Objective To investigate the effect of subretinal fluid (SRF) with different grades of proliferative vitreoretinopathy (PVR) on bcl-2 oncoprotein expression in retinal pigment epithelium (RPE) cells and fib roblast (FB)． Methods Using immunohistological staining technique and Western-blotting method to detect the expression of bcl-2 protein in RPE cells and FB under the stimulation of SRF． Results The expression levels of bcl-2 increased in both types of cells to a certain extent compared with those of the control group 4 hours after the cells subjected to SRF； 36 hours later, the expression levels of bcl-2 of experimental groups decreased more quickly than those of the control group， and the control group showed relatively higher bcl-2 protein levels at the end of observation． Conclusions SRF may stimulate the e xpression of bcl-2 in RPE cells and FB under culture at early stage, but accel arate the declining of bcl-2 protein levels a certain time after subjected to SRF． (Chin J Ocul Fundus Dis, 2001,17:58-60)
Objective To identify pancreatic duct-derived stem cells （PDSCs） from the pancreatic duct of rats, and culture in three-dimension cell culture system or two-dimension cell culture, with characterization on their morphology and phenotype. Methods Adult male Wistar rats underwent perfusion with collagenaseⅤvia the pancreatic duct, the pancreas was surgically procured, digested, followed by discontinued density gradient centrifuge to isolate islets from acinar and ductal tissue. The acinar and ductal tissues were cultivated in serum-containing medium in the three-dimension or two-dimension cell culture seperatedly to obtain adherent cells, as PDSCs, which were expanded by consecutive passages. The cell junction, cell cycle, and cell apoptosis rate between the two-dimension and three-dimision cell culture were compared by means of microscope and flow cytometry. Results PDSCs of rats showed direct adherence culture in three-dimension and two-dimision cell culture systerm. PDSCs grew in many layers in three-dimension cell culture system on 2 days after culture, and possessed capacity of high proliferation on 7 days. Cells were adherent to the bottom of flask on 5 days after culture in two-dimision cell culture. Cell junctions were more obvious in three-dimension cell culture system. Compared with the two-dimision cell culture, the cells of G1 phase increased〔（56.46±1.17） % vs.（37.33±1.21）%〕, and the cells of G2 phase decreased〔（14.62±0.15） % vs. （32.40±1.21）%〕in three-dimension cell culture system （P＜0.01）. The early period apoptosis rate was lower in three-dimension cells culture system than that in two-dimision cell culture 〔（1.48±0.39）% vs. （5.24±1.33）%, P＜0.05〕. The late period apoptosis rate was no statistically significant difference between the three-dimension and two-dimension cell culture （P＞0.05）. Conclusions PDSCs of rats could be obtained in the three-dimension cell culture system. There is cell junction in three-dimension culture mode, which shows a significant enhancement on intercellular interaction, and the biological characters are different between the three-dimension and two-dimension cell culture.
Hydrogel is a creative polymeric biomaterial which can resemble extracellular matrix (ECM) in vitro. Hydrogel is also a material with intrinsic bioinert, but it can offer mechanical support and developmental guide for cell growth and new tissue organization by designing physicochemical and biological properties of hydrogels precisely. This review mainly introduces design of hydrogels, properties and applications in tissue engineering and regenerative medicine, drug delivery, stem cell culture and cell therapy.
ObjectiveTo investigate the antibacterial pretreatment protocol for primary fibroblast cell culture from transbronchial biopsies in patients with benign tracheal stenosis (BTS). MethodsFifteen specimens of intratracheal hyperplastic granulation tissue were obtained from 14 BTS patients by transbronchial biopsies. The tissues were divided into 3 groups according to different antibacterial pretreatment with 5 specimens in each group. An usual concentration of antibiotics pretreatment group (group 1) was pretreated by washing with PBS contained 1%-2% penicillin/streptomycin. A high concentration of antibiotics pretreatment group (group 2) was pretreated by washing with PBS contained 6% penicillin/streptomycin. An alchohol and high concentration of antibiotics pretreatment group (group 3) was pretreated by washing with 75% alcohol 3-4 seconds firstly,then by washing with 6% penicillin/streptomycin. After different pretreatment,all tissues were cultured with tissue culture method in the same condition. ResultsThe primary fibroblasts were successfully cultured from the tissue pretreated by method 2 and 3,but not cultured from the tissue pretreated by method 1 with large amount of bacteria. There were significant differences in the furthest radius of cell proliferation between different culture time points in three groups (P<0.01). The differences in the furthest radius of cell proliferation between three groups were not different at 24,48 or 72 h (P>0.05),but were significant between three groups at 96 h (P<0.05). ConclusionAn pretreatment protocol with high concentration of antibiotics or 75% alcohol is feasible in human primary fibroblasts culture from small specimens obtained by transbronchial biopsy.
ObjectiveTo investigate the effect of overexpressing the Indianhedgehog (IHH) gene on the chondrogenic differentiation of rabbit bone marrow mesenchymal stem cells (BMSCs) in a simulated microgravity environment. MethodsThe 2nd generation BMSCs from rabbit were divided into 2 groups: the rotary cell culture system (RCCS) group and conventional group. Each group was further divided into the IHH gene transfection group (RCCS 1 group and conventional 1 group), green fluorescent protein transfection group (RCCS 2 group and conventional 2 group), and blank control group (RCCS 3 group and conventional 3 group). RCCS group cells were induced to differentiate into chondrocytes under simulated microgravity environment; the conventional group cells were given routine culture and chondrogenic induction in 6 well plates. During differentiation induction, the ELISA method was used to detect IHH protein expression and alkaline phosphatase (ALP) activity, and quantitative real-time PCR to detect cartilage and cartilage hypertrophy related gene expressions, and Western blot to detect collagen typeⅡ, agreecan (ANCN) protein expression; and methylene blue staining and Annexin V-cy3 immunofluorescence staining were used to observe cell slide. ResultsAfter transfection, obvious green fluorescence was observed in BMSCs under fluorescence microscopy in RCCS groups 1 and 2, the transfection efficiency was about 95%. The IHH protein levels of RCCS 1 group and conventional 1 group were significantly higher than those of RCCS 2, 3 groups and conventional 2, 3 groups (P < 0.05); at each time point, ALP activity of conventional 1 group was significantly higher than that of conventional 2, 3 groups (P < 0.05); ALP activity of RCCS 1 group was significantly higher than that of RCCS 2 and 3 groups only at 3 and 7 days (P < 0.05). Conventional 1 group expressed high levels of cartilage-related genes, such as collagen typeⅡand ANCN at the early stage of differentiation induction, and expressed high levels of cartilage hypertrophy-related genes, such as collagen type X, ALP, and Annexin V at the late stage (P < 0.05). RCCS 1 group expressed high levels of cartilage-related genes and low levels of cartilage hypertrophy-related genes at all stages. The expression of collagen typeⅡprotein in conventional 1 group was significantly lower than that of conventional 2 and 3 groups at 21 days after induction (P < 0.05); RCCS 1 group expressed high levels of collagen typeⅡand ANCN proteins at all stages (P < 0.05). Methylene blue staining indicated conventional 1 group was stained lighter than conventional 2 and 3 groups at 21 days after induction; while at each time point RCCS 1 group was significantly deeper than RCCS 2 and 3 groups. Annexin V-cy3 immunofluorescence staining indicated the red fluorescence of conventional 1 group was stronger than that of conventional 2 and 3 groups at each time point. The expression of red fluorescence in each RCCS subgroup was weak and there was no significant difference between the subgroups. ConclusionUnder the simulated microgravity environment, transfection of IHH gene into BMSCs can effectively promote the generation of cartilage and inhibit cartilage aging and osteogenesis. Therefore, this technique is suitable for cartilage tissue engineering.
ObjectiveTo summarize current patient-derived organoids as preclinical cancer models, and its potential clinical application prospects. MethodsCurrent patient-derived organoids as preclinical cancer models were reviewed according to the results searched from PubMed database. In addition, how cancer-derived human tumor organoids of pancreatic cancer could facilitate the precision cancer medicine were discussed. ResultsThe cancer-derived human tumor organoids show great promise as a tool for precision medicine of pancreatic cancer, with potential applications for oncogene modeling, gene discovery and chemosensitivity studies. ConclusionThe cancer-derived human tumor organoids can be used as a tool for precision medicine of pancreatic cancer.
Objective To investigate the reliability of culture method of adult human parathyroid cells. Methods Adult human parathyroid tissue was digested by collagenase, then the original generation of cells were cultured and passaged, and their morphological changes were observed and recorded every other day. Part of the passaged cells were observed through electron microscope and its supernatant parathyroid hormone (PTH) was assayed. Meanwhile, the other part of cells were tested the parathyroid markers, including PTH, calcium-sensing receptor (CaSR) and glial cells missing-2 (GCM-2) by PCR. Results Abundant cytoplasm, mitochondria, endoplasmic reticulum and Golgi apparatus from the seventh day's passaged cells were observed by the electron microscopy, as well as, some secretory granules existing in both cytoplasm and intercellular lacuna. Also, the PTH from supernate was detected, and parathyroid specific markers, such as CaSR, PTH, and GCM2 were positive. Conclusions These trials demonstrated the adult human parathyroid cells could be harvested by collagenase digestion and the cultured. Furthermore, the cells remained good shape and kept functioning, making it a potential source for allogeneic cell transplantation to the treatment of permanent hypoparathyroidism.
Objective To culture primary parathyroid cells by mean of simulated microgravity, observe their basic morphological characteristics, study survival rate and secretory function of parathyroid cells, and explore more excellent culture mean of parathyroid cells. Methods There were 37 male Wistar rats, the body weight was 150–200 g. The rat was intraperitoneally injected with 1% pentobarbital sodium (50 mg/kg). The parathyroid glands were surgically excised and identified pathologically. The parathyroid gland cells were got and digested them with collagenase Ⅱ, which were divided into three groups: conventional culture group (simple parathyroid cells were cultured), polyglycolic acid (PGA) scaffold culture group (the parathyroid cells were cultured on the PGA scaffold), and simulated microgravity culture group (the parathyroid cells and PGA scaffolds were cultured in simulated microgravity environment). The parathyroid cells were cultured for 1, 3, 5 or 7 days in different culture conditions, then the parathyroid hormone (PTH) was measured, morphological characteristics of the parathyroid cell was observed under phase contrast microscope, survival rate of the parathyroid cells was calculated by acridine orange/propidium iodide staining. Results The parathyroid cell morphologies of most cells were well and center of part of cell mass was necrosis on day 7 in the conventional culture group. The most parathyroid cells were spreading toward the poles along the PGA cell scaffold in the longitudinal direction and the adjacent stents were connected by extracellular matrix on day 7 in the PGA scaffold culture group. The parathyroid cells cultured under the simulated microgravity were got round and formed clusters on day 7 in the simulated microgravity culture group. Compared with the other two groups on day 7, the PTH and the survival rate of the parathyroid cells were significantly higher in the simulated microgravity culture group (P<0.05). Conclusions Parathyroid cells cultured in simulated microgravity environment could maintain better morphology, survival rate is higher, and secretory function is better. Therefore, parathyroid cells cultured in simulated microgravity could be used as good donor cell for treatment of hypoparathyroidism. PGA scaffold could be used as a good carrier for culture of parathyroid cell.
The purpose of this study is to investigate the effects of self-assembling peptide GFS-4 on three-dimen-sional myocardial cell culture and tissue repair of myocardial infarction. The circular dichroism (CD) spectrum was used to detect secondary structure of GFS-4, and atomic force microscope (AFM) was used to analyze the microstructure of self-assembly. The nanofiber scaffolds self-assembled by GFS-4 were used as the three-dimensional culture material to observe the growth effect of cardiomyocytes. The model of myocardial infarction was established and the effect of GFS-4 on myocardial infarction was studied. The results indicated that self-assembling peptide GFS-4 could form mainly β-sheet structure that can form dense nanofiber scaffolds after 24 hours’ self-assembling. The myocardial cells had a favorable growth status in GFS-4 nanofiber scaffold hydrogel when cells treated in three-dimen-sional cell culture. The experiment of repairing myocardial infarction in vitro proved that peptide GFS-4 hydrogel scaffold could alleviate tissue necrosis in a myocardial infarction area. As a new nanofiber scaffold material, self-assembling peptide GFS-4 can be used for three-dimensional cell culture and tissue repairing in myocardial infarction area.
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.