3-dimensional Cell Culture Model Research Articles

PNU-74654 Enhanced the Antiproliferative Activity of Gemcitabine by Targeting Wnt/β-Catenin Pathway in Pancreatic Cancer

Background: The Wnt/beta-catenin pathway is one of the pathways that is deregulated in pancreatic cancer and is reported to be associated with a poor prognosis. This indicates the need for the identification of novel agents to improve the efficacy of current therapy or have an improved efficacy. Therefore, in the present study, we explored the anticancer activity of PNU- 74654 alone or in combination with gemcitabine in 2 and 3-dimensional cell culture models of pancreatic cancer. Methods: The MTT assay was carried out to determine the viability of PC cancerous cells (PCCs), while the cytotoxicity of this agent was evaluated in a 3D cell culture model (spheroid). The effects of PNU-74654 were investigated in established cell migration/invasion assays. Results: The expression of candidate genes affecting the cell cycle, migration, and Wnt/bcatenin pathway was evaluated at mRNA and/or proteins by RT-PCR or Western blot. PNU- 74654 inhibited the cell growth at IC50 of 122 ± 0.4 umol/L and had a synergistic effect on the antiproliferative properties of gemcitabine by modulating the Wnt pathway. The PNU- 74654/gemcitabine combination reduced the migratory and invasiveness of PC cells, compared to control cells, through perturbation of E-cadherin. Conclusion: Our findings demonstrate the profound antitumor properties of PNU-74654 in in vitro models of pancreatic cancer, supporting further in vivo studies to evaluate the therapeutic impact of this novel therapy to target the Wnt pathway in the treatment of pancreatic cancer.

Targeting ROCK2 improves macromolecular permeability in a 3D fibrotic pancreatic cancer microenvironment model

Pancreatic cancer is characterized by a densely fibrotic stroma. The fibrotic stroma hinders the intratumoral penetration of nanomedicine and diminishes therapeutic efficacy. Fibrosis is characterized by an abnormal organization of extracellular matrix (ECM) components, namely the abnormal deposition and/or orientation of collagen and fibronectin. Abnormal ECM organization is chiefly driven by pathological signaling in pancreatic stellate cells (PSCs), the main cell type involved in fibrogenesis. However, whether targeting signaling pathways involved in abnormal ECM organization improves the intratumoral penetration of nanomedicines is unknown. Here, we show that targeting transforming growth factor-β (TGFβ)/Rho-associated kinase (ROCK) 1/2 signaling in PSCs normalizes ECM organization and concomitantly improves macromolecular permeability of the fibrotic stroma. Using a 3-dimensional cell culture model of the fibrotic pancreatic cancer microenvironment, we found that pharmacological inhibition of TGFβ or ROCK1/2 improves the permeation of various macromolecules. By using an isoform-specific pharmacological inhibitor and siRNAs, we show that targeting ROCK2, but not ROCK1, alone is sufficient to normalize ECM organization and improve macromolecular permeability. Moreover, we found that ROCK2 inhibition/knockdown attenuates Yes-associated protein (YAP) nuclear localization in fibroblasts co-cultured with pancreatic cancer cells in 3D. Finally, pharmacological inhibition or siRNA-mediated knockdown of YAP normalized ECM organization and improved macromolecular permeability. Our results together suggest that the TGFβ/ROCK2/YAP signaling axis may be therapeutically targeted to normalize ECM organization and improve macromolecular permeability to augment therapeutic efficacy of nanomedicines in pancreatic cancer.

C644-0303, a small-molecule inhibitor of the Wnt/β-catenin pathway, suppresses colorectal cancer growth.

The Wnt/β‐catenin signaling pathway plays an important role in tissue homeostasis, and its malignant activation is closely related to the occurrence and development of many cancers, especially colorectal cancer with adenomatous polyposis coli (APC) and CTNNB1 mutations. By applying a TCF/lymphoid‐enhancing factor (LEF) luciferase reporter system, the high‐throughput screening of 18 840 small‐molecule compounds was performed. A novel scaffold compound, C644‐0303, was identified as a Wnt/β‐catenin signaling inhibitor and exhibited antitumor efficacy. It inhibited both constitutive and ligand activated Wnt signals and its downstream gene expression. Functional studies showed that C644‐0303 causes cell cycle arrest, induces apoptosis, and inhibits cancer cell migration. Moreover, transcription factor array indicated that C644‐0303 could suppress various tumor‐promoting transcription factor activities in addition to Wnt/β‐catenin. Finally, C644‐0303 suppressed tumor spheroidization in a 3‐dimensional cell culture model and inhibited xenograft tumor growth in mice. In conclusion, we report a novel structural small molecular inhibitor targeting the Wnt/β‐catenin signaling pathway that has therapeutic potential for colorectal cancer treatment.

Bioinformatics analysis identified CDC20 as a potential drug target for cholangiocarcinoma.

BackgroundCholangiocarcinoma (CCA) is a malignancy that originates from bile duct cells. The incidence and mortality of CCA are very high especially in Southeast Asian countries. Moreover, most CCA patients have a very poor outcome. Presently, there are still no effective treatment regimens for CCA. The resistance to several standard chemotherapy drugs occurs frequently; thus, searching for a novel effective treatment for CCA is urgently needed.MethodsIn this study, comprehensive bioinformatics analyses for identification of novel target genes for CCA therapy based on three microarray gene expression profiles (GSE26566, GSE32225 and GSE76297) from the Gene Expression Omnibus (GEO) database were performed. Based on differentially expressed genes (DEGs), gene ontology and pathway enrichment analyses were performed. Protein-protein interactions (PPI) and hub gene identifications were analyzed using STRING and Cytoscape software. Then, the expression of candidate genes from bioinformatics analysis was measured in CCA cell lines using real time PCR. Finally, the anti-tumor activity of specific inhibitor against candidate genes were investigated in CCA cell lines cultured under 2-dimensional and 3-dimensional cell culture models.ResultsThe three microarray datasets exhibited an intersection consisting of 226 DEGs (124 up-regulated and 102 down-regulated genes) in CCA. DEGs were significantly enriched in cell cycle, hemostasis and metabolism pathways according to Reactome pathway analysis. In addition, 20 potential hub genes in CCA were identified using the protein-protein interaction (PPI) network and sub-PPI network analysis. Subsequently, CDC20 was identified as a potential novel targeted drug for CCA based on a drug prioritizing program. In addition, the anti-tumor activity of a potential CDC20 inhibitor, namely dinaciclib, was investigated in CCA cell lines. Dinaciclib demonstrated huge anti-tumor activity better than gemcitabine, the standard chemotherapeutic drug for CCA.ConclusionUsing integrated bioinformatics analysis, CDC20 was identified as a novel candidate therapeutic target for CCA.

Angiotensin receptor blocker Losartan inhibits tumor growth of colorectal cancer.

The renin-angiotensin system (RAS) is up-regulated in patients with colorectal cancer (CRC) and is reported to be associated with poor prognosis and chemo-resistance. Here we explored the therapeutic potential of targeting RAS in CRC using Losartan, an angiotensin receptor blocker. An integrative-systems biology approach was used to explore a proteome-level dataset of a gene signature that is modulated by Losartan. The anti-proliferative activity of Losartan was evaluated using 2- and 3-dimensional cell culture models. A xenograft model of colon cancer was used to investigate tumor growth with Losartan alone and in combination with 5-FU followed by histological staining (Hematoxylin & Eosin and Masson trichrome staining), biochemical analyses, gene expression analyses by RT-PCR, western blot/IHC, or MMP Gelatin Zymography studies. Effects on cell cycle and cell death were assessed by flow cytometry. Losartan inhibited cell growth and suppressed cell cycle progression, causing an increase in CRC cells in the G1 phase. Losartan significantly reduced tumor growth and enhanced tumor cell necrosis. An impact on the inflammatory response, including up-regulation of pro-inflammatory cytokines and chemokines in CRC cells are potential mechanisms that could partially explain Losartan's anti-proliferative effects. Moreover, metastasis and angiogenesis were reduced in Losartan-treated mice as observed by inhibited matrix metalloproteinase-2 and -9 activities and decreased tumor vasculature. These data demonstrate the therapeutic potential of combining chemotherapeutic regimens with Losartan to synergistically enhance its activity and target the renin-angiotensin system as a new approach in colorectal cancer treatment.

Cytokine Production and Cytotoxicity of Calcium Silicate–based Sealers in 2- and 3-dimensional Cell Culture Models

IntroductionThe aim of the present study was to assess the effects of different silicate-based sealers (ie, BioRoot RCS [Septodont, Saint Maur des Fosses, France], ProRoot ES [Dentsply Sirona, York, PA], and MTA Fillapex [Angelus, Londrina, PR, Brazil]) on cytokine production and viability of human periodontal ligament stem cells (PDLSCs). AH Plus (Dentsply DeTrey GmbH, Konstanz, Germany) was used as a reference material. MethodsPDLSCs were cultured either in 2-dimensional or 3-dimensional conditions (in 0.15%–0.5% PuraMatrix [BD Biosciences, Bedford, MA]) for 24 hours with eluates from set endodontic sealers. Additionally, the toxicity of eluates from endodontic sealers was evaluated using an in vitro root model experimental procedure. PDLSC viability was determined using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay. PDLSC culture medium was used for cytokine quantification (interleukin [IL]-6, IL-8, growth-regulated oncogene, IL,-4 and IL-10) using the HCYTMAG-60K-PX41 Milliplex kit (EMD Millipore, Burlington, MA). ResultsIn 2-dimensional culture conditions, BioRoot RCS revealed a good PDLSC viability rate. ProRoot ES had no effect on PDLSC viability regardless of the dilution. MTA Fillapex was strongly cytotoxic even at the lowest extract dilutions (1:1, 1:2, and 1:4). Encapsulation of PDLSCs in PuraMatrix tended to decrease the cytotoxic effect of the sealers. In the 3-dimensional in vitro root model experimental procedure, BioRoot RCS, ProRoot ES, and MTA Fillapex revealed a cytocompatibility pattern. Different calcium silicate–based sealers exhibited different proinflammatory cytokine production. BioRoot RCS greatly stimulated the release of IL-10 and, to a lesser degree, IL-4 by PDLSCs (P < .05). ConclusionsBioRoot RCS and ProRoot ES did not induce proinflammatory cytokines and promoted anti-inflammatory cytokine secretion by PDLSCs that may have a positive local impact by attenuating an initial inflammatory response.

Anti-PD-1 immunotherapy leads to tuberculosis reactivation via dysregulation of TNF-α.

Previously, we developed a 3-dimensional cell culture model of human tuberculosis (TB) and demonstrated its potential to interrogate the host-pathogen interaction (Tezera et al., 2017a). Here, we use the model to investigate mechanisms whereby immune checkpoint therapy for cancer paradoxically activates TB infection. In patients, PD-1 is expressed in Mycobacterium tuberculosis (Mtb)-infected lung tissue but is absent in areas of immunopathology. In the microsphere model, PD-1 ligands are up-regulated by infection, and the PD-1/PD-L1 axis is further induced by hypoxia. Inhibition of PD-1 signalling increases Mtb growth, and augments cytokine secretion. TNF-α is responsible for accelerated Mtb growth, and TNF-α neutralisation reverses augmented Mtb growth caused by anti-PD-1 treatment. In human TB, pulmonary TNF-α immunoreactivity is increased and circulating PD-1 expression negatively correlates with sputum TNF-α concentrations. Together, our findings demonstrate that PD-1 regulates the immune response in TB, and inhibition of PD-1 accelerates Mtb growth via excessive TNF-α secretion.

Moderate Heat Application Enhances the Efficacy of Nanosecond Pulse Stimulation for the Treatment of Squamous Cell Carcinoma

Nanosecond pulse stimulation as a tumor ablation therapy has been studied for the treatment of various carcinomas in animal models and has shown a significant survival benefit. In the current study, we found that moderate heating at 43°C for 2 minutes significantly enhanced in vitro nanosecond pulse stimulation-induced cell death of KLN205 murine squamous cell carcinoma cells by 2.43-fold at 600 V and by 2.32-fold at 900 V, as evidenced by propidium iodide uptake. Furthermore, the ablation zone in KLN205 cells placed in a 3-dimensional cell-culture model and pulsed at a voltage of 900 V at 43°C was 3 times larger than in cells exposed to nanosecond pulse stimulation at room temperature. Application of moderate heating alone did not cause cell death. A nanosecond pulse stimulation electrode with integrated controllable laser heating was developed to treat murine ectopic squamous cell carcinoma. With this innovative system, we were able to quickly heat and maintain the temperature of the target tumor at 43°C during nanosecond pulse stimulation. Nanosecond pulse stimulation with moderate heating was shown to significantly extend overall survival, delay tumor growth, and achieve a high rate of complete tumor regression. Moderate heating extended survival nearly 3-fold where median overall survival was 22 days for 9.8 kV without moderate heating and over 63 days for tumors pulsed with 600, 100 ns pulses at 5 Hz, at voltage of 9.8 kV with moderate heating. Median overall survival in the control groups was 24 and 31 days for mice with untreated tumors and tumors receiving moderate heat alone, respectively. Nearly 69% (11 of 16) of tumor-bearing mice treated with nanosecond pulse stimulation with moderate heating were tumor free at the completion of the study, whereas complete tumor regression was not observed in the control groups and in 9.8 kV without moderate heating. These results suggest moderate heating can reduce the necessary applied voltage for tumor ablation with nanosecond pulse stimulation.

Cellular Changes of Stem Cells in 3-Dimensional Culture

During various operations and procedures, such as distraction osteogenesis and orthodontics, skeletal tissues use mechanotransduction. Mechanotransduction is important for maintaining bone health and converting mechanical forces into biochemical signals. We hypothesized that cells put under mechanical stress would adapt and change morphologically and respond with a decrease in cellular proliferation to accommodate the stress differences. These differences will be measured at the molecular and genetic level. We also wanted to test the practicality of an invitro 3-dimensional gel model system. We implemented a 3-dimensional cell culture model. The sample was composed of isolated mouse mesenchymal prefibroblast bone marrow cells from the femurs and tibias of 6- to 8-week-old wild-type C57BL6 mice. The cells were seeded on fibronectin-coated hydrogels along with fibrin and nodulin growth factors. The variables tested were a no-force model (control) and a force model. The force model required two 0.1-mm suture pins put through one 0.25-cm length of cell-gel matrix. After the experiments were run to completion, the samples were fixed with 4% paraformaldehyde and embedded in paraffin. Serial sections were cut at a thickness of 5μm along the long axis for the force construct and encompassing the entire circular area of the control construct. Descriptive and bivariate statistics were computed, and the P value was set at 5%. There was a statistically significant difference between the 2 models. The force model had longer and straighter primary cilia, less apoptosis, and an increase in cell proliferation. In addition, the shape of the cells was markedly different after the experiment. The results of the study suggest cells put under tensile stress have the ability to mechanically sense the environment to provide improved adaptation. Our work also confirms the usefulness of the invitro 3-dimensional gel model system to mimic invivo applications.

Nutrient restriction of glucose or serum results in similar proteomic expression changes in 3D colon cancer cell cultures

Nutrient restriction, also known as caloric restriction, has been extensively examined for its positive impact on lifespan, immune system boost, and aging. In addition, nutrient restriction is implicated in decreasing cancer initiation and progression. Given the phenotypic changes associated with nutrient restriction, we hypothesized significant protein expression alterations must be associated with caloric restriction. To compare the molecular and phenotypic changes caused by glucose restriction and fetal bovine serum restriction there is need for an efficient model system. We establish 3-dimensional cell culture models, known as spheroids, in the HCT 116 colorectal cancer cell line as a high throughput model for studying the proteomic changes associated with nutrient restriction. Flow cytometry was used to assess apoptosis and autophagy levels in the spheroids under nutrient restriction. Isobaric tags for relative and absolute quantification and liquid chromatography tandem mass spectrometry were used to determine differential protein abundances between the nutrient restriction conditions. We identified specific proteins that have implications in cancer progression and metastasis that are differentially regulated by restriction of either glucose or serum. These proteins include the up-regulation of sirtuin 1 and protein inhibitor of activated STAT 1 and down-regulation of multi-drug resistance protein and Zinc finger and BTB domain-containing protein 7A. The results indicate nutrient restriction causes lower apoptotic and higher autophagy rates in HCT 116 spheroids. In addition, proteins shown to be differentially regulated by both glucose and serum restriction were similarly regulated.

Transcriptional profiling of radiation damage and preventive treatments in a 3-dimensional (3D) human cell culture model of oral mucositis.

Cancer patients who receive radiation are often afflicted by oral mucositis, a debilitating disease, characterized by mouth sores and difficulty in swallowing. Oftentimes, cancer patients afflicted with mucositis must stop life-saving therapies. Thus it is very important to prevent mucositis before it develops. Using a validated organotypic model of human oral mucosa, a 3-dimensional cell culture model of human oral keratinocytes, it has been shown that a mixture (NAC–QYD) of N-acetyl cysteine (NAC) and a traditional Chinese medicine, Qingre Liyan decoction (QYD), prevented radiation damage (Lambros et al., 2014). Here we provide detailed methods and analysis of microarray data for non-irradiated and irradiated human oral mucosal tissue with and without pretreatment with NAC, QYD and NAC-QYD. The microarray data been deposited in Gene Expression Omnibus (GEO): GSE62397. These data can be used to further elucidate the mechanisms of irradiation damage in oral mucosa and its prevention.

The Extracellular Matrix Regulates Granuloma Necrosis in Tuberculosis

A central tenet of tuberculosis pathogenesis is that caseous necrosis leads to extracellular matrix destruction and bacterial transmission. We reconsider the underlying mechanism of tuberculosis pathology and demonstrate that collagen destruction may be a critical initial event, causing caseous necrosis as opposed to resulting from it. In human tuberculosis granulomas, regions of extracellular matrix destruction map to areas of caseous necrosis. In mice, transgenic expression of human matrix metalloproteinase 1 causes caseous necrosis, the pathological hallmark of human tuberculosis. Collagen destruction is the principal pathological difference between humanised mice and wild-type mice with tuberculosis, whereas the release of proinflammatory cytokines does not differ, demonstrating that collagen breakdown may lead to cell death and caseation. To investigate this hypothesis, we developed a 3-dimensional cell culture model of tuberculosis granuloma formation, using bioelectrospray technology. Collagen improved survival of Mycobacterium tuberculosis-infected cells analyzed on the basis of a lactate dehydrogenase release assay, propidium iodide staining, and measurement of the total number of viable cells. Taken together, these findings suggest that collagen destruction is an initial event in tuberculosis immunopathology, leading to caseous necrosis and compromising the immune response, revealing a previously unappreciated role for the extracellular matrix in regulating the host-pathogen interaction.

Molecular signatures in the prevention of radiation damage by the synergistic effect of N-acetyl cysteine and qingre liyan decoction, a traditional chinese medicine, using a 3-dimensional cell culture model of oral mucositis.

Qingre Liyan decoction (QYD), a Traditional Chinese medicine, and N-acetyl cysteine (NAC) have been used to prevent radiation induced mucositis. This work evaluates the protective mechanisms of QYD, NAC, and their combination (NAC-QYD) at the cellular and transcriptional level. A validated organotypic model of oral mucosal consisting of a three-dimensional (3D) cell tissue-culture of primary human keratinocytes exposed to X-ray irradiation was used. Six hours after the irradiation, the tissues were evaluated by hematoxylin and eosin (H and E) and a TUNEL assay to assess histopathology and apoptosis, respectively. Total RNA was extracted and used for microarray gene expression profiling. The tissue-cultures treated with NAC-QYD preserved their integrity and showed no apoptosis. Microarray results revealed that the NAC-QYD caused the upregulation of genes encoding metallothioneins, HMOX1, and other components of the Nrf2 pathway, which protects against oxidative stress. DNA repair genes (XCP, GADD45G, RAD9, and XRCC1), protective genes (EGFR and PPARD), and genes of the NFκB pathway were upregulated. Finally, tissue-cultures treated prophylactically with NAC-QYD showed significant downregulation of apoptosis, cytokines and chemokines genes, and constrained damage-associated molecular patterns (DAMPs). NAC-QYD treatment involves the protective effect of Nrf2, NFκB, and DNA repair factors.

The importance of being a lumen.

Advances in tissue engineering and microtechnology have enabled researchers to more easily generate in vitro tissue models that mimic the tissue geometry and spatial organization found in vivo (e.g., vessel or mammary duct models with tubular structures). However, the widespread adoption of these models for biological studies has been slow, in part due to the lack of direct comparisons between existing 2-dimensional and 3-dimensional cell culture models and new organotypic models that better replicate tissue structure. Using previously developed vessel and mammary duct models with 3-dimensional lumen structures, we have begun to explore this question. In a direct comparison between these next generation organotypic models and more traditional methods, we observed differences in the levels of several secreted growth factors and cytokines. In addition, endothelial vessel geometry profoundly affects the phenotypic behavior of carcinoma cells, suggesting that more traditional in vitro assays may not capture in vivo events. Here, we seek to review and add to the increasing evidence supporting the hypothesis that using cell culture models with more relevant tissue structure influences cell fate and behavior, potentially increasing the relevance of biological findings.

Microcavity substrates casted from self-assembled microsphere monolayers for spheroid cell culture.

Multicellular spheroids are an important 3-dimensional cell culture model that reflects many key aspects of in vivo microenvironments. This paper presents a scalable, self-assembly based approach for fabricating microcavity substrates for multicellular spheroid cell culture. Hydrophobic glass microbeads were self-assembled into a tightly packed monolayer through the combined actions of surface tension, gravity, and lateral capillary forces at the water-air interface of a polymer solution. The packed bead monolayer was subsequently embedded in the dried polymer layer. The surface was used as a template for replicating microcavity substrates with perfect spherical shapes. We demonstrated the use of the substrate in monitoring the formation process of tumor spheroids, a proof-of-concept scale-up fabrication procedure into standard microplate formats, and its application in testing cancer drug responses in the context of bone marrow stromal cells. The presented technique offers a simple and effective way of forming high-density uniformly-sized spheroids without microfabrication equipment for biological and drug screening applications.

Visualising the hypoxia selectivity of cobalt(iii) prodrugs

Hypoxic cancer cells have a more aggressive phenotype than cells in a normoxic environment and are often refractory to current chemotherapy protocols due to an altered proliferation rate. They also represent a means of targeting cancer chemotherapy. We have developed a 3-dimensional cell culture model that readily identifies live hypoxic cells by expression of the Eos photo-convertible green fluorescent protein. Using this model, we have examined the diffusion and hypoxia selectivity of two model Co(III) based compounds. The Co(III) compounds have been designed to target hypoxic cells within a tumour and have fluorescent axial ligands. The fluorescent ligands function as model cytotoxins and their fluorescence is quenched on binding to Co(III). The ligands are released upon the reduction of Co(III) to Co(II) and the recovery of fluorescence enables the detection of the released ligands by confocal microscopy. Despite similarities in electrochemical potential and cytotoxicity, the penetration of the compounds into spheroids and uptake into hypoxic cells was found to differ depending on the properties of the released ligand.

Matrix Rigidity Regulates Cancer Cell Growth and Cellular Phenotype

BackgroundThe mechanical properties of the extracellular matrix have an important role in cell growth and differentiation. However, it is unclear as to what extent cancer cells respond to changes in the mechanical properties (rigidity/stiffness) of the microenvironment and how this response varies among cancer cell lines.Methodology/Principal FindingsIn this study we used a recently developed 96-well plate system that arrays extracellular matrix-conjugated polyacrylamide gels that increase in stiffness by at least 50-fold across the plate. This plate was used to determine how changes in the rigidity of the extracellular matrix modulate the biological properties of tumor cells. The cell lines tested fall into one of two categories based on their proliferation on substrates of differing stiffness: “rigidity dependent” (those which show an increase in cell growth as extracellular rigidity is increased), and “rigidity independent” (those which grow equally on both soft and stiff substrates). Cells which grew poorly on soft gels also showed decreased spreading and migration under these conditions. More importantly, seeding the cell lines into the lungs of nude mice revealed that the ability of cells to grow on soft gels in vitro correlated with their ability to grow in a soft tissue environment in vivo. The lung carcinoma line A549 responded to culture on soft gels by expressing the differentiated epithelial marker E-cadherin and decreasing the expression of the mesenchymal transcription factor Slug.Conclusions/SignificanceThese observations suggest that the mechanical properties of the matrix environment play a significant role in regulating the proliferation and the morphological properties of cancer cells. Further, the multiwell format of the soft-plate assay is a useful and effective adjunct to established 3-dimensional cell culture models.

Comparison of two different methods for rat adrenocortical cells culture

Objective To investigate a novel 3-dimensional cell culture model in collagen gel, and its influence on morphology and function of adrenocortical cells. Methods Adrenocortical cells of ne-onate rats were isolated,and then cultured in self-made collagen type Ⅰ gel for 14 days. This 3-dimension-al cell culture model was compared with the classic plane culture model on the morphology, cell growth rate,protein expression and secretory functions. Results Adrenocortical cells expressed specific cyto-chrome P450 :CYP11 B1 and CYP11 B2 under both culture models. At the 8th day after primary culture,the growth rate of adrenocortical cells in 3-dimensional cell culture model was higher than the classic plane culture model significantly. At the 14th day, the growth rate in 3-dimensional cell culture model and the classic plane culture model was 16.6% and 2.9% respectively. The secretory functions of adrenocortical cells were more sustainable in 3-dimensional collagen gel. At the 14th day, corticosterone and aldosterone levels in the medium of 3-dimensional cell culture model were (278.33 ± 38.20) μg/L and (133.85 ±38.20) ng/L,while those were (160.35±28.12) μg/L and (74.03 ± 12.12) ng/L in the medium of plane euhure model, with the differences being statistically significant (P ＜ 0.05). Conclusion Three-dimensional culture in collagen gel is favorable for the maintenance of adrenocortical ceils functions. Key words: Collagen Type Ⅰ ; Adrenocortical cells; Culture

Design of a Microscopy System for Quantitative Spatial and Temporal Analysis of Multicellular Interactions

Abstract The challenge of the post-genomic era is functional genomics, i.e. understanding how the genome is expressed to produce myriad cell phenotypes. A phenotype is the result of selective expression of the genome in response to the microenvironment. to use genomic information to understand the biology of complex organisms, the biological responses and signaling pathways in cells need to be studied in context, i.e. within the proper tissue structure. Nonetheless, most current biology is conducted using cells cultured in monolayers on traditional tissue culture plastic. These non-physiological models impede the ability to predict in vivo responses from model systems. The same cells cultured in 2-dimensions (i.e. monolayers) vs. 3-dimensions (e.g. multicellular tumor spheroids) differ in their responses to external stimuli such as ionizing radiation, viral infection, cytotoxic drugs, and chemotherapeutic agents. Our laboratory has led the way in promoting and developing 3-dimensional cell culture models that more accurately reflect in vivo biology, beginning with the establishment 15 years ago of physiologically functional reconstituted mammary acini in culture. Quantitation of spatial and temporal concurrent behavior of multiple markers in these 3-dimensional cell cultures is hampered by the currently routine mode of sequential image acquisition, measurement and analysis of specific targets. This precludes the detailed analysis of multi-dimensional, time sequence responses and fails to relate features in novel and meaningful ways that will further our understanding of basic biology. Thus new methodology was needed for high-throughput, dynamic evaluations of large numbers of live multicellular specimens. Rather than using confocal microscopy methods, which interfere with live cell systems due to photo-damage, optical sectioning of the 3-dimensional structures is achieved with structured light illumination using the Wilson grating in an implementation described by Lanni.