTS-silk fibroin microcarrier was spherical, with a diameter between 300 and 500 μm, a porous surface, and a porosity of 35.63%±3.51%. CTS could be released slowly in microcarrier for a long time. Under microgravity, the chondrocytes attached to the surface of the two microcarriers increased gradually with the extension of culture time, and the proliferation activity of chondrocytes at 24 hours after co-culture was significantly higher than that of normal chondrocytes (P<0.05). There was no significant difference in proliferation activity of chondrocytes between the two microcarriers (P>0.05). In vivo experiment in animals showed that the levels of MMP-9 and MMP-13 in group C were significantly lower than those in groups A and B (P<0.05), and the level of TIMP-1 in group C was significantly higher (P<0.05). Compared with group A, the cartilage defects in groups B and C were filled with repaired tissue, and the repaired surface of group C was more complete and better combined with the surrounding cartilage. Histological observation and Western blot analysis showed that the International Cartilage Repair Scoring (ICRS) and the relative expression levels of collagen type Ⅱ and proteoglycan in groups B and C were significantly better than those in group A, and group C was significantly better than group B (P<0.05). The histological observation showed that the infiltration of synovial inflammatory cells and hyperplasia of small vessels significantly reduced in group C compared with groups A and B. iNOS immunohistochemical staining showed that the expression of iNOS in group C was significantly lower than that in groups A and B (P<0.05).Conclusion CTS-silk fibroin microcarrier has good CTS sustained release effect and biocompatibility, and can promote the repair of rabbit cartilage defect by carrying chondrocyte proliferation in microgravity environment.
ObjectiveTo explore the morphological and functional features of tissue engineered composite constructed with bone mesenchymal stem cells (BMSCs) as seeding cells, thermosensitive collagen hydrogel (TCH) and poly-L-lactic acid (PLLA) as the extracellular matrix (ECM) scaffolds in the dynamic culture system. MethodsBMSCs were separated from long bones of Fischer344 rat, and cultured; and BMSCs at the 3rd generation were seeded on the ECM scaffold constructed with braided PLLA fiber and TCH. The BMSCs-ECM scaffold composite was cultured in the dynamic culture system which was designed by using an oscillating device at a frequency of 0.5 Hz and at swing angle of 70° (experimental group), and in the static culture system (control group) for 7 days. The general observation and scanning electron microscopy (SEM) observation were performed; total DNA content was measured at 0, 1, 3, and 7 days. ResultsPLLA was surrounded by collagen to form translucent gelatiniform in 2 groups; and compact membrane developed on the surface of PLLA. SEM observation showed that BMSCs had high viability and were fusiform in shape with microvilli on the surface of cells, and arranged in line; collagen and cells filled in the pores of PLLA fiber in the experimental group. The cells displayed a flat shape on the surface; there were less cells filling in the pores of PLLA fiber in the control group. At 1, 3, and 7 days, total DNA content in the experimental group was significantly higher than that in control group (P < 0.05). The total DNA content were increased gradually with time in 2 groups, showing significant difference between at 0 day and at 7 days (P < 0.05). ConclusionThe ECM constructed with TCH and PLLA has good biocompatibility. The dynamic cultivation system can promote the cell proliferation, distribution, and alignment on the surface of the composite, so it can be used for tissue engineered composite in vitro.
Objective To observe the chondrogenic differentiation of adipose-derived stem cells (ADSCs) by co-culturing chondrocytes and ADSCs. Methods ADSCs and chondrocytes were isolated and cultured from 8 healthy 4-month-old New Zealand rabbits (male or female, weighing 2.2-2.7 kg). ADSCs and chondrocytes at passage 2 were used. The 1 mL chondrocytes at concentration 2 × 104/mL and 1 mL ADSCs at concentration 2 × 104/mL were seeded on the upper layer and lower layer of Transwell 6-well plates separately in the experimental group, while ADSCs were cultured alone in the control group. The morphology changes of the induced ADSCs were observed by inverted phase contrast microscope. The glycosaminoglycan and collagen type II synthesized by the induced ADSCs were detected with toluidine blue staining and immunohistochemistry staining. The mRNA expressions of collagen type II, aggrecan, and SOX9 were detected with real-time fluorescent quantitative PCR. Results ADSCs in the experimental group gradually became chondrocytes-like in morphology and manifested as round; while ADSCs in the control group manifested as long spindle in morphology with whirlool growth pattern. At 14 days after co-culturing, the results of toluidine blue staining and immunohistochemistry staining were positive in the experimental group, while the results were negative in the control group. The results of real-time fluorescent quantitative PCR indicated that the expression levels of collagen type II, aggrecan, and SOX9 mRNA in the experimental group (1.43 ± 0.07, 2.13 ± 0.08, and 1.08 ± 0.08) were significantly higher than those in the control group (0.04 ± 0.03, 0.13 ± 0.04, and 0.10 ± 0.02) (P lt; 0.05). Conclusion ADSCs can differentiate into chondrocytes-like after co-culturing with chondrocytes.
Based on the current study of the influence of mechanical factors on cell behavior which relies heavily on experiments in vivo, a culture chamber with a large uniform strain area containing a linear motor-powered, up-to-20-Hz cell stretch loading device was developed to exert mechanical effects on cells. In this paper, using the strain uniformity as the target and the substrate thickness as the variable, the substrate bottom of the conventional incubation chamber is optimized by using finite element technique, and finally a new three-dimensional model of the incubation chamber with “M” type structure in the section is constructed, and the distribution of strain and displacement fields are detected by 3D-DIC to verify the numerical simulation results. The experimental results showed that the new cell culture chamber increased the accuracy and homogeneous area of strain loading by 49.13% to 52.45% compared with that before optimization. In addition, the morphological changes of tongue squamous carcinoma cells under the same strain and different loading times were initially studied using this novel culture chamber. In conclusion, the novel cell culture chamber constructed in this paper combines the advantages of previous techniques to deliver uniform and accurate strains for a wide range of cell mechanobiology studies.
Objective To predict clinical chemotherapy sensitivity of primary non-small cell lung cancer（NSCLC） by methylthiazal （MTT） tumor chemosensitivity assay method in vitro and detection of multidrug resistance gene1 （MDR1）, and provide reference for clinical individualized treatment. Methods We selected 80 fresh primary NSCLC samples from NSCLC patients who underwent surgical resection in Zibo Central Hospital Affiliated to Binzhou Medical College between January 2009 and December 2011. There were 46 male patients and 34 female patients with their median age of 54 （29 to 81）years. Viable NSCLC cells obtained from malignant tissue were tested for their sensitivity to cisplatin （DDP）, gemcitabine （GEM）, docetaxe （DOC）, etoposide （VP-16） ,and vinorelbine （NVB） using MTT assay in vitro. Fluorescent quantitative reverse transcriptase-polymerase chain reaction （RT-PCR） was used to analysis the expression level of multidrug resistance gene1 （MDR1）. Results After exposure to antitumor drugs, morphologic changes, decrease of metabolic activity, and apoptosis were detected in NSCLC cells. MTT results showed that different individual cancer cells had different chemosensitivity to antitumor drugs, and cancer cells also had different chemosensitivity to different antitumor drugs. Inhibitory rates of cancer cells exposed to DOC, GEM, and VP-16 were significantly higher than those of cancer cells exposed to DDP and NVB （42.5%±9.5%, 40.5%±6.5%, 38.4%±7.6% versus 31.5%±8.5%，32.5%±7.8%, P＜0.05）.The positive rate of MDR1 in tumor tissues was 40.0% （32/80）. The expression of MDR1 was not associated with tumor histological type, degree of differentiation, lymph node metastasis and TNM stage. The expression of MDR1 was associated with resistance to NVB （χ2=5.209，P=0.022），GEM （χ2=4.769，P=0.029），VP-16 （χ2=4.596，P=0.032），and DDP（χ2=6.086，P=0.014）, but not associated with resistance to DOC（χ2=0.430，P=0.512）. Conclusion MTT chemosensitivity assay can effectively predict clinical chemotherapy sensitivity. Detection of MDR1, together with MTT chemosensitivity assay, can more accurately predict NSCLC chemosensitivity and be a guide for individualized chemotherapy of NSCLC.
Purpose:To evaluate the function of gap junction-mediated intercellular communication in cultured cells of retinal pigment epithelial(RPE) cells from porcine eyes. Methods:The cultured RPE cells were previously stained by a fluorescent probe 5, 6-carboxy fluorescein diacetate (CFDA) ,and then photobleach the fluorescent molecule in chosed cells. Using laser scanning confocal microscope (LSCM)to observe fluorescence recovery rate of the RPE cells which located in different condition. The function of gap junction communication was evaluated according to the fluorescence recovery rate. Results:The fluorescence recovered after photobleached and the fluorescent density of cells which touching to them descend. The recovery rate per minnte of the cells which the cell number it adjacent to was 1,2 and 3 respectively was 1. 997plusmn;0. 665, 4. 378plusmn;0. 811 and 8. 736plusmn;2. 084. Conclusion:The cultured porcine RPE cells have the function of gap junction communication,and its function proportion is associate to its adjoining cells number. （Chin J Ocul Fundus Dis,1996,12: 41-42）
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.