Collagen Gel Culture Research Articles

Composition of culture medium is more important than co-culture with hepatic non-parenchymal cells in albumin production activity of primary rat hepatocytes, and the effect was enhanced by hepatocytes spheroid culture in collagen gel

Co-culture of primary rat hepatocytes with hepatic non-parenchymal cells or sinusoidal endothelial cells for albumin production activity as an index of liver-specific function was studied. The co-cultures were effective for the expression and maintenance of albumin production activity. However, the co-culture effect was not observed when we used a suitable culture medium, which had already been reported to be sufficient for albumin production activity. Albumin production of dispersed cells in collagen gel culture was higher than that of spheroid culture. In addition, albumin production of spheroids in collagen gel culture was higher than that of spheroid culture and dispersed cell collagen gel culture with a suitable culture medium. We found that culture medium composition was more important than co-culture for expression and maintenance of albumin production. Furthermore, we found that cell–cell interaction was effective for the expression of albumin production, but heterotypic cell–cell interaction was not necessary.

Mesenchymal origin of hepatic stellate cells, submesothelial cells, and perivascular mesenchymal cells during mouse liver development #

We thank Dr. Ramadori and Dr. Mansuroglu for their comments on our recent publication1 reporting the classification of fetal liver mesenchymal cells into three populations based on the location and immunostaining: hepatic stellate cells (HSCs) in the liver parenchyma, perivascular mesenchymal cells around the veins, and submesothelial cells beneath the basal lamina near the liver surface. As they pointed out, we found that there are rare desmin-positive alpha smooth muscle actin (αSMA)–positive mesenchymal cells near the veins in the developing mouse liver. We assume that this minor population represents heterogeneity of fetal HSCs as previously reported in the human liver2 or a transitional cell type between HSCs and perivascular mesenchymal cells. It is also possible that this minor population may become the cells within the arteriole wall in the adult liver. A cell lineage analysis of this minor perivascular cell population is important, but this requires identification of new and definitive molecular markers. We identified activated leukocyte cell adhesion molecule (ALCAM) as a marker for submesothelial cells and mesothelial cells in the developing mouse liver. Unfortunately, fetal HSCs and perivascular mesenchymal cells do not express ALCAM and therefore cannot be separated with fluorescence activated cell sorting. We are looking for new distinct cell surface markers to achieve this goal. Using anti-ALCAM antibodies, we separated an ALCAMhigh population containing submesothelial cells and mesothelial cells.1 The cultured ALCAMhigh population rapidly acquired a myofibroblastic phenotype on a plastic dish; the expression of αSMA messenger RNA (mRNA) showed a nearly 400-fold increase already at day 1, and this was maintained on day 8. When the cells were cultured in collagen gel, αSMA mRNA was increased 25- and 157-fold on days 1 and 8, respectively; this suggests that the myofibroblastic conversion is delayed in collagen gel (unpublished data, 2009). We are currently examining gene expression of these cells upon retinol and palmitic acid treatment. Although we did not test ALCAM immunostaining on the cultured ALCAMhigh cells in collagen gel, quantitative reverse-transcription polymerase chain reaction revealed reduced mRNA expression in a collagen gel culture (unpublished data, 2009). Kinji Asahina Ph.D.*, Hidekazu Tsukamoto D.V.M., Ph.D.*, * Southern California Research Center for Alcoholic Liver and Pancreatic Disease and Cirrhosis, Department of Pathology Keck School of Medicine, University of Southern California, Los Angeles, CA.

Decidualization Attenuates the Contractility of Eutopic and Ectopic Endometrial Stromal Cells: Implications for Hormone Therapy of Endometriosis

Decidualization of the endometrium involves the morphological and biochemical reprogramming of the estrogen-primed proliferative endometrial stromal compartment under the continuing influence of progesterone. The aim of this study was to evaluate the involvement of the extracellular matrix contractility of eutopic and ectopic endometrial stromal cells during the tissue remodeling processes associated with decidualization. The effect of decidualization on the contractile profile of the endometriotic cyst stromal cells and eutopic endometrial stromal cells with or without endometriosis in the three-dimensional collagen gel culture was investigated using laser scanning microscopy, collagen gel contraction assays, and Western blot analysis. Decidualized ectopic and eutopic endometrial stromal cells in the three-dimensional collagen gel culture mimicked the morphology of decidual tissue in vivo. In vitro decidualization inhibited the contractility of these eutopic and ectopic endometrial stromal cells. Down-regulation of integrin alpha1beta1 and alpha2beta1 expression, suppression of Ras homology A (Rho A), Rho-associated coiled-coil-forming protein kinase (ROCK)-I and ROCK-II expression, inhibition of the differentiation into the myofibroblastic phenotype, and induction of differentiation into epithelioid decidual phenotype were observed in these cells during decidualization. It is suggested that the attenuation of eutopic endometrial stromal cell-mediated contractility by decidualization is a novel and integral mechanism of the physiological endometrial tissue remodeling process during menstrual cycles. Although ectopic endometrial stromal cells have enhanced contractile profile, decidualization can attenuate the contractility of these cells. These findings may be one of the action mechanisms by which oral contraceptives and progestins ameliorate endometriosis.

Ang‐1 and MCP‐1 cooperate in pericyte recruitment during angiogenesis

Rat and mouse aortic rings produced angiogenic outgrowths in collagen gel cultures in response to the injury of the dissection procedure. Neovessels were composed of endothelial cells and surrounding pericytes. Pericyte recruitment along endothelial sprouts was enhanced by treating cultures with angiopoeitin‐1 (Ang‐1). Analysis of gene expression in Ang‐1 stimulated cultures revealed upregulated expression of several inflammatory cytokines, including monocyte chemotactic protein‐1 (MCP‐1). Recombinant MCP‐1 dose‐dependently increased pericyte number while anti‐MCP‐1 antibody depleted vessels of pericytes and abrogated Ang‐1 mediated pericyte recruitment. Mouse aortas deficient for MCP‐1 or its receptor CCR2 had fewer pericytes than controls. MCP‐1 deficiency also impaired the pericyte recruitment activity of Ang‐1. These results implicate MCP‐1 as a mediator of Ang‐1 effects on the development and maturation of the vessel wall during angiogenesis following injury. Supported by NHLBI‐HL52585, and a Merit Review Grant from the Department of Veterans Affairs.

Alignment of angiogenic microvessels and the surrounding collagen matrix under anchored boundary conditions

Previously we have observed an alignment of newly formed microvessels when 3D collagen gel cultures were axially anchored to their culture chamber . In contrast, microvessels in free‐floating gels exhibit a random orientation. The objective of this study was to examine changes in collagen alignment that occur during microvessel outgrowth over time in free‐floating and anchored 3D cultures. Microvessel cultures were fixed after 1 to 6 days in culture and endothelial cells were stained with Isolectin IB4‐Alexa 488. 3D image stacks of microvessels and collagen fibrils were obtained using confocal and confocal reflection microscopy, respectively. Microvessel and collagen fibril organization were quantified using standard image processing algorithms. There was significant alignment of vessels in anchored cultures, starting at 3‐4 days in culture. Similarly, the organization of collagen went from a random organization (anisotropy < 0.1) to a progressively more aligned orientation (anisotropy > 0.25) in a direction consistent with the microvessels. These results suggest that the orientation of microvessel outgrowth and matrix realignment follow similar patterns during angiogenesis. Although these events were highly correlated, it is unclear which event leads in the process of alignment.

Effects of Three-Dimensional Culture and Cyclic Stretch Stimulation on Expression of Contractile Proteins in Freshly Isolated Rat Aortic Smooth Muscle Cells

The effects of three-dimensional (3-D) culture and cyclic stretch stimulation on the expression of contractile proteins were investigated in freshly isolated rat aortic smooth muscle cells (FSMC). Primary cells were cultured statically on cell culture dishes (two-dimensional (2-D) culture) or in type I collagen gel matrix (3-D culture). Changes in their expression level of actin filaments (AFs) and smooth muscle myosin heavy chain (SM-MHC) were measured quantitatively using an accurately-calibrated fluorescent microscopy. The expression of AFs and SM-MHC decreased in both cultures in their early stages. Cell morphology was quite different between the two cultures: the cells had a flattened and irregular shape in the 2-D culture, while they had a fusiform shape with a well-defined long axis in the 3-D. Nineteen-day culture in the gel significantly increased the expression levels of AFs and SM-MHC while the expression levels remained low in the 2-D. Further and early increase in the expression levels was observed in the cells cultured in the gel with cyclic stretch of ∼8% amplitude and 1 Hz frequency. The cyclic stretch also induced alignment of FSMCs in the gel parallel to the stretch direction, and the cell alignment was observed earlier than the increase in their contractile proteins. These results indicate that the 3-D culture in collagen gel may increase the expression level of contractile proteins in FSMCs while maintaining their fusiform morphology, and cyclic stretch may efficiently increase the expression levels when the cells aligned in the stretch direction.

Latent Transforming Growth Factor-β-Binding Protein-4 Regulates Transforming Growth Factor-β1 Bioavailability for Activation by Fibrogenic Lung Fibroblasts in Response to Bleomycin

Recent evidence suggests that subsets of lung fibroblasts differentially contribute to fibrogenic progression. We have previously shown that a subset of rat lung fibroblasts with fibrogenic characteristics [Thy-1 (-) fibroblasts] responds to stimuli (bleomycin, interleukin-4, etc) with increased latent transforming growth factor (TGF)-beta activation, whereas non-fibrogenic Thy-1-expressing [Thy-1 (+)] fibroblasts do not. Activation of latent TGF-beta1 by interstitial lung fibroblasts is critical for fibrogenic responses. To better understand the susceptibility of fibrogenic fibroblasts to the stimulation of TGF-beta activation, we examined the role of latent TGF-beta-binding proteins (LTBPs), key regulators of TGF-beta bioavailability and activation, in TGF-beta1 activation by these fibroblasts. Treatment of fibroblasts with bleomycin up-regulated LTBP-4 mRNA, protein, and soluble LTBP-4-bound large latent TGF-beta1 complexes in Thy-1 (-) fibroblasts to significantly higher levels than in Thy-1 (+) fibroblasts. Bleomycin-induced TGF-beta1 activation required LTBP-4, since lung fibroblasts deficient in LTBP-4 did not activate TGF-beta1. Expression of LTBP-4 restored TGF-beta1 activation in response to bleomycin, but expression either of LTBP-4 lacking the TGF-beta-binding site or only the TGF-beta-binding domain did not. Bleomycin treatment of mice increased LTBP-4 expression in the lung. Thy-1 knockout mice had increased levels of both LTBP-4 expression and TGF-beta activation, as well as enhanced Smad3 phosphorylation compared with wild-type mice. Together, these data identify a critical role for LTBP-4 in the regulation of latent TGF-beta1 activation in bleomycin-induced lung fibrosis.

Distribution and bioactivity of the Ret-specific D4 aptamer in three-dimensional collagen gel cultures

The success of tyrosine kinase inhibitors in cancer therapy prompted intensive research efforts addressed to the development of new specific diagnostics and therapeutics. Targeting large transmembrane molecules, including receptor tyrosine kinases, is a major pharmacologic challenge. The D4 RNA-aptamer, isolated applying the Systematic Evolution of Ligand by Exponential Enrichment procedure on living cells, has been proven a specific inhibitor of the human receptor tyrosine kinase Ret. In our attempts to generate new powerful probes for in vivo applications, in the present study, we addressed the ability of D4 to preserve its biological activity in cells embedded in three-dimensional collagen gels. These matrices provide a microenvironment mimicking the cell organization as seen in vivo, thus representing a suitable tool to approach the use of the aptamer in vivo. By taking advantage of transformed fibroblasts expressing Ret as a model system, we showed that the cells maintain normal phenotype and growth patterns when cultured in three-dimensional matrices and that the D4 aptamer preserves its ability to inhibit Ret on the surface of the cells embedded in collagen. Because the biological activity of RNA aptamers is largely dictated by their folded structure, the results indicate that a folded conformation of D4 responsible of its inhibiting function is preserved in the three-dimensional constructs, thus supporting its use in tumors in vivo.

Survival and developmental competence of buffalo preantral follicles using three-dimensional collagen gel culture system

The aim of the present study was to develop a three-dimensional (3D) collagen gel culture system for the in vitro growth and survival of buffalo preantral follicles with or without growth factors. Buffalo ovaries were collected from a local abattoir and preantral follicles were isolated through microdissection. Isolated preantral follicles were put either in collagen gel coated culture dish or embedded in a microdrop of collagen gel. The culture medium was TCM-199 fortified with fetal calf serum (10%), insulin transferin selenium solution (ITS, 1%), epidermal growth factor (EGF, 20 ng/ml) and follicle stimulating hormone (FSH, 0.5 μg/ml). Follicles were divided into three groups and cultured in the medium described above (group a, control), with addition of insulin like growth factor (IGF-I, 100 ng/ml, group b), or with addition of IGF-I and basic fibroblast growth factor (bFGF, 10 ng/ml, group c). Preantral follicles were incubated at 38.5 °C in 5% CO 2 and maximum humidity. Culture medium was replenished after every 72 h and spent medium was stored at −30 °C for hormone analysis. We found that the extracellular matrix of collagen gel maintained follicle viability and growth by providing surface interaction and increasing attachment of follicles. Preantral follicles embedded in collagen gel droplets had better antrum formation and development as compared to the whole surface coated culture method. Follicles cultured with IGF-I on collagen gel matrix showed a significantly ( P < 0.05) higher survival rate and larger mean diameter of follicles on day 10 of culture with improved growth and mucification as compared to the control group. However, follicles cultured in the combination of IGF-I with bFGF had decreased survival rate and smaller mean follicles diameter than the IGF-I group (b). Progesterone (P 4) accumulation was greater on day 9 of culture in follicles cultured in IGF-I as compared to control; whereas, P 4 was markedly decreased in the combination of IGF-I with bFGF. Follicles of the control group could survive for up to 10–15 days before degenerating, but follicles cultured with growth factors were able to survive up to 20 days and showed signs of early antrum formation. In summary, we have shown that collagen gel was a novel and efficacious 3D microenvironment for the extended culture of buffalo preantral follicles. Supplementation of culture medium with growth factors was found to be essential for antrum formation.

Contrasting effect of exercise and angiotensin II hypertension on in vivo and in vitro cardiac angiogenesis in rats

Cardiac vessel density (beta-actin immunolabeling) and angiogenic capacity of coronary artery explants (culture in collagen gel) was determined in hypertrophied heart obtained by exercise training (10 wk) or ANG II infusion for 10 days. A group of rats received ANG II the last 10 days of training. The heart weight index was similarly elevated after exercise, and ANG II-hypertension compared with controls (3.16 +/- 0.09 and 3.11 +/- 0.11 vs. 2.68 +/- 0.08 mg/g, respectively), whereas tail cuff pressure (TCP) increased only in sedentary rats infused with ANG II. Vessel density was increased by 36% in trained rats and reduced by 30% in ANG II-infused rats. The number of sprouts generated by coronary rings was reduced by 50% in ANG II-infused rats and increased by 50% in exercise trained rats compared with controls (35 +/- 4 and 113 +/- 5 vs. 71 +/- 1 sprouts per ring, respectively). Exercise-training partly prevented the hypertensive effect of ANG II (TCP of 141 +/- 5 mmHg), whereas heart weight index (3.66 +/- 0.06 mg/g body wt) was not lowered. Myocardial vessel density was normalized, and sprouting from coronary rings increased by 50% in trained rats infused with ANG II compared with sedentary normotensive rats. Cardiac VEGF (Western blot analysis) was higher in hypertensive rats and not affected by exercise. Facing a similar increase in cardiac mass, intense training, but not ANG II hypertension, is accompanied by an increase in vascular density of the heart. The effect of training is unlikely related to changes in resting VEGF and may represent enhanced angiogenic capacity of the coronary vascular bed.

Involvement of RhoA, ROCK I and myosin II in inverted orientation of epithelial polarity

In multicellular epithelial tissues, the orientation of polarity of each cell must be coordinated. Previously, we reported that for Madin-Darby canine kidney cells in three-dimensional collagen gel culture, blockade of beta1-integrin by the AIIB2 antibody or expression of dominant-negative Rac1N17 led to an inversion of polarity, such that the apical surfaces of the cells were misorientated towards the extracellular matrix. Here, we show that this process results from the activation of RhoA. Knockdown of RhoA by short hairpin RNA reverses the inverted orientation of polarity, resulting in normal cysts. Inhibition of RhoA downstream effectors, Rho kinase (ROCK I) and myosin II, has similar effects. We conclude that the RhoA-ROCK I-myosin II pathway controls the inversion of orientation of epithelial polarity caused by AIIB2 or Rac1N17. These results might be relevant to the hyperactivation of RhoA and disruption of normal polarity frequently observed in human epithelial cancers.

A New Organotypic Culture of Adipose Tissue Fragments Maintains Viable Mature Adipocytes for a Long Term, Together with Development of Immature Adipocytes and Mesenchymal Stem Cell-Like Cells

Adipose tissue that consists of mature and immature adipocytes is suggested to contain mesenchymal stem cells (MSCs), but a culture system for analyzing their cell types within the tissue has not been established. Here we show that three-dimensional collagen gel culture of rat sc adipose tissue fragments maintained viable mature adipocytes for a long term, producing immature adipocytes and MSC-like cells from the fragments, using immunohistochemistry, ELISA, and real time RT-PCR. Bromodeoxyuridine uptake of mature adipocytes was detected. Adiponectin and leptin, and adipocyte-specific genes of adiponectin, leptin, and PPAR-gamma were detected in culture assembly, whereas the lipogenesis factor insulin (20 mU/ml) and inflammation-related agent TNF-alpha (2 nm) increased and decreased, respectively, all of their displays. Both spindle-shaped cell types with oil red O-positive lipid droplets and those with expression of MSC markers (CD105 and CD44) developed around the fragments. The data indicate that adipose tissue-organotypic culture retains unilocular structure, proliferative ability, and some functions of mature adipocytes, generating both immature adipocytes and CD105+/CD44+ MSC-like cells. This suggests that our method will open up a new way for studying both multiple cell types within adipose tissue and the cell-based mechanisms of obesity and metabolic syndrome.

A Two-Step Culture System for Development of Mouse Early Preantral Follicles.

Background: Application of ovarian tissue cryopreservation followed by in-vitro culture of isolated follicles is a feasible method for fertility preservation in young female patients with cancer. For this purpose it is important to develop culture systems that can be used for various stages of follicles. However, only a few reports describe in-vitro growth (IVG) and in-vitro maturation (IVM) for isolated small follicles. This study examines the effectiveness of a two-step culture system for producing fertilizable eggs from early secondary follicles. Materials and Methods: Early preantral follicles constituted of small oocytes and 2-3 layers of granulosa cells were collected from 7-day-old BDF1 mice by mechanical treatment. The isolated follicles were divided into three groups based on their diameters, (Group A: 80-85μm, Group B: 70-90μm, Group C: 60-70μm) and mounted in collagen gel for initial in-vitro growth (IVG-I). The medium was composed of EGF, ITS, FSH, 5% FCS in α-MEM. At day 9, granulosa cell proliferation and follicle diameters were comparatively assessed in the three groups. Oocyte-granulosa cell complexes were isolated from grown follicles by collagenase treatment and transferred to a second in-vitro growth (IVG-II) condition. They were cultured on a collagen-coated membrane for a further 6 days. At day 15 of the whole culture, the grown OGCs were subjected to IVM to assess granulosa cell and oocyte maturation. An additional experiment was carried out to examine fertilization ability and embryo development of in-vitro matured oocytes. Results: The follicle diameters increased from 88.1μm to 160.1μm (Group A), from 78.3μm to 128.5μm (Group B) and from 63.1μm to 128.1μm (Group C) during IVG-I. The oocyte diameters also increased from 47.2 μm to 62.7 μm (Group A), from 46.3 μm to 60.0 μm (Group B) and from 41.8μm to 59.4μm (Group C). At day 9, OGCs having oocytes of more than 55 μm diameter (Group A:67; B:28; C:10) were transferred to the second 6-day culture (IVG-II). After IVG-II, 42/67 (62.7%), 16/28 (57.1%) and 16/28 (57.1%) of OGCs from Groups A, B and C respectively grew successfully. GVBD occurred by IVM in 41/42 (97.6%), 10/16 (62.5%) and 1/2 (50.0%) from Groups A, B and C, respectively. Fertilization rates were A: 6/41 (14.6%), B: 2/10 (20.0%) and C: 0/1 (00.0%) and presumptive embryos in Group A developed into a blastocyst stage in 2/6 (33.3 %). Conclusion: In this study we showed that early secondary follicles of diameter: 80-95μm successfully developed into mature oocytes having fertilizing ability in a two-step IVG culture system. This shows that a combination of collagen gel and collagen-coated membrane cultures is useful for growth and development of small ovarian follicles, although further improvement is necessary for less-developed follicles.

A New Role for the Renin-angiotensin System in the Development of the Ureteric Bud and Renal Collecting System

The renin-angiotensin system (RAS) plays a critical role in kidney development. Mutations in the genes encoding components of the RAS or pharmacological inhibition of RAS in mice or humans cause a spectrum of congenital abnormalities of the kidney and urinary tract (CAKUT). The observed defects include renal vascular abnormalities, abnormal glomerulogenesis, renal papillary hypoplasia, hydronephrosis, aberrant ureteric bud (UB) budding, duplicated collecting system and renal tubular dysgenesis. Little is known about the potential role of Ang II and its receptors in the morphogenesis of the UB and renal collecting system. This review emphasizes a novel role for the RAS in the development of the UB, collecting ducts and renal medulla. We observe that UB and surrounding stroma express angiotensinogen and Ang II AT1 receptors (AT1R) in vivo. Ang II stimulates UB cell branching in collagen gel cultures in vitro and induces UB morphogenesis in intact whole embryonic metanephroi grown ex vivo. In contrast, treatment of metanephroi with the AT1R antagonist candesartan inhibits UB branching. In addition, Ang II induces tyrosine phosphorylation of the epidermal growth factor receptor (EGFR) in UB cells. Furthermore, Ang II-stimulated UB morphogenesis is abrogated by inhibition of EGFR tyrosine kinase activity. In summary: 1) Ang II, acting via the AT1R, stimulates UB branching; 2) This process depends on tyrosine phosphorylation of the EGFR. Together, these data indicate that cooperation of AT1R and EGFR signaling performs essential functions during renal collecting system development via control of UB branching morphogenesis.

DNA Micro-Array Gene Expression Profiling of Angiogenesis in Collagen Gel Culture

We examined angiogenesis-related gene expression profiles using collagen gel culture and a DNA chip. After isolation of total RNA from cultures before and after capillary tube formation, a mouse whole-genome array study was performed. Seventy-three out of over 35,000 transcripts were expressed after capillary tube formation. The majority of genes did not show any significant differences between before and after capillary tube formation. However, there were 7 upregulated genes; tumor necrosis factor alpha-induced protein (Tnfαip) 2, vascular endothelial growth factor b (Vegfb), corticotropin releasing hormone receptor (Crhr) 2, vascular endothelial growth factor C (Vegfc), fi broblast growth factor (Fgf) 6, protein O-fucosyltransferase (Pofut) 1, hypoxia inducible factor 1 alpha subunit (Hif-1a). Eleven down-regulated genes were identified; ras homolog gene family member B (RhoB), Fgf-2, Thrombospondin (Thbs) 1, Angiogenin (Ang) 3, guanine nucleotide binding protein alpha (Gna) 13, tumor necrosis factor receptor superfamily member 12a (Tnfrsf12a), plasminogen (Plg), Elk3, upstream binding protein (Ubp) 1, integrin alpha V (Itgav), mitogen activated protein kinase (Mapk) 14. These up- and down-regulated genes identified in this study offer the strategies which improve the myocardial substrate during and following a myocardial infarction, such as the regrowth of functional blood vessels to the ischemic myocardium.

Three-Dimensional Microenvironments Retain Chondrocyte Phenotypes During Proliferation Culture

Although autologous chondrocyte implantation has already been in clinical use, chondrocyte dedifferentiation is problematic during proliferation culture. We attempted a three-dimensional (3D) collagen gel culture under chondrocyte proliferation with repeated passaging to prevent the chondrocytes dedifferentiation. Human auricular chondrocytes were cultured in 3D or conventional monolayer conditions, which reached a 1000-fold increase in cell numbers at passages 3 and 4, respectively. During multiplication, the chondrocytes in 3D culture showed greater suppression of collagen type I (COL1) and preservation of collagen type II (COL2) than those in monolayer. Tissue-engineered cartilage made of 3D cells also abundantly accumulated COL2 or proteoglycan and possessed favorable mechanical properties. The advantage of 3D cells may result from the similarity of microenvironments in cell-to-matrix adhesion or cell-to-cell contacts with that of native cartilage. The up-regulation of integrins and down-regulation of cadherins in the 3D cells mimicked the expression pattern of native cartilage, rather than that of monolayer cells. The silencing of integrin beta1 and Ob-cadherin expression by small interfering ribonucleic acid in the cultured chondrocytes led to the promotion of dedifferentiation and redifferentiation, respectively, indicating that the 3D collagen gel culture provided sufficient cell preparation and reduced chondrocyte dedifferentiation, which is regarded as a feasible strategy in autologous chondrocyte implantation.

Adipocytes and preadipocytes promote the proliferation of colon cancer cells in vitro

Obesity, a risk factor for colon cancer, is associated with elevated serum levels of leptin, a protein produced by adipocytes. The aim of the present study was to clarify the effects of adipose tissue on colon cancer proliferation by using cultured cell lines. To achieve this, colon cancer cells (CACO-2, T84, and HT29) were cocultured with adipose tissue, isolated mature adipocytes, and isolated preadipocytes in a three-dimensional collagen gel culture system. The adipocytes and preadipocytes used were isolated from C57BL/6J and leptin-deficient ob/ob mice. Proliferation of the cancer cells was evaluated by nuclear bromodeoxyuridine uptake. The adipose tissue, mature adipocytes, and preadipocytes isolated from C57BL/6J mice significantly increased the proliferation of the colon cancer cells. This trophic effect of mature adipocytes on the cancer cell lines was observed only for cells from lean littermates and not for those from ob/ob mice. In contrast, the trophic effect of preadipocytes was not abolished in ob/ob mice, and this finding was supported by the result that leptin had a trophic effect on cancer cells. In conclusion, adipocytes were able to enhance the proliferation of colon cancer cells in vitro, partly via leptin, suggesting that adipose tissues, including mature adipocytes and preadipocytes, may promote the growth of colorectal cancer.

Establishment and characterization of a human adenoid cystic carcinoma cell line forming colonies cultured in collagen gel and transplantable in nude mice

We established a new cell line (ACCNS) from human adenoid cystic carcinoma (AdCC) of the maxilla, using tissue culture techniques and it has been successfully subcultured for more than 100 passages during 2 years. The population doubling time was approximately 39.2 h on a plastic dish. In type I collagen gel culture, the cells formed spherical colonies by day 7 after seeding. The colonies showed tubular and solid structures, and eosinophilic material stained with mucicarmine was revealed in the inner space. Immunohistochemically, ACCNS cells demonstrated expressions of keratin, alpha-smooth muscle actin, vimentin and S-100 protein, similar to those of original AdCC. These findings indicate that ACCNS cells possess the characteristics of AdCC. In addition, inoculation of 4.0x10(6) cells into nude mice developed tumors that were histologically confirmed as undifferentiated carcinoma. Therefore, ACCNS is the first AdCC cell line with tumorigenicity in nude mice. Based on these results, ACCNS provides a useful culture model of AdCC to analyze the biological characteristics and behavior of this tumor.

Culture system for studying thyroid biology

Abstract. The thyroid is composed of many ball-like structures called thyroid follicles, which are supported by the interfollicular extracellular matrix and a dense network of fenestrated capillaries. The follicles consist of many thyrocytes and a few parafollicular cells (C cells). In this microenvironment, the component thyrocytes are highly integrated in their specific structural and functional polarization. In conventional monolayer and floating culture systems, thyrocytes are unable to organize themselves into the follicles with normal cellular polarity. By contrast, in three-dimensional collagen gel culture, thyrocytes easily form stable follicle structures with normal polarity. However, these culture systems cannot reconstruct follicle structures that consist of both thyrocytes and C cells. To overcome this issue, we developed a new organotypic culture system of thyroid tissue fragments, which contain the two cell types, using three-dimensional collagen gel culture with air–liquid interface. Our thyroid tissue-organotypic culture system retains three-dimensional follicle structures with both thyrocytes and C cells for a long term. In relation to our method, we propose a new approach to the cell type-specific culture systems on the basis of in vivo microenvironments of various cell types. We review here four culture systems above to study thyroid biology.

Collagen gel contractility is enhanced in human endometriotic stromal cells: a possible mechanism underlying the pathogenesis of endometriosis-associated fibrosis

Excessive fibrosis is frequently associated with endometriosis. To evaluate the involvement of the extracellular matrix contractility of endometriotic stromal cells (ECSCs) in the pathogenesis of endometriosis-associated fibrosis, we compared the collagen gel contractility of cultured ECSCs with that of normal endometrial stromal cells. To clarify the mechanism underlying collagen gel contraction by ECSCs, we also evaluated the effect of (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide dihydrochloride, monohydrate (Y-27632), a selective Rho-associated coiled-coil-forming protein kinase (ROCK) inhibitor, on the collagen gel contraction by ECSCs. ECSCs showed enhanced collagen gel contractility in comparison with NESCs. Myofibroblastic differentiation and the increased expression of fibronectin, RhoA, ROCK-I and ROCK-II proteins were observed with ECSCs using the 3D culture. Y-27632 significantly inhibited the collagen gel contractility of ECSCs without cytotoxicity. The present findings suggest that the enhanced collagen contractility in ECSCs is associated with myofibroblastic differentiation, the increased expression of fibronectin and the activation of the Rho-ROCK-mediated signalling pathway, all of which may be involved in the pathogenesis of endometriosis-associated fibrosis. These results suggest that the inhibition of the Rho-ROCK-mediated signalling pathway may provide a novel strategy for the treatment of this disease. In addition, our experimental system of ECSCs using 3D collagen gel culture would be suitable for evaluating novel treatments for endometriosis.