DME Medium Research Articles

In Vitro Angiogenesis: Co-Culture Model of the Retina in Microfluidic Devices

Background Age-related macular disease (AMD) is a common eye condition and the most severe form of a disease that is the leading cause of vision loss among people age 50 and above. Simple tasks such as driving or reading become impossible without a functional central vision, which is impaired as the result of damages to the macula. Wet form AMD is responsible for 90% of the blindness out of all AMD patients; it is marked by the development of subretinal choroidal neovascularization and degeneration of retinal pigmental epithelial (RPE) cells. Wet macular degeneration occurs when abnormal blood vessels disrupt the retina and leak blood or fluid that blurs the central vision. Recently, MEMS technology which integrates multidisciplinary field of studies offers a new direction in medical investigation and diagnosis. Here we utilized microfabrication technology to reconstruct ocular fundus tissues (OF) in vitro and to simulate neovascularization in the hope of building a disease model for the retina. Method Human retinal pigment epithelial (ARPE-19) and human umbilical vein endothelial cells (HUVECs), representing the retinal cells and the choroid, were co-cultured in the microfluidic devices. Two different approaches are developed in fabricating microfluidic devices resulting in 2D and 3D models. 2D model:the microfluidic device was made up of 2 layers, on each a channel (width 500 µm; height 150 µm) was embedded. A porous membrane (thickness 6.5 µm, hole diameter 10 µm) was sandwiched between the layers. Microchannels and membranes were made of poly(dimethylsiloxane) (PDMS). Retinal pigment epithelial (RPE) cells were introduced into the upper channel of the device and umbilical vein endothelial cells (HUVECs) were loaded into the lower channel. 3D model: the wire with a 0.2 mm diameter was pretreated before embedded in collagen (6.5 mg/ml). After the collagen had polymerized, the needle was removed resulting in an empty channel where HUVECs were to be seeded. RPE cells were seeded on the top of the collagen gel to initiate co-culture experiments. Data analysis: RPE cells and HUVEC were dyed respectively with Cell TrackerTM red and green and cell growth was recorded at certain time intervals. RPE cells were under hypoxia and hypoglycemia 24hr prior the start of the co-culture experiment. Before the aforementioned co-culture model, RPE were cultured alone on the upper channel and the VEGF concentration was collected and measured by ELIZA after replacing DME medium High Glucose (20 mM) with DME medium Low Glucose (3 mM). HUVECs were loaded into the lower channel and cultured alone. The upper channel was perfused with 50 ng/ml of VEGF-A165and the number of HUVECs migrating to the upper channel was analyzed using the same monitoring system. Characteristics of cells in the 3D model were characterized following the same methodology. Results Validation of the devices: The devices were validated prior culturing cells inside the devices. Confocal images of the cross-sections of channel length and diameter were taken after perfusion of fluorescent microbeads. ARPE characterization: Results show that RPE cells responded to the lowered glucose microenvironment by increasing VEGF secretion (58.8 % compared to the control). In addition, RPE cells exhibited a polar secretion of VEGF which is consistent to the cell physiology. HUVEC characterization: The increased number of HUVEC migrating through the porous membrane in response to VEGF gradient signifies neovascularization. Comparing to the control, there was a 31.3 % increase of cell migration. Conclusions and Future work The physiology of RPE cells and HUVECs is reproduced in the microfluidic devices. The preliminary results suggest that the alteration of the microenvironment induces responses of RPE cells and the significance of VEGF in neovascularization. In the near future, we anticipate studying responses of the co-culture model to hypoxia to characterize the AMD pathology.

Microfluidic cell culture model of the ocular fundus

Event Abstract Back to Event Microfluidic cell culture model of the ocular fundus Li Jiun Chen1, Shunichi Tsunajima1, Matsuhiko Nishizawa1, Nobuhiro Nagai2, Toshiaki Abe2 and Hirokazu Kaji1 1 Tohoku University, Bioengineering and Robotics, Japan 2 Tohoku University, Clinical Cell Therapy, United Centers for Advanced Research and Translational Medicine, Japan Introduction: Age-related macular disease (AMD) is a common eye condition and a leading cause of vision loss among people age 50 and above[1]. Simple tasks such as driving or reading become impossible without a functional central vision, which is impaired as the result of damages to the macula. The causes leading to AMD includes, but not limited to, family history, and changes in the cellular microenvironments. Wet form AMD is responsible for 90% of the blindness out of all AMD patients[1]; it is marked by the development of subretinal choroidal neovascularization and degeneration of retinal pigmental epithelial (RPE) cells[2]. Although there are many studies aiming at using microdevices to reproduce organ physiologies, knowledge of on-chip models of the eye is limited. Here, microfabrication technology is used to reconstruct ocular fundus tissues (OF) in vitro and to simulate neovascularization in the microfluidic devices. Materials and Methods: The microfluidic device was made up of 2 layers, on each a channel (width 500 µm; height 150 µm) was embedded. A porous membrane (thickness 6.5 µm, hole diameter 10 µm) was sandwiched between the layers. Microchannels and membranes were made of poly(dimethylsiloxane) (PDMS). Retinal pigment epithelial (RPE) cells were introduced into the upper channel of the device and cultured until they were confluent. Thereafter, human umbilical vein endothelial cells (HUVECs) were loaded into the lower channel. The device was turned upside down to allow cells to adhere onto the lower side of the membrane. RPE cells and HUVEC were dyed respectively with Cell TrackerTM green and red and cell growth was recorded under Real-Time Cultured Cell Monitoring System (Fig. 1,2). Before the aforementioned co-culture model, RPE were cultured alone on the upper channel and the VEGF concentration was collected and measured by ELIZA after replacing DME medium High Glucose (20 mM) with DME medium Low Glucose (3 mM). HUVECs were loaded into the lower channel and cultured alone. The upper channel was perfused with 50 ng/ml of VEGF-A165 and the number of HUVECs migrating to the upper channel was analyzed using the same monitoring system. Results and Discussion: Results show that RPE cells responded to the lowered glucose microenvironment by increasing VEGF secretion (58.8 % compared to the control). In addition, RPE cells exhibited a polar secretion of VEGF which is consistent to the cell physiology. The increased number of HUVEC migrating through the porous membrane in response to VEGF gradient signifies neovascularization. Comparing to the control, there was a 31.3 % increase of cell migration (Fig. 3). Conclusions: The physiology of RPE cells and HUVECs is reproduced in the microdevice. The preliminary results suggest that the alteration of the microenvironment induces responses of RPE cells and the significance of VEGF in neovascularization. In the near future, we anticipate studying responses of the co-culture model to hypoxia to characterize the AMD pathology. Figure 1. Schematic representation of the placement of cells. Figure 2. RPE cells were stained and imaged by fluorescent microscopy. Figure 3. Number of cells migrated through the porous membrane were counted and compared against the control.

Abstract 1074: Epigenetic silencing of DACH1 in triple negative breast cancer contributes to the tumorigenesis

Abstract BACKGROUND: Increasing evidence indicates that cell fate determination factor DACH1 functions as a tumor suppressor. Specifically, in estrogen receptor positive (luminal), highly expression of DACH1 predicted longer cancer specific survival and disease free interval, as well as reduced metastasis formation. Our analysis of over 2,000 patients demonstrated increased nuclear DACH1 expression correlated inversely with cellular mitosis and predicted 40 months survival advantage in breast cancer patients. But, endogenous DACH1 was dramatically reduced in breast cancer cell lines with high expression of tumor initiation cell markers and in patient samples of triple negative breast cancer, the most aggressive subtype. We proposed that epigenetically change might contribute to the loss expression of DACH1 in breast cancer. MATERIALS AND METHODS: Clinical samples included 10cases of normal breast tissues, 26 cases of luminal breast cancer(ER+,PR+,HER1-2+), and 32 cases of triple negative breast cancer(ER-,PR-,HER-). Genomic DNA was extracted from formalin-fixed tissue and methylation-specific PCR of DACH1 promoter was performed. DACH1 protein expression was detected in human breast cancer tissue arrays by immunohistochemistry. Human triple negative breast cancer cell lines MDA-MB-231 and sum159 were cultured in DME medium supplied with 10% fetal bovine serum and growth factors. Stable cell lines expressing DACH1 were established in MDA-MB-231 and Sum159 though lentivirus transfection. In vitro cell growth was evaluated by MTT assay and colony formation. In vivo tumor growth was measured in xenograft mice model. RESULTS: There is no methylation of DACH1 promoter region was found in normal breast tissues, but methylation was found in 2 out of 26 (7.7%) in luminal breast cancer and 10 of 32(32%) in triple negative breast cancer. Consisting with previous report, immunohistochemical stain revealed nuclear stain of DACH1 in all normal and majority of luminal type breast cancer tissues, but lost or very weak expression of DACH1 in triple negative cancer tissues. Correspondingly, expression microarrays demonstrated that DACH1 mRNA levels in triple negative breast cancer cell lines were dramatically less than that of normal breast cell line and luminal breast cancer cell lines. Treatment of MDA-MB-231 and Sum159 cells with 5-Aza-2′-deoxyazacytidine induced the expression of DACH1 and decreased cellular proliferation. Ectopic expression of DACH1 inhibited in vitro cell growth and in vivo tumor formation. CONCLUSION: Promoter hypermethylation is a main factor leading to the inactivation of DACH1 in triple negative breast cancer; Re-expression of DACH1by epigenetic modification might provide a novel therapeutic strategy for this aggressive cancer. Citation Format: Qian Chu, Na Han, Yan Dong, Xun Yuan, Mingzhou Guo, Hanxiao Xu, Shiying Yu, Kongming Wu. Epigenetic silencing of DACH1 in triple negative breast cancer contributes to the tumorigenesis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1074. doi:10.1158/1538-7445.AM2015-1074

Use of a silver ion selective electrode to assess mechanisms responsible for biological effects of silver nanoparticles

For a detailed analysis of the biological effects of silver nanoparticles, discrimination between effects related to the nano-scale size of the particles and effects of released silver ions is required. Silver ions are either present in the initial particle dispersion or released by the nanoparticles over time. The aim of this study is to monitor the free silver ion activity {Ag+} in the presence of silver nanoparticles using a silver ion selective electrode. Therefore, silver in the form of silver nanoparticles, 4.2 ± 1.4 nm and 2–30 nm in size, or silver nitrate was added to cell culture media in the absence or presence of A549 cells as a model for human type II alveolar epithelial cells. The free silver ion activity measured after the addition of silver nanoparticles was determined by the initial ionic silver content. The p {Ag+} values indicated that the cell culture media decrease the free silver ion activity due to binding of silver ions by constituents of the media. In the presence of A549 cells, the free silver ion activity was further reduced. The morphology of A549 cells, cultivated in DME medium containing 9.1% (v/v) FBS, was affected by adding AgNO3 at concentrations of ≥30 μM after 24 h. In comparison, silver nanoparticles up to a concentration of 200 μM Ag did not affect cellular morphology. Our experiments indicate that the effect of silver nanoparticles is mainly mediated by silver ions. An effect of silver on cellular morphology was observed at p {Ag+} ≤ 9.2.

Abstract 3073: Rab25 expression, loss and therapeutic implications in human breast cancer

Abstract Introduction: Our previous work has shown that the loss of Rab25 occurs in estrogen receptor (ER) and progesterone receptor (PR) negative breast cancer cell lines and human tumor samples and seems to correlate with an active ras pathway. We have expressed Rab25 in several hormonally insensitive breast cancer cell lines and have demonstrated marked reduction in tumor growth. Histologically, the tumor cells appear smaller, and angiogenesis is significantly reduced. We have investigated the mechanism by which Rab25 exerts its anti-tumorigenic effect. Loss of Rab25 cooperates with mutant Ras to promote tumorigenesis in primary human mammary epithelial cells (HMEC). HIF1-alpha levels are markedly increased with loss of Rab25 if Ras is overexpressed. Rab25 is expressed in both luminal and basal mammary epithelium at similar levels. The loss of rab25 enhances VEGF-A secretion and VEGFR1 expression. Monoclonal antibodies can be potentially used to target these pathways which appear to be active in Rab25 negative tumors. Materials and Methods: Cell culture - MDA-MB-231 cells were grown in DME medium with 10% FBS, and Rao-3 cell line and HMEC were grown in DFCI-1 medium. Cells were transduced with retroviral supernatant and selected. Expression of RAB25 in transduced cells was determined by RT-PCR and western blot. Silencing of Rab25-Rab-25 was silenced in RAO-2 cells by pENTRTM/H1/TO siRNA system (Invitrogen, Carlsbad, CA). Our shRNA sequences are designed using the BLOCK-iTTM RNAi Designer. The most effective silencer of Rab25 was selected on the basis of testing 3 sequence pairs. The double-stranded oligos were inserted into the pENTR/H1/TO vector and transfected into cells. Silencing of Rab25 in transduced cells was confirmed by RT-PCR and Western blot. qPCR analysis- Expression of HIF1-alpha was quantified using real-time PCR with fluorescence detection. Western blot- Western blot was performed on cell extracts for rab25 and ras. Immunohistochemistry - CK5/6 served as a basal maker, and CK18 served as a luminal maker for co-staining with Rab25 in human mammary gland. In vivo tumor formation assay − 5 × 106 cells in 0.1 ml PBS were injected into the left mammary fat pad of five week-old female nude mice. Mice were monitored for the following 7 weeks for tumor volume. Therapeutic assays- Cell lines were incubated with anti-VEGF-A, anti-VEGFR1, or both, and cell numbers were tabulated over 5 days. Results: 1) Rab25 loss cooperated with Ras to promote tumorigenesis in several donors. 2) HIF1α levels rose significantly with loss of Rab25 and Ras co-expression. 3) Rab25 is expressed in both basal and luminal normal mammary epithelium. 4) Both anti-VEGF-A and anti-VEGFR1 antibodies have therapeutic implications for Rab25 negative breast cancer cell lines. Conclusions: Rab25 is a tumor suppressor that antagonizes Ras and may be part of an important therapeutic pathway. Its loss may serve as therapeutic marker for certain types of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3073.

Immobilization of Bioactive Fibroblast Growth Factor-2 into Cubic Proteinous Microcrystals (Bombyx mori Cypovirus Polyhedra) That Are Insoluble in a Physiological Cellular Environment

The supramolecular architecture of the extracellular matrix and the disposition of its specific accessory molecules give rise to variable heterotopic signaling cues for single cells. Here we have described the successful occlusion of human fibroblast growth factor-2 (FGF-2) into the cubic inclusion bodies (FGF-2 polyhedra) of the Bombyx mori cytoplasmic polyhedrosis virus (BmCPV). The polyhedra are proteinous cubic crystals of several microns in size that are insoluble in the extracellular milieu. Purified FGF-2 polyhedra were found to stimulate proliferation and phosphorylation of p44/p42 mitogen-activated protein kinase in cultured fibroblasts. Moreover, cellular responses were blocked by a synthetic inhibitor of the FGF signaling pathway, SU5402, suggesting that FGF-2 polyhedra indeed act through FGF receptors. Furthermore, FGF-2 polyhedra retained potent growth stimulatory properties even after desiccation. We have demonstrated that BmCPV polyhedra microcrystals that occlude extracellular signaling proteins are a novel and versatile tool that can be employed to analyze cellular behavior at the single cell level.

Inhibitory effects of a nutrient mixture on human testicular cancer cell line NT 2/DT matrigel invasion and MMP activity

Current treatment of testicular cancer is associated with secondary malignancy, infertility, and cytotoxicity. Based on reported antimetastatic potential, we investigated the effect of a nutrient mixture (NM) containing lysine, proline, arginine, ascorbic acid, and green tea extract on human testis cancer cell line NT 2/DT by measuring cell proliferation/cytotoxicity, modulation of MMP-2 and MMP-9 secretion, and cancer cell invasive potential. Human testis cancer cells NT 2/DT (ATCC) were grown in DME medium. At near confluence, the cells were treated with NM dissolved in media and tested at 0,10, 50, and 100 microg/mL in triplicate at each dose. Cells were also treated with PMA 200 ng/mL to study enhanced secretion of MMP-9. Cell proliferation/cytotoxicity was evaluated by MTT assay, MMP activity by gelatinase zymography, and invasion through Matrigel. The nutrient mixture showed no significant effect on testis cancer cell growth. Zymography demonstrated secretion of MMP-2 by untreated human testis cancer cells and MMP-9 with PMA induction. NM inhibited secretion of both MMPs in a dose-dependent fashion with virtual total inhibition of MMP-9 at 100 microg/mL. Invasion of human testis cancer cells through Matrigel was reduced by 84% at 50 microg/mL and at 100 microg/mL (p = 0.004). NM significantly inhibited MMP secretion and matrix invasion in testicular cancer cells without toxic effect, indicating potential as an anticancer agent.

Hsp90 restrains ErbB‐2/HER2 signalling by limiting heterodimer formation

ErbB-2/HER2 is an oncogenic tyrosine kinase that regulates a signalling network by forming ligand-induced heterodimers with several growth factor receptors of the ErbB family. Hsp90 and co-chaperones regulate degradation of ErbB-2 but not other ErbB members. Here, we report that the role of Hsp90 in modulating the ErbB network extends beyond regulation of protein stability. The capacity of ErbB-2 to recruit ligand-bound receptors into active heterodimers is limited by Hsp90, which is dissociated from ErbB-2 following ligand-induced heterodimerization. We show that Hsp90 binds a specific loop within the kinase domain of ErbB-2, thereby restraining heterodimer formation and catalytic function. These results define a role for Hsp90 as a molecular switch regulating the ErbB signalling network by limiting formation of ErbB-2-centred receptor complexes.

Inhibition of p38 MAP kinase decreases the production of MCP-1 and IL-8 in endometrial stromal cells

Mitogen-activated protein kinase (MAPK) pathway is one of the most ubiquotus signaling pathways. This pathway is activated by a variety of cellular stimuli and regulates numerous physiological processes. It has been proposed that p38 MAPK functions in the regulation of cytokine and chemokine expression. Given the role of p38 MAPK in cellular inflammatory responses, we aimed to determine whether p38 MAPK pathway is involved in the production of interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) in endometrial stromal cells. A prospective in vitro study to assess the effect of p38 MAPK inhibition on IL-8 and MCP-1 expression in endometrial stromal cells. Endometrial tissues were obtained from women undergoing surgery for benign gynecologic conditions. Endometrial stromal cells were isolated and cultured in Ham’s F-12: DME medium that contained 10% fetal bovine serum, and grown to confluence. Experiments were performed after incubating cells in serum-free, phenol red-free medium for 24 h. Statistical analysis of the data was performed using ANOVA. Cells were treated for 24 h with a specific p38 MAPK inhibitor, SB203580 (10 μM) or with vehicle (control), and supernatants were collected. IL-8 and MCP-1 levels in culture media were quantified using a specific ELISA for each chemokine. Treatment of cells with p38 MAPK inhibitor resulted in a 37±2% (mean±SEM; p<0.001) decrease in IL-8 levels and a 29±2% (mean±SEM; p=0.003) decrease in MCP-1 levels. Inhibition of p38 MAPK signaling pathway significantly inhibited IL-8 and MCP-1 production in endometrial stromal cells. Therefore, we propose that IL-8 and MCP-1 expression in these cells, is at least partially regulated through p38 MAPK pathway. We speculate that this pathway may be involved in the pathogenesis of endometriosis, a proinflammatory disease where high levels of these chemokines are observed.

Studies on cytotoxic effect of nickel ions on three cultured fibroblasts.

Cytotoxicity of Ni ions on three fibroblasts such as L929, Balb/3T3 clone A31 and MC3T3-E1 were examined by cell count (CC) and Neutral Red assay (NR). Three cells were incubated for 6 days in 1 ml DME medium containing Ni ions which ranged from 0 to 2 mM/l. The results clarified that Ni ions had dose-dependent cytotoxicity. L929 possessed the largest TC50 values (the amount of Ni ion that caused 50% cell death or 50% cell viability) of 0.12 mM/l (CC) and 0.32 mM/l (NR), and Balb/3T3 clone A31 had the least values of 0.05 mM/l (CC) and 0.09 mM/l (NR), whilst MC3T3-E1 had the intermediate values of 0.08 mM/l (CC) and 0.15 mM/l (NR). The dissolution of Ni ions from Ni-containing metallic restorations must be lower than these concentration levels so that body tissues might not be severely damaged.

Studies on cytotoxicity of nickel ions using C3H10T1/2 fibroblast cells.

Cytotoxicity of Ni ions were examined using C3H mouse derived 10T1/2 fibroblast cells. It became evident that Ni ions had dose-dependent cytotoxicity. The cell number incubated in DME medium containing 0.04 mM/L Ni ion for 6 days was reduced to half that in control DME medium without Ni. The cell totally disappeared in DME medium containing 2 mM/L Ni ion. The dissolution of Ni ions from Ni-containing metallic restorations must be lower than these concentration levels so that oral tissues might not be damaged. No neoplastic transformation was found on all cells examined.

The Effect of Non Steroidal Anti-Inflammatory Drugs (Naproxen Sodium) on Endometrial Stromal Cell Proliferation

The Effect of Non Steroidal Anti-Inflammatory Drugs (Naproxen Sodium) on Endometrial Stromal Cell Proliferation

Sensory and Motor Neuron-derived Factor Is a Transmembrane Heregulin That Is Expressed on the Plasma Membrane with the Active Domain Exposed to the Extracellular Environment

The beta-heregulin sensory and motor neuron-derived factor (SMDF) has been suggested to be an important regulator of Schwann cell development and proliferation. In the present study, human SMDF was expressed in cultured cell lines. The cells and the recombinant protein were used to examine the membrane association and biological activity of the growth factor. Transfection of cells with SMDF cDNA constructs bearing FLAG epitope tags at either the amino- or carboxyl-terminal ends of the polypeptide resulted in expression of anti-FLAG immunoreactive polypeptides of approximately 44 and 83 kDa. The 83-kDa polypeptide was the major form expressed on the cell surface, as demonstrated by sensitivity to proteolysis in intact cells and surface biotinylation. SMDF was tightly associated with membranes isolated from transfected cells but was solubilized by Triton X-100. Immunofluorescent staining and immunoprecipitation experiments using cells expressing amino- or carboxyl-terminal tagged SMDF revealed that only the carboxyl-terminal end of the protein is exposed on the cell surface. Membranes from SMDF-transfected cells stimulated tyrosine phosphorylation of the beta-heregulin receptor ErbB3 in Schwann cells. Conditioned medium from transfected cells contained a similar activity, suggesting that SMDF is subject to proteolytic release from the plasma membrane. In contrast with other beta-heregulin isoforms, SMDF failed to bind heparin. Stimulation of Schwann cell ErbB3 receptor phosphorylation by SMDF was not affected by inhibition of Schwann cell heparan sulfate proteoglycan synthesis. These results demonstrate that SMDF is a type II transmembrane protein. This orientation places the active epidermal growth factor homology domain, which is located near the carboxyl-terminal end of the polypeptide, on the cell surface, where it can function as a membrane-anchored growth factor.

Characterization and fed-batch culture of hybridoma overexpressing apoptosis suppressing gene bcl-2.

Mouse hybridoma 2E3 transfected with human bcl-2 gene survived longer with increasing expression level of bcl-2 when cultured in DME medium supplemented with 9% serum. One of the transfectants, 2E3BCMGbcl-2, overexpressed bcl-2 and could maintain viable cell density higher than the initial density for more than four days at a low 0.5% serum concentration. In comparison a mock transfectant 2E3BCMG remained viable for only one day. However, both hybridomas died out within a day in serum-free medium. These results suggested that bcl-2 needed a small amount of some serum components to suppress apoptosis of the hybridoma. Overexpression of bcl-2 also suppressed apoptosis of the hybridoma induced by glutamine deprivation. When hybridoma 2E3BCMGbcl-2 was inoculated in DME medium supplemented with 9% serum and cultured for 10 d with additional 2% serum feed at day 4 of the culture, viable cell density increased 2-fold and antibody produced 3-fold, in comparison with mock transfected 2E3 cultured in the same manner. The mock transfectant with additional feed of serum at day 4 of the culture showed no difference in viable cell density and antibody production. These results suggested that the mock transfectant committed to apoptosis before day 4 of the culture and the additional serum at day 4 could not reverse the commitment.

An optimal hormonally defined serum-free medium for the differentiation of a colic adenocarcinoma cell line

The aim of this work was to design a serum-free medium which can promote both growth and differentiation of an adenocarcinoma cell line (HT29-D4). Undifferentiated HT29-D4 cells growing as multilayers in 10% FBS DME medium, were first subcultured in glucose-free medium containing decreasing concentrations of serum and then in a hormonally defined serum-free medium constituted essentially of glucose-deprived DME supplemented with galactose, selenous acid, hormones (hydrocortisone and triiodothyronine), growth factors (insulin and epidermal growth factor), transferrin, fibronectin and bovine serum albumin. HT29-D4 cells grew in monolayer and displayed a highly differentiated enterocytic phenotype at the ultrastructural level. The carcinoembryonic antigen was restricted within the apical membrane domain whereas HLA class 1 molecules and transferrin receptors remained within the basolateral membrane domain. These findings demonstrate that the HT29-D4 cells grown in the serum-free medium were highly differentiated.

In vitro differentiation inhibits the migration of cultured neonatal rat cortical astrocytes transplanted to the neonatal rat cerebrum

Neonatal rat astrocytes transplanted into the rat cerebrum migrate extensively. However, few of the molecular signals determining this migration have been defined. In the present study, in vitro modifications were designed to examine whether differentiation prior to transplantation would affect the magnitude or pattern of astrocyte migration in the neonatal host brain. Here, cortical astrocytes were collected from the brains of rats 1–3 days postpartum and purified by culturing them in DME medium supplemented with 10% calf serum. After 14–21 days, astrocytes were labelled with fluorescein-tagged latex microspheres for 16 hr; the label was then removed and replaced with either fresh medium or fresh serum-free medium plus 1 mM dbcAMP. After 48 hr, cells were harvested and then transplanted into the right frontal cerebrum of neonatal rats at 3 days postpartum by injection with a hand-held Hamilton syringe. Animals were sacrificed at 3, 6, 9, 15, 21 and 28 days after inoculation and their brains examined with fluorescence microscopy. Astrocytes not exposed to dbcAMP prior to implantation migrated along the corpus callosum, internal capsule, glial limitans, ventricular linings and the hippocampal structure. They also appeared to migrate in a radial fashion toward the periphery from the ventricular lining. Astrocytes treated with dbcAMP prior to transplantation did not appear to migrate into the neonatal parenchyma, remaining confined to the injection site for at least 6 days. Migration then appeared to commence at a normal rate after 9 days. Thus, neonatal cortical migrate outward in a pattern similar to that defined by the radial glia. Astrocytes differentiated by dbcAMP treatment, however, do not appear to migrate to any large degree in the neonatal brain until the treatment effect diminishes, suggesting that differentiation may represent an end-point to glial migration in the neonatal host brain.

Effect of TGF‐Alpha on growth of normal human breast epithelial cells in serum‐free primary culture using 3‐dimensional collagen gels.

The mitogenic effect of TGF-alpha, acidic-FGF, basic-FGF and lithium on normal human breast epithelial cells was studied in a collagen gel culture system using a serum-free 1:1 mixture of Ham's F12 and DME medium containing insulin, cholera toxin and bovine serum albumin. TGF-alpha elicited a strong mitogenic response in a dose dependent manner. Addition of cortisol to TGF-alpha stimulated growth over and above that achieved with TGF-alpha alone. A consistent observation has been the effect of a combination of TGF-alpha and cortisol on growth stimulation of normal human breast epithelial cells resulting in 3-12 fold growth after 11-13 days in culture. Acidic-FGF, basic-FGF and lithium were not growth promoting.

Modulation of the expression of the VIP receptor by serum factors on the human melanoma cell line IGR39

IGR39 cells, isolated from a human superficial melanoma, display at their surface high and low affinity receptors for the vasoactive intestinal peptide (VIP). When grown in DME medium supplemented with 10% fetal calf serum, cells display 1.6 x 10(5) high affinity (Kd 0.74 nM) and 5.6 x 10(5) low affinity (Kd 55 nM) VIP binding sites per cell. When cultured in a chemically defined medium containing EGF, transferrin, and selenium, IGR39 cells display many neurite-like extensions. Following these morphological changes, the specific [125I]VIP binding is increased four- to fivefold after 6 days in culture. This phenomenon is reversible and is the result of an increased number of VIP binding sites available at the cell surface, without modification of their affinities. The molecular mass of the binding sites is also unchanged whatever cell culture conditions. Increase in [125I]VIP binding is inversely correlated to the serum concentration in the culture medium. When added to the chemically defined medium, sera from various origins as well as some serum substitutes reduce [125I]VIP binding to the same extent as that of the serum. The total cAMP production by VIP-stimulated IGR39 cells is enhanced by a factor of six to seven when cells are cultured in serum-free medium, in good correlation with the increase of VIP binding capacity. These data suggest that factor(s) present in fetal calf serum inhibit(s) the expression of VIP receptor, thus demonstrating the importance of a strict control of cell culture conditions for in vitro studies.

Extra- and intracellular metabolite concentrations for murine hybridoma cells.

Intra- and extracellular metabolite concentrations were determined for hybridoma cells grown in tissue culture flasks in batch culture. Significant differences were found for intracellular lactate and amino acid concentrations depending on the culture medium. AB2-143.2 cells cultivated in Dulbecco's modified Eagle's (DME) medium supplemented with 50 mM lactate exhibited intracellular lactate and glutamine levels of 40 mM and 4 mM, respectively, whereas cells grown in standard DME medium had low intracellular glutamine and 20 mM lactate concentrations. For cells cultivated in medium supplemented with 6 mM pyruvate, intracellular lactate levels were estimated at approx. 40 mM, but these cells also showed an increased intracellular alanine concentration of 22 mM. The higher alanine pools probably result from increased transamination of pyruvate under these conditions.

Effects of bombesin and its antibody on growth of human prostatic carcinoma cell lines

Bombesin (BMBS), a tetradecapeptide isolated from frog skin, and its antibody were evaluated in vitro and in vivo for their growth modulating effects on human prostatic carcinoma cell lines DU-145 and PC-3. The two cell lines were maintained in DME medium containing 2% FCS and 1 microgram/ml insulin in a humidified atmosphere of 5% CO2/95% air at 37 degrees C. BMBS added at 0.1-10.0 nM caused a striking shift of the concentration-dependent increase in cell growth of DU-145 and PC-3 in the absence of any other exogenously added growth factors. The addition of 1:250 antibody versus BMBS (Ab-BMBS; rabbit antiserum) to the medium during the lag phase of growth resulted in specific inhibition of growth of DU-145 and PC-3 with or without BMBS. Nude mice were transplanted with DU-145 cells in order to evaluate the suppressing qualities of Ab-BMBS on carcinoma cells in vivo. After the inoculation of 10(7) cells/mouse DU-145 into nude mice, 20 microliters/mouse Ab-BMBS was intraperitoneally injected into mice three times weekly for three weeks. In mice administrated with Ab-BMBS, the growth of DU-145 was suppressed markedly. By immunocytochemical study, BMBS immunoreactivities were detected on DU-145 and PC-3 cells. These results suggest that BMBS can function as an autocrine growth factor for human prostatic carcinoma cells. Furthermore, on xenografts in mice, Ab-BMBS inhibits the increase of human prostatic carcinoma.