Conventional Monolayer Culture Research Articles

Towards a more representative in vitro method for fish ecotoxicology: morphological and biochemical characterisation of three-dimensional spheroidal hepatocytes

The use of fish primary cells and cell lines offer an in vitro alternative for assessment of chemical toxicity and the evaluation of environmental samples in ecotoxicology. However, their uses are not without limitations such as short culture periods and loss of functionality, particularly with primary tissue. While three-dimensional (spheroid) technology is now established for in vitro mammalian toxicity studies, to date it has not been considered for environmental applications in a model aquatic species. In this study we report development of a reproducible six-well plate, gyratory-mediated method for rainbow trout (Oncorhynchus mykiss) hepatocyte spheroid culture and compare their functional and biochemical status with two-dimensional (2D) monolayer hepatocytes. Primary liver spheroid formation was divided into two stages, immature (1-5 days) and mature (≥6 days) according to size, shape and changes in functional and biochemical parameters (protein, glucose, albumin and lactate dehydrogenase). Mature spheroids retained the morphological characteristics (smooth outer surface, tight cell-cell contacts) previously described for mammalian spheroids as demonstrated by light and scanning electron microscopy. Glucose production and albumin synthesis were significantly higher in mature spheroids when compared to conventional 2D monolayer cultures (P < 0.01) and increased as spheroids matured (P < 0.01). Basal lactate dehydrogenase (LDH) leakage significantly decreased during spheroid formation and was significantly lower than 2D cultures (P < 0.01). It is therefore suggested that mature spheroids can maintain a high degree of functional, biochemical and morphological status over-time in culture that is superior to conventional 2D models and can provide realistic organotypic responses in vitro. Trout spheroids that take ~6-8 days to reach maturity would be suitable for use in acute toxicological tests and since it is possible to culture individual spheroids for over a month, there is potential for this work to lead towards in vitro bioaccumulation alternatives and to conduct high throughput screens of chronic exposure. This is an important step forward for developing alternative in vitro tools in future fish ecotoxicological studies.

Ex vivo mimicry of normal and abnormal human hematopoiesis.

Hematopoietic stem cells require a unique microenvironment in order to sustain blood cell formation; the bone marrow (BM) is a complex three-dimensional (3D) tissue wherein hematopoiesis is regulated by spatially organized cellular microenvironments termed niches. The organization of the BM niches is critical for the function or dysfunction of normal or malignant BM(5). Therefore a better understanding of the in vivo microenvironment using an ex vivo mimicry would help us elucidate the molecular, cellular and microenvironmental determinants of leukemogenesis. Currently, hematopoietic cells are cultured in vitro in two-dimensional (2D) tissue culture flasks/well-plates requiring either co-culture with allogenic or xenogenic stromal cells or addition of exogenous cytokines. These conditions are artificial and differ from the in vivo microenvironment in that they lack the 3D cellular niches and expose the cells to abnormally high cytokine concentrations which can result in differentiation and loss of pluripotency. Herein, we present a novel 3D bone marrow culture system that simulates the in vivo 3D growth environment and supports multilineage hematopoiesis in the absence of exogenous growth factors. The highly porous scaffold used in this system made of polyurethane (PU), facilitates high-density cell growth across a higher specific surface area than the conventional monolayer culture in 2D. Our work has indicated that this model supported the growth of human cord blood (CB) mononuclear cells (MNC) and primary leukemic cells in the absence of exogenous cytokines. This novel 3D mimicry provides a viable platform for the development of a human experimental model to study hematopoiesis and to explore novel treatments for leukemia.

Engineering fibrotic tissue in pancreatic cancer: A novel three-dimensional model to investigate nanoparticle delivery

Pancreatic cancer contains both fibrotic tissue and tumor cells with embedded vasculature. Therefore anti-cancer nanoparticles need to extravasate from tumor vasculature and permeate thick fibrotic tissue to target tumor cells. To date, permeation of drugs has been investigated in vitro using monolayer models. Since three-dimensional migration of nanoparticles cannot be analyzed in a monolayer model, we established a novel, three-dimensional, multilayered, in vitro model of tumor fibrotic tissue, using our hierarchical cell manipulation technique with K643f fibroblasts derived from a murine pancreatic tumor model. NIH3T3 normal fibroblasts were used in comparison. We analyzed the size-dependent effect of nanoparticles on permeation in this experimental model using fluorescent dextran molecules of different molecular weights. The system revealed permeation decreased as number of layers of cultured cells increased, or as molecule size increased. Furthermore, we showed changes in permeation depended on the source of the fibroblasts. Observations of this sort cannot be made in conventional monolayer culture systems. Thus our novel technique provides a promising in vitro means to investigate permeation of nanoparticles in fibrotic tissue, when both type and number of fibroblasts can be regulated.

Application of floating cells for improved harvest in human chondrocyte culture

Cell culture medium, which must be discarded during medium change, may contain many cells that do not attach to culture plates. In the present study, we focused on these floating cells and attempted to determine their usefulness for cartilage regeneration. We counted the number of floating cells discarded during medium change and compared the proliferation and differentiation between floating cells and their adherent counterparts. Chondrocyte monolayer culture at a density of 5 × 103 cells/cm(2) produced viable floating cells at a rate of 2.7-3.2 × 10(3) cells/cm(2) per primary culture. When only the floating cells from one dish were harvested and replated in another dish, the number of cells was 2.8 × 10(4) cells/cm(2) (approximately half confluency) on culture day 7. The number of cells was half of that obtained by culturing only adherent cells (5 × 10(4) cells/cm(2)). The floating and adherent cells showed similar proliferation and differentiation properties. The recovery of floating cells from the culture medium could provide an approximately 1.5-fold increase in cell number over conventional monolayer culture. Thus, the collection of floating cells may be regarded as a simple, easy, and reliable method to increase the cell harvest for chondrocytes.

The phosphoinositide 3-kinase/mammalian target of rapamycin inhibitor NVP-BEZ235 is effective in inhibiting regrowth of tumour cells after cytotoxic therapy

PurposeRegrowth of tumour cells between cycles of chemotherapy is a significant clinical problem. Treatment strategies where antiproliferative agents are used to inhibit tumour regrowth between chemotherapy cycles are attractive, but such strategies are difficult to test using conventional monolayer culture systems. MethodsWe used the in vitro tumour spheroid model to study regrowth of 3-D colon carcinoma tissue after cytotoxic therapy. Colon carcinoma cells with wild-type or mutant phosphatidyl inositol 3-kinase catalytic subunit (PI3KCA) or KRAS alleles were allowed to form multicellular spheroids and the effects of different pharmacological compounds were studied after sectioning and staining for relevant markers of cell proliferation and apoptosis. ResultsStudies using colon cancer cells with gene disruptions suggested that the phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) pathway was essential for proliferation in 3-D culture. The dual PI3K-mTOR inhibitor NVP-BEZ235, currently in clinical trials, was found to inhibit phosphorylation of the mTOR target 4EBP1 in 3-D cultured cells. The ability of NVP-BEZ235 to inhibit tumour cell proliferation and to induce apoptosis was markedly more pronounced in 3-D cultures compared to monolayer cultures. It was subsequently found that NVP-BEZ235 was effective in inhibiting regrowth of 3-D cultured cells after treatment with two cytotoxic inhibitors of the ubiquitin–proteasome system (UPS), methyl-13-hydroxy-15-oxokaurenoate (MHOK) and bortezomib (Velcade®). ConclusionsThe dual PI3K-mTOR inhibitor NVP-BEZ235 was found to reduce cell proliferation and to induce apoptosis in 3-D cultured colon carcinoma cells, NVP-BEZ235 is a promising candidate for use in sequential treatment modalities together with cytotoxic drugs to reduce the cell mass of solid tumours.

Abstract A249: BI 853520, a potent and highly selective inhibitor of protein tyrosine kinase 2 (focal adhesion kinase), shows efficacy in multiple xenograft models of human cancer.

Abstract PTK2/FAK is a widely expressed non-receptor tyrosine kinase located mainly at focal adhesions. Sensing upstream signals from growth factor receptors as well as integrins, the enzyme contributes to activation of multiple downstream signaling pathways involved in the regulation of cell survival, proliferation and motility. PTK2 protein expression is elevated in many human cancers, and dysregulated PTK2 is known to mediate anchorage-independent growth of malignant cells. Inhibition of PTK2 activity thus may interfere with tumor growth and metastasis formation. BI 853520 inhibited the enzymatic activity of PTK2 at low nanomolar concentrations. In a selectivity screen of 264 tyrosine and serine/threonine kinases, BI 853520 inhibited only 4 enzymes at a concentration of 1,000 nM by more than 50%. In contrast to PTK2 inhibitors described previously, the compound showed more than 1,000-fold selectivity against closely related kinases such as PYK2/PTK2B. In human PC-3 prostate carcinoma cells, PTK2 is highly expressed and phosphorylated on Tyr397. Treatment of PC-3 cells with BI 853520 inhibited autophosphorylation with an EC50 of 1 nM. Colony formation of PC-3 cells in a soft agar matrix was inhibited with an EC50 of 3 nM. In contrast, the same cell line growing in conventional monolayer culture showed growth inhibition only at 1,000-fold higher concentration. Similar results were obtained for other carcinoma cell lines. BI 853520 administered to immunodeficient nude mice showed good oral bioavailability (F = 90%) and a half-life of 5 hours. Daily oral treatment at dose levels up to 100 mg/kg over a period of several weeks was well tolerated by the animals. Treatment with BI 853520 resulted in suppression of PTK2 autophosphorylation in tissue samples from both human tumor xenografts and mouse skin. BI 853520 was tested for efficacy in a panel of 18 human tumor xenograft models representing multiple indications, including carcinomas of the lung, ovary, pancreas and prostate as well as sarcomas. At daily oral doses of 50 mg/kg the compound was highly active in a subset of models, including the PC-3 prostate carcinoma, resulting in strong suppression of tumor growth (TGI &amp;gt; 90% compared to controls) or tumor regression in individual animals. Significant inhibition of tumor growth was observed at doses as low as 6 mg/kg, indicating a substantial therapeutic window and opportunities for combination therapy. Another subset of models showed intermediate drug sensitivity (TGI 40 − 80%), whereas several models were resistant to treatment (non-significant TGI). This database is currently being explored to identify biomarkers predicting sensitivity to BI 853520 based on genetic aberrations, gene expression signatures and phosphoproteome analysis of xenograft tumors. In conclusion, BI 853520 is a novel, highly selective PTK2 kinase inhibitor that demonstrates anti-tumor activity in vitro and in vivo. Clinical development of BI 853520 has recently been initiated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A249.

Microstructuring of multiwell plates for three-dimensional cell culture applications by ultrasonic embossing

Since three-dimensional (3D) cell culture models better reflect tissues in vivo in terms of cell shape and microenvironment compared to conventional monolayer cultures, 3D tissue culture substrates gain more importance for a wide range of biological applications like drug discovery, toxicological studies, cancer and stem cell research. In this study we developed a method for the fabrication of 3D cell culture substrates in a multiwell plate format by microstructuring the bottom of 96-well cell culture plates using an ultrasonic embossing process. The resulting microstructured area consists of cubic microcavities in which adherent multicellular aggregates can be formed. We performed the biological evaluation of the system with the liver-derived human cell-line HepG2 and compared the novel substrate with a commercially available 3D culture system comprising porous alginate sponges. Metabolic activity (alamarBlue® reduction) and induction of four biotransformation enzymes (EROD, ECOD, UGT, SULT) were determined by fluorimetry or HPLC. Our results revealed that HepG2 cells in microstructured plates showed a higher mitochondrial activity, as well as enzyme activity of ECOD and UGT after treatment with an inducer when compared to cells cultured in alginate sponges at otherwise comparable conditions. Since we have modified standard cell culture plates, the obtained system is adaptable to automated screening and might be useful for all kinds of cultures including adult, progenitor and stem cells which need a 3D culture configuration to restore or maintain the differentiated status.

Three-Dimensional Cell Grafting Enhances the Angiogenic Efficacy of Human Umbilical Vein Endothelial Cells

Despite the great potential of cell therapy for ischemic disease, poor cell survival after engraftment in ischemic tissue limits its efficacy. Here we tested a hypothesis that three-dimensionally grafted human umbilical vein endothelial cell (HUVEC) spheroids would exhibit improved angiogenic efficacy following transplantation into mouse ischemic limbs compared with HUVECs prepared by conventional two-dimensional monolayer culture. One day after surgical induction of hindlimb ischemia in athymic mice, HUVECs cultured in monolayer or HUVEC spheroids were transplanted intramuscularly into ischemic limbs. Four weeks after the treatment, in the spheroid HUVEC transplantation group, we observed increased hypoxia-inducible factor-1α expression, decreased apoptosis, and increased HUVEC survival in the ischemic tissue compared with the monolayer HUVEC transplantation group. Transplantation of HUVEC spheroids also resulted in enhanced and prolonged secretion of paracrine factors as well as enhanced expression of factors involved in the recruitment of circulating angiogenic progenitor cells. In summary, transplantation of HUVECs as spheroids enhanced cell survival, increased paracrine factor secretion, and showed a potential as a therapeutic method to treat ischemic tissue damages by promoting angiogenesis.

Differential gene expression following ionizing radiation in multicellular spheroid depending on p53 status: identification of potential targets and prediction of responsive signaling pathways

Ionizing radiation (IR) therapy is a potential treatment option of several solid tumors; however, the molecular responses of carcinoma cells to IR are not yet clarified. A multicellular spheroid (MCS)-in vivo mimic model was established in which allow us to in situ study of cellular response to IR in human carcinoma cells with different intrinsic p53 status. Here, relative cell survival rate was determined in p53-wild and-null type cells of MCS in comparison to that of monolayer culture in response to 10 Gy IR by trypan blue exclusion assay. It obviously showed that under MCS culture system radioresistance phenotype was dependent on p53 status since p53-null type MCS exhibited significantly higher cell survival rate, in contrast to p53-wild type MCS. In order to screen molecular targets in p53-proficient cancer cells upon IR exposure, we conducted microarray under 10 Gy IR to observe gene expression pattern change in MCS compared to monolayer cells, with and without p53. In addition, potential molecular network was analyzed using Pathway Studio software to define responsive signaling and interactions. Total 478 genes were notably altered at transcript level toward the IR treatment. Discovered genes were participated in several cellular major processes, including apoptosis (both caspase-dependent and-independent pathways), cell migration and proliferation. Among them ANXA11, C2, KCNE2, KIF3C, MSH5, and OSCAR were differentially expressed in p53-proficient MCS but constant in p53-deficient MCS. These genes might be considered as target molecules for evaluation of IR efficiency in p53-proficient MCS as a typical 3D-in vivo mimic model. Our findings demonstrate complex responses of p53-carcinoma cells following IR exposure including putative signaling pathways, leading to emphasis on the importance of in vivo mimic MCS model rather than conventional monolayer culture system.

Scaffold-free culture of mesenchymal stem cell spheroids in suspension preserves multilineage potential

While traditional cell culture methods have relied on growing cells as monolayers, three-dimensional (3D) culture systems can provide a convenient in vitro model for the study of complex cell-cell and cell-matrix interactions in the absence of exogenous substrates and may benefit the development of regenerative medicine strategies. In this study, mesenchymal stem cell (MSC) spheroids, or "mesenspheres", of different sizes, were formed using a forced aggregation technique and maintained in suspension culture for extended periods of time thereafter. Cell proliferation and differentiation potential within mesenspheres and dissociated cells retrieved from spheroids were compared to conventional adherent monolayer cultures. Mesenspheres maintained in growth medium exhibited no evidence of cell necrosis or differentiation, while mesenspheres in differentiation media exhibited differentiation similar to conventional 2D culture methods based on histological markers of osteogenic and adipogenic commitment. Furthermore, when plated onto tissue culture plates, cells that had been cultured within mesenspheres in growth medium recovered morphology typical of cells cultured continuously in adherent monolayers and retained their capacity for multi-lineage differentiation potential. In fact, more robust matrix mineralization and lipid vacuole content were evident in recovered MSCs when compared to monolayers, suggesting enhanced differentiation by cells cultured as 3D spheroids. Thus, this study demonstrates the development of a 3D culture system for mesenchymal stem cells that may circumvent limitations associated with conventional monolayer cultures and enhance the differentiation potential of multipotent cells.

Hepatocyte spheroid arrays inside microwells connected with microchannels

Spheroid culture is a preferable cell culture approach for some cell types, including hepatocytes, as this type of culture often allows maintenance of organ-specific functions. In this study, we describe a spheroid microarray chip (SM chip) that allows stable immobilization of hepatocyte spheroids in microwells and that can be used to evaluate drug metabolism with high efficiency. The SM chip consists of 300-μm-diameter cylindrical wells with chemically modified bottom faces that form a 100-μm-diameter cell adhesion region surrounded by a nonadhesion region. Primary hepatocytes seeded onto this chip spontaneously formed spheroids of uniform diameter on the cell adhesion region in each microwell and these could be used for cytochrome P-450 fluorescence assays. A row of microwells could also be connected to a microchannel for simultaneous detection of different cytochrome P-450 enzyme activities on a single chip. The miniaturized features of this SM chip reduce the numbers of cells and the amounts of reagents required for assays. The detection of four cytochrome P-450 enzyme activities was demonstrated following induction by 3-methylcholantlene, with a sensitivity significantly higher than that in conventional monolayer culture. This microfabricated chip could therefore serve as a novel culture platform for various cell-based assays, including those used in drug screening, basic biological studies, and tissue engineering applications.

Expansion and preservation of multipotentiality of rabbit bone-marrow derived mesenchymal stem cells in dextran-based microcarrier spin culture

The use of mesenchymal stem cells (MSCs) in tissue repair and regeneration despite their multipotentiality has been limited by their cell source quantity and decelerating proliferative yield efficiency. A study was thus undertaken to determine the feasibility of using microcarrier beads in spinner flask cultures for MSCs expansion and compared to that of conventional monolayer cultures and static microcarrier cultures. Isolation and characterization of bone marrow derived MSCs were conducted from six adult New Zealand white rabbits. Analysis of cell morphology on microcarriers and culture plates at different time points (D0, D3, D10, D14) during cell culture were performed using scanning electron microscopy and bright field microscopy. Cell proliferation rates and cell number were measured over a period of 14 days, respectively followed by post-expansion characterization. MTT proliferation assay demonstrated a 3.20 fold increase in cell proliferation rates in MSCs cultured on microcarriers in spinner flask as compared to monolayer cultures (p < 0.05). Cell counts at day 14 were higher in those seeded on stirred microcarrier cultures (6.24 ± 0.0420 cells/ml) × 10(5) as compared to monolayer cultures (0.22 ± 0.004 cells/ml) × 10(5) and static microcarrier cultures (0.20 ± 0.002 cells/ml) × 10(5). Scanning electron microscopy demonstrated an increase in cell colonization of the cells on the microcarriers in stirred cultures. Bead-expanded MSCs were successfully differentiated into osteogenic and chondrogenic lineages. This system offers an improved and efficient alternative for culturing MSCs with preservation to their phenotype and multipotentiality.

Microcarrier bioreactor culture system promotes propagation of human intervertebral disc cells

Cell-based tissue engineering has emerged as a potential therapy for intervertebral disc degeneration. However, propagating and maintaining high quantity and quality of the seed cells remains a challenge. To investigate the feasibility of culturing human disc cells using a microcarrier bioreactor system. Cell counts, growth patterns, cell cycles and cellular viability were examined during the course of cell cultivation and compared between the microcarrier bioreactor culture system and the conventional monolayer culture. Cultures in the microcarrier bioreactor resulted in enhanced disc cell growth and satisfactory cell viability in comparison with the conventional monolayer culture. The cells in the microcarrier bioreactor cultivation exhibited higher S phase ratios, elevated mitotic index and persistent exponential growth. The microcarrier bioreactor culture system appears suitable for human disc cell propagation and may provide considerably more seeding cells for the tissue engineering process of intervertebral discs.

Role of Amblyomin-X on microcirculatory vessels, endothelial cell migration and tumor growth

Role of Amblyomin-X on microcirculatory vessels, endothelial cell migration and tumor growth

Co-culture of primary rat hepatocytes with rat liver epithelial cells enhances interleukin-6-induced acute-phase protein response.

Three different primary rat hepatocyte culture methods were compared for their ability to allow the secretion of fibrinogen and albumin under basal and IL-6-stimulated conditions. These culture methods comprised the co-culture of hepatocytes with rat liver epithelial cells (CC-RLEC), a collagen type I sandwich culture (SW) and a conventional primary hepatocyte monolayer culture (ML). Basal albumin secretion was most stable over time in SW. Fibrinogen secretion was induced by IL-6 in all cell culture models. Compared with ML, CC-RLEC showed an almost three-fold higher fibrinogen secretion under both control and IL-6-stimulated conditions. Induction of fibrinogen release by IL-6 was lowest in SW. Albumin secretion was decreased after IL-6 stimulation in both ML and CC-RLEC. Thus, cells growing under the various primary hepatocyte cell culture techniques react differently to IL-6 stimulation with regard to acute-phase protein secretion. CC-RLEC is the preferred method for studying cytokine-mediated induction of acute-phase proteins, because of the pronounced stimulation of fibrinogen secretion upon IL-6 exposure under these conditions.

Abstract LB-167: Identification of ionizing radiation (IR)-responsive mRNA in p53-deficient human non-small cell lung cancer (NSCLC) grown in three-dimensional cell culture system

Abstract Non-small cell lung cancer (NSCLC) has been known as the most common human malignancy. The p53 tumor suppressor gene is mutated in approximately 50 % of NSCLC. For treatment of lung cancer, radiotherapy using -ray is general treatment. However, many patients suffer from recurred disease. Therefore, the definite mechanism of radiation based on in vivo mimic system is required for effective remedical value to NSCLC. Most of radiation biology studies were performed in conventional monolayer culture system which can not represent in vivo tumor environment. Hence, study applying in vivo mimic three-dimensional culture system is necessary to elucidate the molecular mechanism of solid tumor. Here, we analyzed gene expression profile induced by ionizing radiation (IR) in H1299 cells grown as spheroid in three-dimensional cell culture system. Our data showed that different gene expression profiles among monolayer culture system, three-dimensional small spheroid and three-dimensional large spheroid system. Particularly, genes related with microenvironment including adhesion junction and hypoxia had different expression level in three-dimensional spheroid system compare with monolayer culture system. This study suggested that in vivo mimic three-dimensional cell culture engineering approach might provide critical clues for understanding of cellular responses in p53 mutated or deleted lung cancer cells against IR. (* This work was supported by Nuclear Research Development Program of the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korean government (MEST). (Grand code: 2007-2001431, 2008-2001694, 2009-0078295) from Korea Science and Engineering Foundation) Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 LB-167.

Alterations in the content and physiological role of sphingomyelin in plasma membranes of cells cultured in three-dimensional matrix

The three-dimensional (3D) cell culture approach offers a means to study cells under conditions that mimic an in vivo environment, thus avoiding the limitations imposed by the conventional two-dimensional (2D) monolayer cell cultures. By using this approach we demonstrated significant differences in the plasma membrane phospholipid composition and susceptibility to oxidation in cells cultured in three-dimensional environment compared to conventional monolayer cultures. The plasma membrane sphingomyelin (SM), which is a functionally active membrane phospholipid, was markedly increased in plasma membranes of 3D cells. To analyze the mechanisms underlying SM accumulation, we determined the activities of sphingolipid-metabolizing enzymes like neutral sphingomyelinase and ceramidase, which are also related to cellular redox homeostasis and to oxidative stress. Fibroblasts cultured in three-dimensional environment showed different redox potential and lower lipid susceptibility to oxidative damage compared to monolayer cells. The relative content of unsaturated fatty acids, which serve as targets of oxidative attack, was observed to be higher in major phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, in plasma membranes of 3D cells. The possibility that the higher level of SM, might be responsible for the lower degree of oxidation of 3D phospholipids was tested by selective reduction of SM through treatment with exogenous sphingomyelinase. The results showed that the decrease of plasma membrane SM was accompanied by an increase of the lipid peroxides in both 2D and 3D cells. We presume that culturing as a monolayer is stressful for the cells and leads to activation of certain stress-related enzymes, resulting in reduction of the SM level. Our results show that the lower content of plasma membrane SM in cells cultured as a monolayer renders the phospholipid molecules more susceptible to oxidative stress.

Evaluation of Cartilage Specific Matrix Synthesis of Human Articular Chondrocytes after Extended Propagation on Microcarriers by Image Analysis

Background Cell-based technologies for the repair of cartilage defects usually rely on the expansion of low numbers of chondrocytes isolated from biopsies of healthy cartilage. Proliferating chondrocytes are known to undergo dedifferentiation characterized by downregulation of collagen type II and proteoglycan production, and by upregulation of collagen type I synthesis. Re-expression of cartilage specific matrix components by expanded chondrocytes is therefore critical for successful cartilage repair. Methods Human articular chondrocytes were expanded on microcarriers Cytodex 3. The growth area was increased by adding empty microcarriers. Added microcarriers were colonized by bead-to-bead transfer of the cells. The chondrocytes were harvested from the microcarriers and characterized by their ability to synthesize collagen type II when cultivated in alginate beads using chondrogenic growth factors. A semi-automatic image analysis technique was developed to determine the fractions of collagen type II and type I positive cells. Results The expansion of human articular chondrocytes on microcarriers yielded high cell numbers and propagation rates compared to chondrocytes expanded in flask culture for one passage. The proportion of collagen type II positive cells compared to collagen type I synthesizing cells was increased compared to chondrocytes expanded using conventional methods. The matrix synthesis upon treatment with chondrogenic factors IGF-I and BMP-7 was enhanced whereas TGF-β had an inhibitory effect on microcarrier expanded chondrocytes. Conclusions Expanding human articular chondrocytes on microcarriers omitting subcultivation steps leads to superior ratios of collagen type II to type I forming cells compared to the expansion in conventional monolayer culture.

The contribution of Roberto Montesano to the study of interactions between epithelial sheets and the surrounding extracellular matrix

The interactions between epithelial cells and the surrounding extracellular matrix are a central issue in morphogenesis studies. Roberto Montesano and colleagues at the University Medical Center of Geneva, Switzerland, have extensively investigated the mechanisms underlying two morphogenetic processes: the formation of new capillary blood vessels from pre-existing ones (angiogenesis) and the generation of branching epithelial tubules (tubulogenesis), which are crucial events in the development of most parenchymal organs. Dr. Montesano has contributed to clarifying some cellular and molecular mechanisms of angiogenesis and tubulogenesis using an original three-dimensional cell culture system which replicates key events of angiogenesis and tubulogenesis, thereby facilitating molecular analysis. A major advance of this technique over conventional monolayer cultures is that cells can be embedded within a lattice of reconstituted collagen fibrils which mimics the three-dimensional organization of connective tissue matrices.The results of these studies support the notion that cell interactions with the surrounding extracellular matrix are crucial determinants of cell responses to growth factors and that epithelial tissues morphogenesis is governed by the interplay of two different classes of signaling molecules, i.e. paracrine-acting growth factors and insoluble extracellular matrix components.

Droplet-based microfluidic system for multicellular tumor spheroid formation and anticancer drug testing

Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since it may provide a better model than monolayer culture of tumor cells. Moreover, continuous dynamic perfusion allows the establishment of long term cell culture and subsequent multicellular spheroid formation. A droplet-based microfluidic system was used to form alginate beads with entrapped breast tumor cells. After gelation, the alginate beads were trapped in microsieve structures for cell culture in a continuous perfusion system. The alginate environment permitted cell proliferation and the formation of multicellular spheroids was observed. The dose-dependent response of the tumor spheroids to doxorubicin, and anticancer drug, showed multicellular resistance compared to conventional monolayer culture. The microsieve structures maintain constant location of each bead in the same position throughout the device seeding process, cell proliferation and spheroid formation, treatment with drug, and imaging, permitting temporal and spatial tracking.