Heterogeneous Cell Cultures Research Articles

PO-081 Notch signalling pathway plays a crucial role in maintaining the cancer stem cell population in lung and colorectal cancer

IntroductionThere is increasing evidence that cancer stem cells (CSCs) could mediate resistance to chemo- and radiotherapy, metastasis and recurrence in a wide range of solid tumours. Notch signalling pathway is involved in CSCs maintenance. Notch influences intra- and intercellular communications of various cell types in embryogenesis and adult organs. The oncogenic role of Notch signalling in survival, proliferation, self-renewal, differentiation and migration of cancer cells is demonstrated in most solid tumours and leukaemia. This study aims to explore the effect of Notch1 suppression on CSCs in human lung (A549) and colorectal (HCT116) adenocarcinoma cell lines.Material and methodsKnockdown of Notch1 by shRNA was confirmed by PCR and western-blot analyses. PCR was used for estimation of stemness, epithelia-to-mesenchyma transition (EMT) markers and proto-oncogene c-MYC expression. The proportion of CD133+ cells in different subpopulations was identified by Flow cytometry. Cell migration potential was examined by Boyden chamber assay. The effect of Notch1 inhibition on CSCs was established using classical detection methods of CSCs in heterogeneous cancer cell cultures: tumourisphere formation, tumourigenicity in vivo and quantitative assessment of ABC-transporter activity assays.Results and discussionsNotch1 inhibition resulted in stemness (SOX2, OCT3/4, NES), EMT (TWIST1, HES1, SNAIL1), CSCs (CD133) markers, c-MYC expression decrease and CDH1 (E-cadherin) expression increase suggesting that Notch pathway is involved in EMT. Loss of Notch1 did not influence cell proliferation in vitro but reduced migration potential. It was confirmed that Notch signalling interruption leads to reduction of stemness features: decreased drug resistance (A549 but not HCT116), ability to form tumourispheres and tumourigenic potential in vivo. ABC-transporter activity in HCT116 could be upregulated by TAZ/YAP signalling pathway and not by Notch. Moreover, reduced Notch1 expression increased the minimum inoculum cell dose for xenograft formation. The xenograft vascularisation assay revealed that Notch1 inhibition is not necessary for neovascularizaton but it is essential for angiogenesis but in tumour.ConclusionThe Notch signalling pathway is important for maintaining the CSCs population, stemness and migration so Notch1 could be considered as a potential therapeutic target in lung and colorectal cancer. The study was supported Russian Science Foundation (RSCF), grant No. 14-15-00467.

A correlative and quantitative imaging approach enabling characterization of primary cell-cell communication: Case of human CD4+ T cell-macrophage immunological synapses

Cell-to-cell communication engages signaling and spatiotemporal reorganization events driven by highly context-dependent and dynamic intercellular interactions, which are difficult to capture within heterogeneous primary cell cultures. Here, we present a straightforward correlative imaging approach utilizing commonly available instrumentation to sample large numbers of cell-cell interaction events, allowing qualitative and quantitative characterization of rare functioning cell-conjugates based on calcium signals. We applied this approach to examine a previously uncharacterized immunological synapse, investigating autologous human blood CD4+ T cells and monocyte-derived macrophages (MDMs) forming functional conjugates in vitro. Populations of signaling conjugates were visualized, tracked and analyzed by combining live imaging, calcium recording and multivariate statistical analysis. Correlative immunofluorescence was added to quantify endogenous molecular recruitments at the cell-cell junction. By analyzing a large number of rare conjugates, we were able to define calcium signatures associated with different states of CD4+ T cell-MDM interactions. Quantitative image analysis of immunostained conjugates detected the propensity of endogenous T cell surface markers and intracellular organelles to polarize towards cell-cell junctions with high and sustained calcium signaling profiles, hence defining immunological synapses. Overall, we developed a broadly applicable approach enabling detailed single cell- and population-based investigations of rare cell-cell communication events with primary cells.

NKX6.1 induced pluripotent stem cell reporter lines for isolation and analysis of functionally relevant neuronal and pancreas populations

Recent studies have reported significant advances in the differentiation of human pluripotent stem cells to clinically relevant cell types such as the insulin producing beta-like cells and motor neurons. However, many of the current differentiation protocols lead to heterogeneous cell cultures containing cell types other than the targeted cell fate. Genetically modified human pluripotent stem cells reporting the expression of specific genes are of great value for differentiation protocol optimization and for the purification of relevant cell populations from heterogeneous cell cultures. Here we present the generation of human induced pluripotent stem cell (iPSC) lines with a GFP reporter inserted in the endogenous NKX6.1 locus. Characterization of the reporter lines demonstrated faithful GFP labelling of NKX6.1 expression during pancreas and motor neuron differentiation. Cell sorting and gene expression profiling by RNA sequencing revealed that NKX6.1-positive cells from pancreatic differentiations closely resemble human beta cells. Furthermore, functional characterization of the isolated cells demonstrated that glucose-stimulated insulin secretion is mainly confined to the NKX6.1-positive cells. We expect that the NKX6.1-GFP iPSC lines and the results presented here will contribute to the further refinement of differentiation protocols and characterization of hPSC-derived beta cells and motor neurons for disease modelling and cell replacement therapies.

Prevention of hepatocellular carcinoma by targeting MYCN-positive liver cancer stem cells with acyclic retinoid

Hepatocellular carcinoma (HCC) is a highly lethal cancer that has a high rate of recurrence, in part because of cancer stem cell (CSC)-dependent field cancerization. Acyclic retinoid (ACR) is a synthetic vitamin A-like compound capable of preventing the recurrence of HCC. Here, we performed a genome-wide transcriptome screen and showed that ACR selectively suppressed the expression of MYCN, a member of the MYC family of basic helix-loop-helix-zipper transcription factors, in HCC cell cultures, animal models, and liver biopsies obtained from HCC patients. MYCN expression in human HCC was correlated positively with both CSC and Wnt/β-catenin signaling markers but negatively with mature hepatocyte markers. Functional analysis showed repressed cell-cycle progression, proliferation, and colony formation, activated caspase-8, and induced cell death in HCC cells following silencing of MYCN expression. High-content single-cell imaging analysis and flow cytometric analysis identified a MYCN+ CSC subpopulation in the heterogeneous HCC cell cultures and showed that these cells were selectively killed by ACR. Particularly, EpCAM+ cells isolated using a cell-sorting system showed increased MYCN expression and sensitivity to ACR compared with EpCAM- cells. In a long-term (>10 y) follow-up study of 102 patients with HCC, MYCN was expressed at higher levels in the HCC tumor region than in nontumor regions, and there was a positive correlation between MYCN expression and recurrence of de novo HCC but not metastatic HCC after curative treatment. In summary, these results suggest that MYCN serves as a prognostic biomarker and therapeutic target of ACR for liver CSCs in de novo HCC.

Abstract 4591: Specific elimination of invasive and multidrug-resistant cancer cells by an antibody-drug conjugate targeting AXL

Abstract Upon therapeutic pressure, cancers commonly select for drug-resistant, invasive subpopulations with elevated expression of the receptor tyrosine kinase AXL. Besides the correlation between high AXL expression and induction of epithelial-to-mesenchymal transition, a process known to support metastasis, ample evidence also links AXL to resistance against a variety of targeted therapies, including inhibitors of the MAPK pathway in malignant melanoma and the EGFR pathway in lung cancer. AXL-107-MMAE (HuMax-AXL-ADC) is a therapeutic antibody-drug conjugate specific for AXL, containing the microtubule disrupting agent monomethyl auristatin E as the cytotoxic payload. AXL-107-MMAE was previously shown to induce potent cytotoxicity in vitro and in vivo, which was dependent expression of AXL on the cell surface. We evaluated the capacity of AXL-107-MMAE to target AXL-positive MAPK pathway inhibitor resistant tumor cells, using malignant melanoma as a clinically relevant example. First, AXL-107-MMAE was shown to induce cytotoxicity in BRAF-mutant tumor cell lines that showed AXL expression upon acquired resistance to BRAF-inhibitors. In contrast, no cytotoxicity was observed in the BRAF-inhibitor-sensitive, AXL-negative parental cell lines. In heterogeneous tumor cell cultures, treatment with a BRAF-inhibitor selected for AXL-high, MAPK pathway inhibitor-insensitive melanoma cells, which was prevented by combined BRAF-inhibitor and AXL-107-MMAE treatment. Interestingly, we observed marked AXL upregulation in biopsies obtained from patients after they developed resistance to MAPK pathway inhibitors compared to paired pre-treatment biopsies. The in vivo potential of AXL-107-MMAE in malignant melanoma was demonstrated using BRAFV600E-mutated xenograft model derived from a patient who developed resistance to the BRAF inhibitor vemurafenib in the clinic. This indicates that AXL expression levels in treatment-resistant malignant melanoma are sufficient to induce tumor regression with AXL-107-MMAE, at least in this model system. These findings merit clinical validation of the targeting of both treatment-naïve and drug-resistant cancers with AXL-107-MMAE, either alone or in combination with other targeted therapies. Citation Format: Julia Boshuizen, Louise A. Koopman, Esther C. Breij, David Satijn, Daniel Peeper, Paul W. Parren. Specific elimination of invasive and multidrug-resistant cancer cells by an antibody-drug conjugate targeting AXL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4591. doi:10.1158/1538-7445.AM2017-4591

Selection of the cell culture to accumulate in vitro the sacbrood virus of bees

Bees, like other living organisms are exposed to various pathogenic microorganisms, such as bacteria, fungi, protozoans, parasites and viruses. Viruses are of particular threat to beekeeping. Some diseases caused by viruses that lead to the collapse of the bee colonies, which leads to loss of a huge number of bees and it causes a recession in the industry. One of the major bees diseases caused by viruses is a sacculated brood. This pathology causes significant damage to beekeeping, because sick families without assistance does not give the commodity honey and not able to provide themselves food, then farms lose the opportunity to sell queens and bee packages. Problem of prompt diagnosis is inaccessibility of bees culturing cells method in vitro virus, which is why understanding of the biological mechanisms underlying viral diseases is limited. These difficulties in the virologists works leads to late diagnosis of disease and as a result, there are large losses in farms. Currently, there is very little information about the cultivation of viruses bees on heterogeneous cell cultures, which give after passage of the pathogen any degenerative changes. However, in last few years there is an active debate on the cultivation of bees viruses in cell cultures. It is known that the highest sensitivity to the saccular brood virus are explants of ovarian nymphs queens bees, chick fibroblast culture, bee fibroblasts tissue of the pectoral muscles of adult bees, culture of the tissue reproductive organs, muscle and egg bee primary trypsinized culture of monkey kidney, primary cell culture, obtained from the larvae of worker bees, culture PC-15, IBRS-2. The major problem of cultivation of bees viruses - a small amount of data on heterologous type of sensitive cell cultures. However, research in this area continues, and scientists around the world are trying to solve a serious problem of cultivation of bee viruses.

RiboTag is a flexible tool for measuring the translational state of targeted cells in heterogeneous cell cultures.

Primary neuronal cultures are a useful tool for measuring pharmacological- and transgene-regulated gene expression; however, accurate measurements can be confounded by heterogeneous cell types and inconsistent transfection efficiency. Here we describe our adaptation of a ribosomal capture strategy that was designed to be used in transgenic mice expressing tagged ribosomal subunits (RiboTag) in specific cell types, thereby allowing measurement of translating RNAs from desired cell types within complex tissues. Using this strategy we were able to isolate and analyze neuron-specific RNA despite the presence of glia by co-transfecting experimental plasmids with plasmids that selectively express RiboTag in neurons. RiboTag immunoprecipitation was capable of recovering high integrity RNA from small numbers of transfected cells that can then be interrogated by a variety of methods (e.g., RT-qPCR, PCR array, RNA-Seq) and compared with basal RNA expression of the entire culture. Additionally, we demonstrate how co-transfection of RiboTag with small hairpin RNA (shRNA) constructs can validate and accurately assess the degree of gene expression knockdown, and how RiboTag can be used to measure receptor-mediated gene regulation with transiently expressed designer receptors exclusively activated by designer drugs (DREADDs). RiboTag co-transfection represents a convenient and powerful tool to isolate RNA from a specific subset of cultured cells with a variety of applications for experiments in vitro.

Chemotherapy in heterogeneous cultures of cancer cells with interconversion

Recently, the interconversion between differentiated and stem-like cancer cells has been observed. Here, we model the in vitro growth of heterogeneous cell cultures in the presence of interconversion from differentiated cancer cells to cancer stem cells (CSCs), showing that, by targeting only CSC with cytotoxic agents, it is not always possible to eradicate cancer. We have determined the kinetic conditions under which cytotoxic agents in in vitro heterogeneous cultures of cancer cells eradicate cancer. In particular, we have shown that the chemotherapeutic elimination of in vitro cultures of heterogeneous cancer cells is effective only if it targets all cancer cell types, and if the induced death rates for the different subpopulations of cancer cell types are large enough. The quantitative results of the model are compared and validated with experimental data.

Time- and Concentration-Dependent Effects of Exogenous Serotonin and Inflammatory Cytokines on Mast Cell Function

Mast cells play a significant role in both the innate and adaptive immune response; however, the tissue-bound nature of mast cells presents an experimental roadblock to performing physiologically relevant mast cell experiments. In this work, a heterogeneous cell culture containing primary culture murine peritoneal mast cells (MPMCs) was studied to characterize the time-dependence of mast cell response to allergen stimulation and the time- and concentration-dependence of the ability of the heterogeneous MPMC culture to uptake and degranulate exogenous serotonin using high performance liquid chromatography (HPLC) coupled to an electrochemical detector. Additionally, because mast cells play a central role in asthma, MPMCs were exposed to CXCL10 and CCL5, two important asthma-related inflammatory cytokines that have recently been shown to induce mast cell degranulation. MPMC response to both allergen exposure and cytokine exposure was evaluated for 5-HT secretion and bioactive lipid formation using ultraperformance liquid chromatography coupled to an electrospray ionization triple quadrupole mass spectrometer (UPLC-MS/MS). In this work, MPMC response was shown to be highly regulated and responsive to subtle alterations in a complex environment through time- and concentration-dependent degranulation and bioactive lipid formation. These results highlight the importance of selecting an appropriate mast cell model when studying mast cell involvement in allergic response and inflammation.

Isotope-dilution UPLC-MS/MS determination of cell-secreted bioactive lipids

Secreted bioactive lipids play critical roles in cell-to-cell communication and have been implicated in inflammatory immune responses such as anaphylaxis, vasodilation, and bronchoconstriction. Analysis of secreted bioactive lipids can be challenging due to their relatively short lifetimes and structural diversity. Herein, a method has been developed using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to quantify five cell-secreted, structurally and functionally diverse bioactive lipids (PGD2, LTC4, LTD4, LTE4, PAF) that play roles in inflammation. Sample analysis time is 5 min, and isotopically labeled internal standards are used for quantification. This method was applied to an immortal secretory cell line (RBL-2H3), a heterogeneous primary cell culture containing peritoneal mast cells, and murine platelets. In RBL cell supernatant samples, intrasample precisions ranged from 7.32-21.6%, averaging 17.0%, and spike recoveries in cell supernatant matrices ranged from 88.0-107%, averaging 97.0%. Calibration curves were linear from 10 ng mL(-1) to 250 ng mL(-1), and limits of detection ranged from 0.0348 ng mL(-1) to 0.803 ng mL(-1). This method was applied to the determination of lipid secretion from mast cells and platelets, demonstrating broad applicability for lipid measurement in primary culture biological systems.

Human Stromal (Mesenchymal) Stem Cells: Basic Biology and Current Clinical Use for Tissue Regeneration

Human stromal (mesenchymal) stem cells (hMSC) represent a group of non-hematopoietic stem cells present in the bone marrow stroma and the stroma of other organs including subcutaneous adipose tissue, placenta, and muscles. They exhibit the characteristics of somatic stem cells of self-renewal and multi-lineage differentiation into mesoderm-type of cells, e.g., to osteoblasts, adipocytes, chondrocytes and possibly other cell types including hepatocytes and astrocytes. Due to their ease of culture and multipotentiality, hMSC are increasingly employed as a source for cells suitable for a number of clinical applications, e.g., non-healing bone fractures and defects and also non-skeletal degenerative diseases like heart failure. Currently, the numbers of clinical trials that employ MSC are increasing. However, several biological and biotechnological challenges need to be overcome to benefit from the full potential of hMSC. In this current review, we present some of the most important and recent advances in understanding of the biology of hMSC and their current and potential use in therapy.

Morphological and electrophoretic data about heterogeneous primary skin cells cultures obtained from normal and Ovalbumin-Challenged Wistar rats after treatment by speleotherapy in the Cacica and Dej Romanian Salt Mines

Objective: To investigate the influence of salt mine medium from the Romanian Cacica and Dej Salt Mines upon the cell morphology and electrophoretic expression of heterogeneous skin cell cultures obtained from Wistar rats’ abdominal skin, on normal and Ovalbumin-sensitized animals. Materials and methods: Heterogeneous skin cell cultures were prepared from Wistar abdominal skin. Cultures derived from skin rat develop with a monolayer of fibroblasts and epithelial cells attached to the culture dish. Before cultures initiation, Wistar rats of 75-100 g weight were divided in two lots: control and ovalbumin challenged animals. Ten animals of each lot were send to Cacica and Dej Salt Mine for 14 days and maintained in the salt mine medium, as in speleotherapy treatment. Results: Speleotherapy of Wistar rats had induced significant differences in cell morphology and electrophoretic expression of primary dermal cells cultures. The data obtained support the protective effects of speleotherapy by comparing with ovalbumin sensibilised animals. Conclusions: The results of this study indicate the fact that speleotherapy induces changes on the morphology and protein expression of dermal cells in vitro, and these changes support the beneficial effects of speleotherapy.

Toxin–antitoxin based transgene expression in mammalian cells

Long-term, recombinant gene expression in mammalian cells depends on the nature of the transgene integration site and its inherent properties to modulate transcription (epigenetic effects). Here we describe a method by which high transgene expression is achieved and stabilized in extensively proliferating cultures. The method is based on strict co-expression of the transgene with an antitoxin in cells that express the respective toxin. Since the strength of antitoxin expression correlates with an advantage for cell growth, the cells with strong antitoxin expression are enriched over time in cultures of heterogeneous cells. This principle was applied to CHO cell lines that conditionally express the toxin kid and that are transduced to co-express the antitoxin kis together with different transgenes of interest. Cultivation of pools of transfectants that express the toxin steadily increase their transgene expression within several weeks to reach a maximum that is up to 120-fold over the initial status. In contrast, average transgene expression drops in the absence of toxin expression. Together, we show that cells conditionally expressing kid can be employed to create overexpressing cells by a simple coupling of kis to the transgene of interest, without further manipulation and in absence of selectable drugs.

Kinetic validation of 6‐NBDG as a probe for the glucose transporter GLUT1 in astrocytes

In recent years, the use of fluorescent glucose analogs has allowed the study of rapid transport modulation in heterogeneous cell cultures and complex tissues. However, the kinetic behavior of these tracers is not conventional. For instance, the fluorescent glucose analog 6-NBDG permeates the cell 50-100 times slower than glucose but the uptake of 6-NBDG is almost insensitive to glucose, an observation that casts doubts as to the specificity of the uptake pathway. To investigate this apparent anomaly in cultured astrocytes, which are rich in the glucose transporter GLUT1, we first estimated the kinetic parameters of 6-NBDG uptake, which were then incorporated into the kinetic model of GLUT1. The main outcome of the analysis was that 6-NBDG binds to GLUT1 with 300 times higher affinity than glucose, which explains why its uptake is not efficiently displaced by glucose. The high binding affinity of 6-NBDG also explains why cytochalasin B is less effective at inhibiting 6-NBDG uptake than at inhibiting glucose uptake. We conclude that 6-NBDG, used at low concentrations, permeates into astrocytes chiefly through GLUT1, and advise that the exofacial GLUT1 inhibitor 4,6-ethylidine-D-glucose be used, instead of glucose, as the tool of choice to confirm the specificity of 6-NBDG uptake.

Evaluation of Intracellular Signaling by Automatic Relative Immunofluorescence in Heterogeneous Cell Cultures

Evaluation of Intracellular Signaling by Automatic Relative Immunofluorescence in Heterogeneous Cell Cultures

Synchronization phenomena in mixed media of passive, excitable, and oscillatory cells

We study collective phenomena in highly heterogeneous cardiac cell culture and its models. A cardiac culture is a mixture of passive (fibroblasts), oscillatory (pacemakers), and excitable (myocytes) cells. There is also heterogeneity within each type of cell as well. Results of in vitro experiments are modelled by Luo-Rudy and FitzHugh-Nagumo systems. For oscillatory and excitable media, we focus on the transitions from fully incoherent behavior to partially coherent behavior and then to global synchronization as the coupling strength is increased. These regimes are characterized qualitatively by spatiotemporal diagrams and quantitatively by profiles of dependence of individual frequencies on coupling. We find that synchronization clusters are determined by concentric and spiral waves. These waves arising due to the heterogeneity of medium push covered cells to oscillate in synchrony. We are also interested in the influence of passive and excitable elements on the oscillatory characteristics of low- and high-dimensional ensembles of cardiac cells. The mixture of initially silent excitable and passive cells shows the transitions to oscillatory behavior. In the media of oscillatory and passive or excitable cells, the effect of oscillation death is observed.

Isolation, culture and adenoviral transduction of parietal cells from mouse gastric mucosa

Here we describe a method for the isolation of intact gastric glands from mice and primary culture and transfection of mouse gastric epithelial cells. Collagenase digestion of PBS-perfused mouse stomachs released large intact gastric glands that were plated on a basement membrane matrix. The heterogeneous gland cell cultures typically contain ∼60% parietal cells. Isolated mouse parietal cells remain viable in culture for up to 5 days and react strongly with an antibody specific to the gastric H+/K+ ATPase. Isolated intact mouse gastric glands and primary cultures of mouse parietal cells respond to the secretagogue, histamine. Typical morphological changes from a resting to an acid-secreting active parietal cell were observed. In resting cultures of mouse parietal cells, the H+/K+ ATPase displayed a cytoplasmic punctate staining pattern consistent with tubulovesicle element structures. Following histamine stimulation, an expansion of internal apical vacuole structures was observed together with a pronounced redistribution of the H+/K+ ATPase from the cytoplasm to the apical vacuoles. A reproducible procedure to express genes of interest exogenously in these cultures of mouse parietal cells was also established. This method combines recombinant adenoviral transduction with magnetic field-assisted transfection resulting in ∼30% transduced parietal cells. Adenoviral-transduced parietal cells maintain their ability to undergo agonist-induced activation. This protocol will be useful for the isolation, culture and expression of genes in parietal cells from genetically modified mice and as such will be an invaluable tool for studying the complex exocytic and endocytic trafficking events of the H+/K+ ATPase which underpin the regulation of acid secretion.

Polymethylmethacrylate particles inhibit osteoblastic differentiation of MC3T3‐E1 osteoprogenitor cells

Orthopedic wear debris has been implicated as a significant inhibitory factor of osteoblast differentiation. Polymethylmethacrylate (PMMA) particles have been previously shown to inhibit the differentiation of osteoprogenitors in heterogeneous murine marrow stromal cell cultures, but the effect of PMMA particles on pure osteoprogenitor populations remains unknown. In this study, we challenged murine MC3T3-E1 osteoprogenitor cells with PMMA particles during their initial differentiation in osteogenic medium. MC3T3-E1 cultures challenged with PMMA particles showed a gradual dose-dependent decrease in mineralization, cell number, and alkaline phosphatase activity at low particle doses (0.038-0.150% v/v) and complete reduction of these outcome parameters at high particle doses (> or =0.300% v/v). MC3T3-E1 cultures challenged with a high particle dose (0.300% v/v) showed no rise in these outcome parameters over time, whereas cultures challenged with a low particle dose (0.075% v/v) showed a normal or reduced rate of increase compared to controls. Osteocalcin production was not significantly affected by particles at all doses tested. MC3T3-E1 cells grown in conditioned medium from particle-treated MC3T3-E1 cultures showed a significant reduction in mineralization only. These results indicate that direct exposure of MC3T3-E1 osteoprogenitors to PMMA particles results in suppression of osteogenic proliferation and differentiation.

Localized delivery of DNA to the cells by viral collagen-loaded silica colloidal crystals

Low-molecular-weight colloidal crystals with enhanced biocompatibility and ordered porous structure are used in drug-delivery systems. The objective of our study is to demonstrate the use of silica nanoscale colloid particles for localized recombinant DNA release. The colloids were coated with collagen-containing viral vector constructs of lentiviral green fluorescent protein (GFP), and solidified at 37 degrees C. The colloid-collagen-viral vector platform (CCP) was transferred to cell monolayer cultures of human lung fibroblasts. Results show specific infection of cells directly beneath the platform, as evidenced by positive GFP in their cytoplasm, while neighboring cells show no cytoplasmic GFP The infection of specific cells is probably due to the gradual release of viral particles from the collagen matrix by cell-secreted collagenase, which avoids overdosing the cells with viral particles, resulting from the cytopathic effect often seen with high-titer viral infection. Cells infected with the lentiviral-GFP or lentivirus alone, not incorporated into the colloid-collagen device, show caspase 3-associated apoptotic cell death. This suggests that colloidal crystal-coated collagen may be used as a powerful platform to deliver genes of choice to localized subgroups of specific cells of interest. This specificity in the delivery mode is beneficial for functional studies of gene-directed impact on a particular cell population of interest in a heterogeneous cell culture.

Microgenomics: Identification of new expression profiles via small and single-cell sample analyses.

Since the sequencing of the human genome has been finished, microgenomics has been booming, employing highly sophisticated, high-throughput platforms. But these mainly chip-based methods can only generate biologically relevant data if the samples investigated consist of homogeneous cell populations, in which no unwanted cells of different specificity and/or developmental stage obscure the results. Different sampling methods have been routinely applied to overcome the problem presented by heterogeneous samples, e.g., global surveys, cell cultures, and microdissection. Various methods of laser-assisted microdissection, employing either positive or negative selection of tissue areas or even single cells, are available. These laser-assisted microdissection methods allow for fast and precise procurement of extremely small samples. Through subsequent application of recently developed methods of linear mRNA amplification in a pool of isolated total RNA, it has now become possible to perform complex high-throughput RNA expression profiling by microdissecting and processing even single-cell samples. Studies using the tools and methods of microgenomics have shed light on how those new approaches will eventually aid in the development of a new generation of diagnostics, e.g., leading to new patient-specific drugs tailored to the requirements assessed by assaying only a few biopsy cells.