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BACKGROUND: Corneal epithelial stem cells, also known as limbal stem cells, are distributed in the basal layer of limbal epithelium. It is extremely difficult to deal with limbal stem cell deficiency or dysfunction that is caused by severe thermal burn, chemical burn, and chronic inflammation of ocular surface. At present, in vitro culture of corneal epithelial stem cells using tissue engineering technology followed by clinical transplantation is a new and effective therapeutic direction. OBJECTIVE: To explore the feasibility of serum-free culture of human corneal epithelial stem cells in vitro using modified explant culture method. METHODS: The remaining donor corneal tissues after keratoplasty (less than 8 mm in diameter) were obtained from Henan Eye Bank, and the outer and middle limbus were dissected under surgical microscope. Two culture methods were used to culture human corneal epithelial stem cells. In the conventional explant culture group, the limbal tissues were adhered to the dish with the epithelium being upward, then Keratinocyte-serum free medium (K-SFM) was added into dishes, followed by incubation at 37 °C in a 5% CO2 incubator. In the modified explant culture group, limbal tissues were dissected to immerse in the K-SFM culture medium and incubated at 37 °C in the 5% CO2 incubator for 12 hours. The limbal tissues were then adhered to the dish with the epithelium being downward. The day whenever the cells from the limbal tissues adhered to the dish was marked as the 1st day of culture, and changes in cell morphology and growth were recorded by phase contrast microscopy every day. Immunofluorescent staining was used to detect the expression of K3 and p63 in primary cells on the 5th, 10th and 14th day of the modified explant culture. RESULTS AND CONCLUSION: The mean early stage of growth in the modified explant culture group was shorter than that of the conventional explant culture group (P < 0.05), and the mean growth rate of the modified explant culture group was higher than that of the conventional explant culture group (P < 0.05). In the modified explant culture group, cells had a good growth state, and many cells with small size gathered together on the 10th day of culture. On the 14th day, cell clones were formed, and the cells in the clone showed uniform morphology. On the 5th day, K3 highly expressed, while p63 lowly expressed in primary cells. On the 10th day, both of K3 and p63 had an increased expression. On the 14th day, there was no significant increase in the K3 expression, but the expression of p63 increased significantly. In the in vitro serum-free culture condition, the modified explant culture could significantly promote the growth of corneal epithelial stem cells, and expand corneal epithelial stem cells in vitro, which could provide sufficient seed cells for enriching corneal epithelial stem cells and constructing human limbal multilayered epithelial sheets.
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[Abstract] Objective To establish a new, simple method for isolation and culture of fibroblast-like synoviocytes (FLSs) from adjuvant arthritis (AA) rabbits using suspended explant culture method. Methods Sixteen healthy New Zealand white rabbits, weighing 2.0-2.5 kg, were randomly divided into two groups: the normal control group (n＝8) and the model group (n＝8). Rabbit models of AA were prepared by injection with complete Freund’s adjuvant at multiple sites and time points. The joint synovial layers were obtained. The FLSs of rabbits with AA were cultured by suspended explant culture method and explant culture method. The growth and morphological characteristics of cells were observed, and the cell viability was measured by CCK-8 assay. The expression of vimentin was detected by immunocytochemistry. Results The tarsometatarsal joints and toes of rabbits in the model group were swollen more obviously than those in the normal control group. After the toes were dissected, obvious microvascular proliferation and inflammation were observed in the model group. The results of H-E staining showed that synoviocytes in the normal control group were arranged in a regular manner like beads, while those in the model group were arranged in a disordered manner, suggesting that the AA model was successfully prepared. The operating durations required for suspended explant culture method and explant culture method were about 25 min and 1 h, respectively. The morphology and viability were similar for the cells obtained by the two methods. The positive rates of vimentin in the suspended explant culture method and explant culture method group were 98.01% and 98.35%, respectively. Conclusion Isolation and culture of FLSs by suspended explant culture method has been successfully established, and the method is simple and needs a short time.
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Objective To compare and analyze the time of cell eruption formation and the rate of cell eruption of human corneal epithelial cells cultured by different explant culture methods under serum-free conditions.Methods Explant tissues were randomly divided into A,B,C and D groups.In A group,the cornea epithelial cells were put side up after stripping corneal endothelium and scraping the corneal epithelium;in B group,the corneal epithelium was put side down after stripping corneal endothelium and scraping the corneal epithelium;in C group,the corneal epithelium was put side up after only stripping corneal endothelium;and in D group,the corneal epithelium was put side down after only stripping corneal endothelium.The morphological changes of the cells in the 4 groups were observed by phase-contrast microscope,and both aforementioned variable were recorded.Results The average time of cell eruption formation and the rate of cell eruption in A,B,C and D group was (7.00 ± 3.00) d and 47.50％ (19/40),(7.55 ±2.58)d and 45.83％ (11/24),(8.43 ±3.32)d and 63.64％ (14/22),(7.49 ± 2.20) d and 72.22％ (39/54),respectively.The chi-square test showed that there was significant difference in the cell eruption rate between A and D group as well as B and D group (all P ＜ 0.05),but there was no significant difference between the other groups (all P ＞ O.05).Conclusion The time of cell eruption formation in the four different explant culture methods was about one week in free-serum conditions.And D group has higher cell eruption rate,and pure plus stable cultured cells when compared with the other three groups.
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AIM:To explore a novel method to isolate human nucleus pulposus mesenchymal stem cells (hNP-MSCs) in vitro and to identify their biological characteristics.METHODS:The explant culture method was employed to isolate hNP-MSCs from nucleus pulposus tissue obtained by percutaneous endoscopic lumbar discectomy (PELD).The isolated cells were passaged for purification and cultured in vitro followed by morphological observation.The cell proliferation ability was detected by CCK-8 assay.Growth curves of the cells were drawn and surface antigens were detected by flow cytometry.The cells at the 3rd~6th passages were induced for adipogenic, osteogenic and chondrogenic differentiation, and examined by oil red O staining, alizarin red staining and Alcian blue staining.RESULTS:The cells with self-renewal were obtained from nucleus pulposus tissue obtained by PELD.The results of flow cytometry analysis revealed that the cells were positive for CD29, CD44, CD90, CD73 and CD105, but negative for CD34 and CD45.The proliferative capacity was consistent with the growth characteristics of MSCs and multilineage differentiation potential was identified.CONCLUSION:A novel method to efficiently isolate and culture hNP-MSCs,PELD combined with explant culture method,was established, which would promote the study of regenerative medicine based on hNP-MSCs.
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Objective:To isolate SD rat adipose-derived stem cells(ASCs)by suspended explant culture method.Methods: The healthy rat inguinal fat pads were obtained.The SD rat ASCs were isolated by suspended explant culture method and explant adherent culture method.The growth status and morphology were observed.The growth curve and cell surface markers CD29,CD44,CD45 of the 3rd passage cells were analyzed respectively by CCK-8,immunocytochemistry;the 3rd passage cells were induced individually by adipogenic differentiation medium and osteogenic differentiation medium,and cells were examined by oil red O staining and alizarin red staining.Results: The SD rat ASCs obtained by the two methods exhibited a spindle-shaped appearance and could rapidly expand.The cell growth curves were typical of S type.Immunocytochemistry analysis revealed that the third passage of SD rat ASCs were positive for CD29,CD44,but were negative for CD45.SD rat ASCs were positive for oil red O staining at 14 days after adipogenic induction,and positive for alizarin red staining at 14 days after osteogenic induction.Conclusion: Isolation of SD rat ASCs by suspended explant culture method is successfully established.The method is simple.It provides a new method for the isolation of SD rat ASCs in vitro.
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Objective To establish an easier and better method for primary culture of rabbit fibroblast-like synoviocytes(FLS) by modified tissue cell culture.Method Healthy rabbit joint synovial layers were cut into small pieces and siphoned off water attached on the tissue with sterile filter paper and then cultured.The growth status and morphology were observed.The growth curve of the 3rd passage cells was measured by CCK-8 assay, and the expression of vimentin was detected by immunocytochemistry.Results The FLS cultured in vitro exhibited a spindle-shaped appearance and could rapidly expand.The cell growth curve was typical of S type, and the cells highly expressed vimentin.Conclusions Primary culture of rabbit FLS by the improved explant culture method is successfully established.The method is simple and highly efficient.It provideds a new method for isolation of FLS.
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Objective:To isolate rabbit fibroblast-like synoviocytes(FLSs) by suspended explant culture method. Methods:The healthy rabbit joint synovial layers were obtained. The cells were cultured by suspended explant culture method and compared with the explants adherent culture method. The growth status and morphology were observed. The growth curve of the 3rd passage cells was measured by CCK-8 assay,and the expression of vimentin was tested by immunocytochemistry. Results: The FLS obtained by the two methods exhibited a spindle-shaped appearance and could rapidly expand. The cell growth curve was typical of S type, and the cells highly expressed vimentin. Conclusion:Primary culture of rabbit fibroblast-like synoviocytes by suspended explant culture method were successfully established. The method was simple and highly efficient. It provided a new method for the isolation of FLS in vitro.
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AIM: To compare cell-suspension and explant culture of mouse corneal epithelial cells (MCEC).METHODS: MCEC were cultured by cell-suspension culture and explant culture, respectively. Colony forming efficiency (CFE) and cell proliferation were determined. The expression of corneal epithelial progenitor cell marker p63 and K19, as well as differentiation marker K12 was investigated by Western blotting.RESULTS: Twenty of 25 (80％) cornea explant were successfully subcultured to passage1 (P1), while only 12％ cell-suspension culture were successfully subcultured to P1. There were statistical significance between explant culture and cell-suspension culture (P<0.01). Up to 55％ of P1 cells in explant culture were passaged over P10 and were stably subcultured though at least 25 passages. However, cells cultured in suspension culture never achieved confluence in P2. CEF of P1 in explant culture was higher than P1 in cell-suspension culture (P=0.02) and CEF of P20 in explant culture was higher than P1 in explant (P=0.001). Immunostaining images showed expression of p63 and K19 in cell-suspension culture P1 and explant culture P1 and P20. K12 was expressed in P1 of both cell-suspension culture and explant culture, however, there was not K12 expressed in P20 of explant culture.CONCLUSION: In MECE culture, compared with cell-suspension culture, the explant culture is a preferable option.
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OBJECTIVE: In vitro study systems for research of placental hypoxia are needed, among which human placental villous explant culture technique under experimentally variable condition is commonly used. So we performed this study to assess the viability of placental villous explant in normoxic and hypoxic culture that can provide validity for that system. METHOD: Placental villous explant tissues obtained from 9 cases of normal term pregnancies were incubated in normoxic (20% O2) and hypoxic (2~5% O2) condition for 72 hours. The viability of tissue was evaluated morphologically by microscopic examination and biochemically by LDH assay at variable time interval (0, 6, 12, 24, 48, 72 hours). The apoptosis of the tissue was assessed by TUNEL assay. RESULT: By light microscope, all of H&E stained placental explant tissue sections in normoxic and hypoxic culture showed intact villous integrities without definitive syncytial sloughing and fibrinoid necrosis as time elapsed. Tissue viability of LDH assay during 6, 24, 48, 72 hours of placental villous explants showed over all 52.3~67.6% and didn't show statistically significant difference between normoxic and hypoxic culture. Tissue viability in both groups maintained 61.2~67.6% for the first 24 hours and eventually decreased with time. TUNEL assay showed over all negative findings in normoxic and hypoxic culture at different time periods. CONCLUSION: In vitro human placental explant culture system can be a useful and feasible technique for research of placental hypoxia which is related to development of obstetrical complications such as preeclampsia, intrauterine fetal growth restriction and preterm labor and so on. But our in vitro placental explant system needs some modification in culture condition and technique for maximizing viability of the tissue.
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Female , Humans , Pregnancy , Hypoxia , Apoptosis , Culture Techniques , Fetal Development , In Situ Nick-End Labeling , Necrosis , Obstetric Labor, Premature , Pre-Eclampsia , Tissue SurvivalABSTRACT
BACKGROUND: Recent advance in tissue engineering in the biomedical field shed light on the replacement or regeneration of various organs with synthetic substitutes. Currently emerging cartilage tissue engineering therapies involve artificial cartilage fabricated from three dimensional cultures using appro-priate scaffolds. It is mandatory to expand or proliferate the chondrocytes in vitro to prepare the artificial cartilage. The purpose of this study was to find out the most favorable culture conditions for chon-drocyte viability in vitro. METHODS: Articulr chondrocytes or cartilage explants were isolated from the patellofemoral groove of adult pigs. And then we standardized the size and thickness of the cartilage explants as well as preparing alginate-chondrocyte beads for three-dimensional cultures. The cartilage explants, including 10% fetal bovine serum for 10 days, 36 days and passage 6. Cellualr viability was measured by methylthiazol tetrazolium (MTT) assay on monolayer, alginate bead and cartilage explant. SPSS 11.5 was used for data anaylsis. RESULTS: Chondrocytes cultured on monolayers in vitro showed no significant difference in cellular viability until passage 6 following isolation from the patellofemoral groove of adult pigs (P>0.05, n=4). Chondrocyte viability was markedly increased by day 16 both in the monolayer (148%) and three dimensional cultures (245%), and then slightly decreased 126% and 200%, respectively, at day 36. Three dimensional cultures using alginate bead were more favorable for chodrocyte viability than monolayer culture in chondrocyte primary culture (P=0.003, n=6). Chondrocyte viability in the algi-nate bead was increased 300% during 36 days' incubation period (P=0.001, n=3). Cellular viability in the cartilage explant culture was decreased after day 4 in both MTT score (P=0.022, n=10) and MTT OD (P=0.039, n=10). CONCLUSIONS: Three dimensional cultures using alginate bead were the most favorable for chon-drocyte viability in chondrocyte primary cultures.
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Adult , Humans , Cartilage , Cartilage, Articular , Chondrocytes , Regeneration , Swine , Tissue EngineeringABSTRACT
PURPOSE: Immaure brain is more resistant to seizure-induced neuronal damage than the adult brain in animal study. Immediate early genes such as c-jun play a critical role in neuronal damage. Therefore, we hypothesized that the difference of constitutive and electrically stimulated c-Jun expression would explain the difference in neuronal damage from seizures between immature and mature explant culture. METHODS: Seven and 14 days-in-vitro(DIV) hippocampal explant cultures derived from 8-day-old rat pups were used. Extracellular field recording was done in cultures. A 1-sec stimulus train(60 Hz, 0.1 msec rectangular pulses) was applied to the Schaffer collaterals, and the afterdischarge was recorded in CA1 pyramidal layer. Cultures were returned to the incubator and observed serially. Intensity of propidium iodide fluorescence indicative of neuronal damage was quantitated as percent of total damage induced by 2 mM NMDA. Proteins extracted from individual cultures were analyzed by Western blot. RESULTS: Constitutive c-Jun protein expression at 7 DIV was higher than that at 14 DIV. There was not a significant difference of c-Jun expression between the 7 DIV and 14 DIV cultures after electrical stimulation. Neuronal damage after electrical stimulation in the hippocampus at 7 DIV was significantly lower than at 14 DIV. CONCLUSION: The results show reduced neuronal injury from seizures in more immature culture. However, constitutive expression of c-Jun protein was higher. Higher constitutive expression may inhibit further induction of c-Jun from seizures and thus result in less severe neuronal injury.
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Adult , Animals , Humans , Rats , Blotting, Western , Brain , Electric Stimulation , Fluorescence , Genes, Immediate-Early , Hippocampus , Incubators , N-Methylaspartate , Neurons , Propidium , SeizuresABSTRACT
PURPOSE: Epileptic patients have a increasing tendency to develop seizure attack in high temperature. This finding suggests that high temperature may have an effect on neuronal hyperexcitability and injury of epileptic brain. Therefore, the influence of high temperature on normal and epileptic brain was studied in organotypic explant cultures of rat. METHODS: Fourteen days-in-vitro cultures from 8 day-old rat pups were perfused with standard aCSF bubbled with 95%/5% O2/CO2 in a microchamber. Stimulus train(0.3 sec, 60 Hz) was applied to Schaffer collaterals in CA3 and extracellular field potential was recorded in the CA1 pyramidal layer. At 36degrees C initially, AD was evoked. In high temperature(HT) group, the cultures were subjected to 39degrees C for a period of 8 min before the second stimulus train was applied. They were then restored to 36degrees C for 10 min. In normal temperature group, temperature was maintained at 36degrees C for the second stimulus train. The cultures were returned to the incubator and observed serially for neuronal damage. Intensity of propidium iodide fluorescence indicative of neuronal injury was quantitated by digital image analysis. The cultures on the same insert that were not stimulated served as the unstimulated groups. RESULTS: There was not a statistically significant difference in neuronal damage between the unstimulated high-temperature(HT) and normal-temperature(NT) group. In CA1 sector, % damage(mean+/-SEM) was 0.42+/-0.20 vs 0.27+/-0.05 at 24 hrs(HT vs NT group, n=16 each, P>0.05, Student t-test); 1.81+/-0.79 vs 1.43+/-0.27 at 48 hrs(P>0.05); 3.50+/-1.32 vs 3.35+/-0.56 at 72 hrs(P>0.05). In CA3 sector, % damage was 0.34+/-0.10 vs 0.20+/-0.03 at 24 hrs(P>0.05); 0.99+/-0.20 vs 0.83+/-0.23 at 48 hrs(P>0.05); 2.00+/-0.38% vs 2.26+/-0.35% at 72 hrs(P>0.05). Neuronal damage on AD induced cultures during febrile setting(n=16) was significantly higher than in nonfebrile setting(n=16). In CA1 sector, % damage was 6.63+/-2.56 vs 0.92+/-0.45 at 24 hrs(febrile setting vs nonfebrile setting, P= 0.036); 26.37+/-7.44 vs 4.99+/-2.23 at 48 hrs(P=0.010); 38.59+/-9.63 vs 6.48+/-2.30 at 72 hrs (P=0.003). In CA3 sector, % damage was 1.23+/-0.48 vs 3.91+/-2.37 at 24 hrs(P=0.277); 13.09+/-5.75 vs 5.93+/-3.27 at 48 hrs(P=0.288); 27.86+/-8.68 vs 7.54+/-3.74 at 72 hrs(P=0.04). CONCLUSION: At high temperature, seizures in epileptic brain may be more injurious than seizures in normal temperature.
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Animals , Humans , Rats , Brain , Fluorescence , Hippocampus , Incubators , Neurons , Propidium , SeizuresABSTRACT
Objective To observe the effect of furosemide on stria vascularis cultivated in vitro from guinea pig, to explore the probable mechanism of furosemide ototoxicity.Methods 20 healthy pigmented guinea pigs was randomly divided into two groups: Furosemide group (n=16), control group (n=4). The stria vascularis explant were cultured for 24 hours using the explant-culture technique. Experimental groups were added into imediately different final concentration of Furosemide (60ug/ml?300ug/ml?600ug/ml?1250ug/ml?2500ug/ml) differently, and continuously cultivated 30 minutes or 90 minutes , then observed structure feature of the cultivated stria vascularis .Results Furosemide group did not show pathological changes such as edema of stria vascularis,shrinkage of the intermediate cells and enlargement of intercellular spaces. No significant changes were found in the stria vascularis structure features between furosemide group and control group. Conclusion Furosemide did not induce remarkable edema on cultivated stria vascularis from guinea pig.These findings suggested that furosemide might induce ototoxicity by indirect mechanism.
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Gonadotropin releasing hormone agonist(GnRHa) has been used as a temporaruy medical management for uterine myoma. Insulin-like growth factors(IGFs) has been supposed to play an etiological role in myoma growth and their action is modulated by IGF-binding proteins(IGFBPs). The purpose of this study is to evaluate the effects of GnRHGa on the IGFBPs production in the explant culture of adjacent myometrium and uterine myoma. The adjacent myometrium and myoma explants from patients treated(n=10) and nontreated(n=16) with GnRHa were cultured under serum free condition.Some explants from untreated patients were also cultured in the presence of GnRHa(10(-7) M/L). Western ligand blot and immunoprecipitation showed the presence of 276 kilodalton IGFBP, IGFBP-2, IGFBP-3 and IGFBP-4 in culture media of these explants and IGFBP-4 only was consistently present in culture media of all explants. There were no significant differences in the frequency of each IGFBP between adjacent myometrium and myoma explant cultures regardless of the type of GnRHa exposure. The relative IGFBP-4 level in explant cultures of myoma from patients treated with GnRHa was significantly higher than that from untreated patients.Addition of GnrHa in the media of myoma explants did not result in an significant change in the relative IGFBP-4 level compared with in vitro untreated explant cultures. Our data suggest that Gnrha may act through indirect involvement in the EGFBP-4 production in myoma tissue.
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Animals , Female , Humans , Mice , Culture Media , Gonadotropin-Releasing Hormone , Gonadotropins , Immunoprecipitation , Insulin-Like Growth Factor Binding Protein 2 , Insulin-Like Growth Factor Binding Protein 3 , Insulin-Like Growth Factor Binding Protein 4 , Insulin-Like Growth Factor Binding Proteins , Leiomyoma , Myoma , MyometriumABSTRACT
Gonadotropin releasing hormone agonist(GnRHa) has been used as a temporaruy medical management for uterine myoma. Insulin-like growth factors(IGFs) has been supposed to play an etiological role in myoma growth and their action is modulated by IGF-binding proteins(IGFBPs). The purpose of this study is to evaluate the effects of GnRHGa on the IGFBPs production in the explant culture of adjacent myometrium and uterine myoma. The adjacent myometrium and myoma explants from patients treated(n=10) and nontreated(n=16) with GnRHa were cultured under serum free condition.Some explants from untreated patients were also cultured in the presence of GnRHa(10(-7) M/L). Western ligand blot and immunoprecipitation showed the presence of 276 kilodalton IGFBP, IGFBP-2, IGFBP-3 and IGFBP-4 in culture media of these explants and IGFBP-4 only was consistently present in culture media of all explants. There were no significant differences in the frequency of each IGFBP between adjacent myometrium and myoma explant cultures regardless of the type of GnRHa exposure. The relative IGFBP-4 level in explant cultures of myoma from patients treated with GnRHa was significantly higher than that from untreated patients.Addition of GnrHa in the media of myoma explants did not result in an significant change in the relative IGFBP-4 level compared with in vitro untreated explant cultures. Our data suggest that Gnrha may act through indirect involvement in the EGFBP-4 production in myoma tissue.
Subject(s)
Animals , Female , Humans , Mice , Culture Media , Gonadotropin-Releasing Hormone , Gonadotropins , Immunoprecipitation , Insulin-Like Growth Factor Binding Protein 2 , Insulin-Like Growth Factor Binding Protein 3 , Insulin-Like Growth Factor Binding Protein 4 , Insulin-Like Growth Factor Binding Proteins , Leiomyoma , Myoma , MyometriumABSTRACT
BACKGROUND: Skin sulfhydryl oxidase(SSO) is the enzyme catalyzing the covalent crosslinking of the protein molecules via disulfide bond formation, which is probably responsible for the formation of tight and stable structures in the skin tissues, However, the lack of its gene cloning and antibody preparation limited the study for the role of the enzyme in the tissues. OBJECTIVE: It is required to study the role of the enzyme in the normal skin tissues and several dermatosis with abnormal keratinization and its regulation by several materials and drugs in the culture models. METHODS: We performed immunohistochemical analysis to determine the localization and expression of the enzyme in the normal skin and mucosa and hair tissues and in the skin explant cultures and artificial skins with irritant- and differentiation modifieradded conditions. RESULTS: In the skin, SSO was most strongly expressed in the granular layers and mostweakly in the basal layers. The similar but weaker expression pattern was observed in the mucosa with stronger expression in the upper flattened layers compared to the lower layers. In the hair, it was expressed in the inner and outer root sheaths at the isthmus portion in weaker stainable patterns. The strong expression of SSO was not observed beneath the Monro microabscess or parakeratosis in the involved aren of psoriasis. In actinic keratosis and Bowen disease, it was expressed more strongly in the dyskeratotic cells or parakeratotic areas. And in the horn pearls of the squamous cell carcinoma, the strong expression of SSO was observed. In the skin ezplant culture, the expression of SSO is induced by treatment with sodium lauryl sulfate or retinoic acid with more extended stained areas compared to the control. In contrast, its expression is not basically modified on the cellular level but inhibited dynamically by a decrease of the granular layers with retinoic acid in the artificial skin(raft culture). CONCLUSION: And the inducibility of the enzyme by the irritating agents in the skin organ explant culture suggests the role of the enzyme as one of skin defense system. The decrease of granular layers by retinoic acid in the artificial skin(raft culture) reflects the fact that its expression can be inhibited indirectly during the keratinization process. Therefore, it can be summarized that SSO is the enzyme involved in the keratinizing process of skin tissues as well as the protective function for the skin tissues.