Variation in Growth Pattern and Morphological Appearance of Primary Monolayer Cultures of Chondrocytes and Neural Cells Isolated from the Chick Embryo at Different Stages.

Aim: This study was an attempt at investigating the variation in growth and morphology of primary avian chondrocyte and neural cell cultures established under standard laboratory conditions. Methods: For establishing chondrocyte cultures, the tibia were dissected from chick embryos that were 12 (E12), 14 (E14) and 15 (E15) days old. For the neural cell culture initiation, the two lobes of the forebrain were dissected from chick embryos that were 4 (E4), 8 (E8) and 12 (E12) days old. The final characterization of the cells was performed by H&E and CFV staining. Results: In the chondrocyte cultures, two forms of morphology were observed which is reversible in process.The primary variation evaluated in chondrocytes cultures was the effect of the media on the state of the cells. Based on whether the cells were cultured in DMEM or MEM, there was a difference in the transformation of the attached cells from the differentiated to de-differentiated forms. The primary variation examined in neuron cultures was the embryonic age of the tissue and the effect that it had on the proliferation and neurite formation of the cells. E8 embryo neural cells cultures result in well-developed cells with neurite formation along with axonal and dendritic outgrowths with highly complex interconnections between them. Conclusion: Ultimately, this study demonstrated the composition of culture media had an effect on the morphological appearance of the chondrocytes, as well as, confirmed that the culture of primary neurons is best performed using cells from an E8 embryo.

Ex Vivo Observation of Human Nucleus Pulposus Chondrocytes Isolated From Degenerated Intervertebral Discs

STUDY DESIGN: We performed an ex vivo study to observe cell morphology and viability of human nucleus pulposus (NP) chondrocytes isolated from degenerated intervertebral discs (IVD). PURPOSE: To better understand the biological behavior of NP chondrocytes in monolayer cultures. OVERVIEW OF LITERATURE: Biological repair of IVDs by cell-based therapy has been shown to be feasible in clinical trials. As one of the most promising transplanting seeds, how the isolated NP chondrocytes behavior ex vivo has not been fully understood. METHODS: Human NP chondrocytes were harvested from 20 degenerated IVDs and cultured in monolayers. Histological and immunochemistry staining was used to detect cell morphology change. Cell viability was studied by analyzing cell cycle distribution and apoptotic rate in the primary and subculuted cells. RESULTS: The round or polygonal primary NP chondrocytes had an average adherence time of 7 days and took nearly 31 days to reach 95% confluence. The spindle-shaped P1 NP chondrocytes increased growth kinetics and took about 12 hours to adhere and 6.6 days to get 95% confluent. Immunochemistry staining of collagen II was positive in the cell cytoplasm. Nearly 90% of the confluent NP chondrocytes stayed in G1 phase while 16% underwent apoptosis. No significant difference of the collagen II expression, cell cycle distribution or the apoptosis indices were detected between the primary and subcultured NP chondrocytes. CONCLUSIONS: Human NP chondrocytes undergo significant morphological change in monolayer cultures. Cell cycle distribution pattern and apoptosis index of the cutured NP chondrocytes potentially influence their clinical transplantation or laboratory use.

Periosteum as a source of mesenchymal stem cells: the effects of TGF-β3 on chondrogenesis

INTRODUCTION: Numerous experimental efforts have been undertaken to induce the healing of lesions within articular cartilage by re-establishing competent repair tissue. Adult mesenchymal stem cells have attracted attention as a source of cells for cartilage tissue engineering. The purpose of this study was to investigate chondrogenesis employing periosteal mesenchymal cells. METHODS: Periosteum was harvested from patients who underwent orthopedic surgeries. Mesenchymal stem cells were characterized through flow cytometry using specific antibodies. The stem cells were divided into four groups. Two groups were stimulated with transforming growth factor β3 (TGF-β3), of which one group was cultivated in a monolayer culture and the other was cultured in a micromass culture. The remaining two groups were cultivated in monolayer or micromass cultures in the absence of TGF-β3. Cell differentiation was verified through quantitative reverse transcription-polymerase chain reaction (RT-PCR) and using western blot analysis. RESULT: In the groups cultured without TGF-β3, only the cells maintained in the micromass culture expressed type II collagen. Both the monolayer and the micromass groups that were stimulated with TGF-β3 expressed type II collagen, which was observed in both quantitative RT-PCR and western blot analysis. The expression of type II collagen was significantly greater in the micromass system than in the monolayer system. CONCLUSION: The results of this study demonstrate that the interactions between the cells in the micromass culture system can regulate the proliferation and differentiation of periosteal mesenchymal cells during chondrogenesis and that this effect is enhanced by TGF-β3.

Culture and identification of chondrocytes from human iliac growth-plate in vitro / 中国矫形外科杂志

[Objective] To establish a viable and convenient method for human iliac growth-plate chondrocyte culture in vitro and study the biological characters of the chondrocyte.[Method] The chondrocytes from 10 human iliac growth-plate were cultured in monolayer and passaged to observe the changes of cell morphology.The growth kinetics was detected by using MTT colorimetric.Immunocytochemistry and RT-PCR were performed to investigate the expression of collagenⅡat the protein and mRNA level,respectively.[Result](1)The phenotype of chondrocyte was changed from original polygonal shape to fusiform at 4th passage.(2)MTT test showed the growth curve of the second passage cells presented inverted "S",and cells were found logarithmic growth at 4th~8th day reached platform stage at 9th~10th day,and decreased at the 11th day.(3)The collagen typeⅡimmunohistochemical staining was extensive positive in cells at P2.(4)RT-PCR confirmed that the mRNA level decreased with the cells passaged after P2.[Conclusion]The modified method of the enzymatic two-step digestion is an effective and simple procedure.The second passage cells maintain the phenotype of chondrocyte well,could be serving as seeding cells.

The Modulation of Integrin Expression by the Extracellular Matrix in Articular Chondrocytes

Normal articular cartilage is composed of chondrocytes embedded within an extracellular matrix (ECM). The patterns of integrin expression determine the adhesive properties of cells by modulating interactions with specific ECMs. Our hypothesis is that chondrocyte integrin expression changes in response to changes in their microenvironment. Porcine articular chondrocytes were encapsulated in alginate beads with several ECMs (collagen type I, collagen type II and fibronectin) for 7 days, subjected to RT-PCR, western blot analysis and immunofluorescence staining. It was found that chondrocytes in different ECMs showed different patterns of integrin expression. Integrin alpha5 and beta1 were strongly expressed in all groups, but integrin alpha1 was strongly expressed only in collagen type I and fibronectin conjugated alginate beads, and integrin alpha2 was strongly expressed only in collagen type II conjugated alginate beads. These findings suggest that the addition of different ECMs to chondrocytes can modulate the patterns and levels of integrin expression possibly through a feedback mechanism. These finding suggest that the modulation of ECM interactions may play a critical role in the pathogenesis of osteoarthritis.

Isolation and Culture of Pig Hepatocyte in Large Scale for the Application of Bioartificial Liver System / 대한간학회지

BACKGROUND/AIMS: Acute hepatic failure is a serious problem. Its mortality reaches up to 80%. Only liver transplantation has been accepted as a definite treatment for patients with hepatic failure but shortage of donor organs is the main obstacle of this approach. A possible solution to this problem is a bioartificial liver system, perfusion of patients blood to isolated hepatocyte. In this study, we performed the isolation and culture of pig hepatocyte in large scale for the application of bioartificial liver system. METHODS: Hepatocyte isolation was performed by two-step collagenase method via portal vein perfusion in 10kg female pigs. After that, we compared the functional differences of the spheroid culture to the monolayer culture of hepatocyte. The viability and the function of hepatocyte were assessed using trypan-blue exclusion test and the measurement of the rate of ureagenesis and ammonia removal. RESULTS: The average viability and yield of hepatocyte were 86.8 +/- 8.0 % and 7.8 +/- 5.4 X 10(9), respectively. The spheroid culture was superior to the monolayer culture in functional aspect of hepatocyte, and their differences, especially for ammonia removal, were more apparent in parallel with culture time. CONCLUSIONS: For hepatocyte isolation, we obtained sufficient viability and yield of hepatocyte for clinical usage of bioartificial liver system. The function of hepatocyte seems to be better in the spheroid culture than in the monolayer culture. Further studies are needed for application of bioartificial liver system in clinical setting.

Matrix Synthesis of Human Intervertebral Disc Cells: Effect of Gene Transfer, Exogenous Growth Factor, Incubation Period, and Culture Methods / 대한척추외과학회지

STUDY DESIGN: In vitro experiment to determine the matrix synthesis of intervertebral disc (IVD) cell to various biologic interventions and conditions. OBJECTIVES: To elucidate biologic responses in terms of matrix synthesis of human IVD cells in vitro to various factors i.e. concentration of adenoviral vector and exogenous growth factor, duration of incubation, and type of culture methods. SUMMARY OF LITERATURE REVIEW: Sophisticated method to delivery of growth factors, in continuous manner, is the genetic modification of disc cells through gene transfer. Direct comparison of gene transfer and exogenous growth factor on matrix synthesis has not been reported. MATERIALS AND METHODS: IVD tissue was obtained from twenty three patients. Isolation and preparation of disc cells in monolayer (D) and alginate beads (3D) culture were performed. Disc cells in 2D and 3D were treated with either Ad/TGF-beta1 or exogenous TGF-beta1. Control cultures were treated with either saline or Ad/luciferase. Matrix synthesis (newly synthesized proteoglycan) was measured in various conditions (concentration of adenoviral vector and exogenous growth factor, duration of incubation, and type of culture methods). Newly synthesized proteoglycan were analyzed using chromatography on Sephadex G-25 in PD-10 columns after S35-sulfate incorporation. RESULTS: Ad/TGF-beta1 showed increase in proteoglycan synthesis (plateau at 75MOI) in 3D culture, (plateau at 25MOI) in 2D culture. In 3D culture, Ad/TGF-beta1 showed significant increase in proteoglycan synthesis on day 1, 2, and 3 of incubation. In 2D culture, Ad/TGF-beta1 showed significant increase in proteoglycan synthesis on day 2 of incubation with significant loss of anabolic effect on day 3. In 3D culture, exogenous TGF-beta1 showed increase in proteoglycan synthesis (plateau at 2ng/ml) while in 2D culture, there is no synthetic response to exogenous TGF-beta1 CONCLUSION: Therapeutic gene transfer provided sustained and increased anabolic responses than exogenous growth factor.

Dose-Dependent Effects of Transforming Growth Factor-beta1 on Cell Proliferation and Matrix Synthesis in Relation to the Degree of Chondrocyte Dedifferentiation in Monolayer Culture / 대한정형외과연구학회지

Transforming growth factor-beta1(TGF-beta1) has been suggested to be a useful growth facto. that could maintain the phenotypic characteristics of cultured chondrocytes. We performed this study to investigate the dose-dependent effects of TGF-beta1 on cell proliferation and matrix synthesis in relation to the degree of chondrocyte dedifferentiation in monolayer culture. Chondrocytes were isolated from the articular cartilage of distal femur of neonatal Sprague-Dawley rats and were cultured in monolayer. When the cell population reached 70-80% of confluence, the cells were subcultured successively for 6 weeks for the following studies. Every weak, the dose dependent effects of rhTGF-beta1 (0ng/ml ,1ng/ml, 5ng/ml, 10ng/ml, 20ng/m1) on DNA synthesis, proteoglycan synthesis and collagen synthesis were evaluated by 3H-thymidine incorporation, 35S-sulfate incorporation and 3H-proline incorporation respectively. The DNA synthesis decreased with time of culture. The DNA synthesis was significantly decreased by the addition of rhTGF-beta1 at all concentrations. The dose-dependent inhibitory action was most prominent at initial 3 weeks. Proteoglycan synthesis was in the highest level at the second week and markedly decreased after 2 weeks. At 2 weeks, TGF-beta1 treatment increased the proteoglycan synthesis at all concentrations and this stimulatory effect was prominent at the concentrations of 5ng/ml and 10ng/ml. There was no significant difference in the level of collagen synthesis during the whole experimental period. TGF- beta1 treatment increased collagen synthesis at all concentrations and this stimulatory effect was prominent at the concentrations at 5ng/ml and 10ng/ml. The results of this study suggest that if the cultured chondrocytes treated by TGF-beta 1 are used for transplantation, the time for TGF-beta1 treatment and transplantation to be recommended is before 3 weeks and the beneficial concentration of TGF-beta1 is 5ng/ml to 10 ng/ml for the maintenance of the phenotypic properties.

In Vitro Culture of Human Nasal Epithelial Cells by Monolayer Culture of Dissociated Cells / 영남의대학술지

Different techniques for culturing respiratory epithelial cells have been developed to overcome the limitations of studies on in vivo and on bioptic material. Traditionally, culture systems are divided into organ cultures, explant cultures and dissociated cell cultures. The first two contain both epithelial and non-epithelial cells. However, in monolayer cultures of dissociated cells only epithelial cells are present, the effects observed are caused by a pure epithelial responses. The purpose of this study is to establish primary culture method of human nasal epithelium (HNEC) by monolayer culture of dissociated cells to evaluate the role of the epithelial cells in the allergic and non-allergic nasal inflammatory reactions. HNEC was prepared by primary culture method of monolayer culture of dissociated cells from human inferior nasal turbinate mucosa of septal deviation patients. Primary cultured cells were characterized by indirect immunofluorescence assay and transmission electron microscopy. The immunoreactivities of cytokeratin-pan and cytokeratin No. 8 were observed in cultured HNEC. However, the immnoreactivities of vimentin and von Willebrand factor were not observed in cultured HNEC. The tonofilaments and desmosome were observed in cultured HNEC. The cultured epithelial cells were identified to be pure nasal epithelial cells. The monolayer culture of dissociated cells could successfully be employed for further study to investigate the role of the epithelial cells in allergic or non-allergic nasal inflammatory diseases.

Transforming growth factor-beta 1 responsiveness of human articular chondrocytes in vitro: normal versus osteoarthritis

The transforming growth factor-beta 1 was known as having the most important influence on chondrocytes among various growth factors, being abundant in articular chondrocytes and osteocytes. We performed in vitro monolayer cultures of human articular chondrocytes from normal and osteoarthritic patients and studied the transforming growth factor-beta 1 responsiveness of those chondrocytes. The cell-growth curve indicated that the primary osteoarthritic chondrocyte culture with transforming growth factor-beta 1 showed a more rapid growth pattern than normal chondrocytes with or without TGF-beta 1 and osteoarthritic chondrocytes without TGF-beta 1. The osteoarthritic group showed a sharp decline in growth pattern with subsequent culture. The shape of osteoarthritic chondrocytes was bigger and more bizarre compared to those of normal chondrocytes. With subsequent culture, this change became prominent. The transforming growth factor-beta 1 increased the [3H]-TdR uptake in each group. The phenotypes of chondrocytes were more clearly expressed in the normal group. The chondrocytes lost their phenotype (production of collagen type II) following subculture in each group. The transforming growth factor-beta 1 could not inhibit or delay the dedifferentiation process (loss of phenotype).

Effects of Chinese Medicinals with Right-supporting and Phlegm-transforming Actions on Albumin Synthesis and Secretion of Fibrous Hepatic Cells of Rat in Vitro / 中成药

Objective:To investigate the effects of Chinese Medicinals with right supporting and phlegm transforming actions on albumin synthesis and secretion of fibrous hepatic cells of rat in vitro. Methods: Amygdalin, the water soluble extract of Radix Salviae Miltiorrhizae, lordyceps, and colchicine were used to act on fibrous hepatic cells of rat produced by primary monolayer culture in vitro. The results were determined by ELISA method. Results: Besides colchicine, the others amygdalin and cordyceps. Cordyceps, the water soluble extract of Radix Salviae Miltiorrhizae) showed the obvious raising actions on albumin synthesis and secretion of fibrous hepatic cells of rat ( P