Quantification Of Cell Numbers Research Articles

Artificial MicroRNAs as Novel Secreted Reporters for Cell Monitoring in Living Subjects

Reporter genes are powerful technologies that can be used to directly inform on the fate of transplanted cells in living subjects. Imaging reporter genes are often employed to quantify cell number, location(s), and viability with various imaging modalities. To complement this, reporters that are secreted from cells can provide a low-cost, in vitro diagnostic test to monitor overall cell viability at relatively high frequency without knowing the locations of all cells. Whereas protein-based secretable reporters have been developed, an RNA-based reporter detectable with amplification inherent PCR-based assays has not been previously described. MicroRNAs (miRNAs) are short non-coding RNAs (18–22 nt) that regulate mRNA translation and are being explored as relatively stable blood-based disease biomarkers. We developed an artificial miRNA-based secreted reporter, called Sec-miR, utilizing a coding sequence that is not expressed endogenously and does not have any known vertebrate target. Sec-miR was detectable in both the cells and culture media of transiently transfected cells. Cells stably expressing Sec-miR also reliably secreted it into the culture media. Mice implanted with parental HeLa cells or HeLa cells expressing both Sec-miR and the bioluminescence imaging (BLI) reporter gene Firefly luciferase (FLuc) were monitored over time for tumor volume, FLuc signal via BLI, and blood levels of Sec-miR. Significantly (p<0.05) higher Sec-miR was found in the blood of mice bearing Sec-miR-expressing tumors compared to parental cell tumors at 21 and 28 days after implantation. Importantly, blood Sec-miR reporter levels after day 21 showed a trend towards correlation with tumor volume (R2 = 0.6090; p = 0.0671) and significantly correlated with FLuc signal (R2 = 0.7067; p<0.05). Finally, we could significantly (p<0.01) amplify Sec-miR secretion into the cell media by chaining together multiple Sec-miR copies (4 instead of 1 or 2) within an expression cassette. Overall, we show that a novel complement of BLI together with a unique Sec-miR reporter adds an in vitro RNA-based diagnostic to enhance the monitoring of transplanted cells. While Sec-miR was not as sensitive as BLI for monitoring cell number, it may be more sensitive than clinically-relevant positron emission tomography (PET) reporter assays. Future work will focus on improving cell detectability via improved secretion of Sec-miR reporters from cells and more sensitive detection platforms, as well as, exploring other miRNA sequences to allow multiplexed monitoring of more than one cell population at a time. Continued development may lead to more refined and precise monitoring of cell-based therapies.

Abstract 334: Novel selective glucocorticoid receptor modulators (SGRMs) in castration-resistant prostate cancer

Abstract (A) Introduction: Because of the central role of androgen receptor (AR) signaling in prostate cancer (PC), AR inhibition is the primary modality of initial PC treatment. While AR-targeted therapies are often initially effective in castration-resistant prostate cancer (CRPC), disease progression is common. AR and glucocorticoid receptor (GR) are structurally similar nuclear receptors, bind the same DNA response elements, and regulate common genes. Previous work from our lab demonstrated that GR expression is upregulated following AR antagonism, GR expression decreased the effectiveness of AR blockade, and inhibiting GR activity reversed these effects. However, the only FDA-approved GR antagonist, mifepristone, is not GR-specific and has significant drug-drug interactions; therefore, we report our initial data with a new generation of selective GR-antagonists (SGRMs) for PC treatment using models of CRPC. (B) Experimental Procedures: The LAPC4, CWR-22Rv1 and VCAP PC cell lines were utilized. GR target gene expression analysis was performed at various time-points by qRT-PCR and protein immunoblots subsequent to treatment with AR agonist (R1881, 1nM), AR antagonist (enzalutamide, 10 μM), GR agonist (dexamethasone, 100nM) and the new SGRMs CORT108297 and CORT118335(1μM). Quantification of cell numbers in vitro in response to these treatments was determined by trypan blue exclusion/live cell counting and the CellTiter Glo 2.0 ATP-luciferase assay. For in vivo studies, PC cells were injected subcutaneously into male nude mice and grown to a predetermined tumor size, mice were then castrated, and fifteen days later treated with mifepristone (12mg/kg), SGRM (16mg/kg, 20mg/kg), or vehicle by daily intra-peritoneal injection to assess time to CRPC. (C) Results: AR antagonism with concurrent GR activation resulted in increased expression of canonical AR/GR-target genes and SGRMs (1μM) effectively inhibited expression of glucocorticoid-mediated genes at the RNA and protein levels. SGRMs did not affect AR-mediated gene expression. When AR was antagonized, activated GR promoted cell survival, which was inhibited by SGRM treatment to varying degrees. Preliminary studies support the hypothesis that these novel SGRMs delay CRPC tumor xenograft progression in vivo. (D) Conclusion: In vitro, new generation SGRMs inhibit GR-mediated, but not AR-selective gene expression and result in decreased tumor cell number. SGRM administration to nude mice bearing PC xenografts can delay castration-resistant tumor growth. Therefore, new SGRMs may be an effective therapy for CRPC treatment worthy of further exploration. Studies are ongoing to determine how SGRMs modulate GR target gene expression globally and whether they decrease expression of tumorigenic pro-survival genes in vivo. Citation Format: Jacob Kach, Phillip Selman, Diana C. West, Donald J. Vander Griend, Suzanne D. Conzen, Russell Z. Szmulewitz. Novel selective glucocorticoid receptor modulators (SGRMs) in castration-resistant prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 334.

The precision of bacterial quantification techniques on different kinds of environmental samples and the effect of ultrasonic treatment

The precision of cell number quantification in environmental samples depends on the complexity of the sample and on the applied technique. We compared fluorescence microscopy after filtration, quantification of gene copies and the cultivation based most probable number technique for their precision. We further analyzed the effect of increasing complexity of the sample material on the precision of the different methods by using pure cultures of Pseudomonas aeruginosa, fresh water samples and sediment slurries with and without ultrasonic treatment for analyses. Microscopy reached the highest precision, which was similar between pure cultures and water samples, but lower for sediment samples due to a higher percentage of cells in clusters and flocks. The PCR based quantification was most precise for pure cultures. Water and sediment samples were similar but less precise, which might be caused by the applied DNA extraction techniques. MPN measurements were equally precise for pure cultures and water samples. For sediment slurries the precision was slightly lower. The applied ultrasonic treatment of the slurries dispersed the cell clusters and flocks, increased the precision of microscopical and MPN measurements and also increased the number of potential colony forming units. However, the culturable cell number decreased by half. For MPN quantification of viable cells in samples with a high proportion of clustered cells we therefore recommend an optimization of ultrasonic treatment and a confirmation by microscopy and cultivation to reach highest possible dispersion of the cells with a minimum of inactivation. As a result of these observations we suggest a correction factor for MPN measurements to consider the effect of sonication on complex samples. The results are most likely applicable to other complex samples such as soil or biofilms.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy.

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (<10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers. This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

Lost signature: progress and failures in in vivo tracking of implanted stem cells.

Stem cell therapy as a part of regenerative medicine provides promising approaches for the treatment of injuries and diseases. The increasing use of mesenchymal stem cells in various medical treatments created the demand for long-term in vivo cell tracking methods. Therefore, it is necessary to analyze post-transplantational survival, biodistribution, and engraftment of cells. Furthermore, stem cell treatment has been discussed controversially due to possible association with tumor formation in the recipient. For therapeutic purpose, stem cells must undergo substantial manipulation such as differentiation and in vitro expansion, and this can lead to the occurrence of genetic aberrations and altered expression of both tumor suppression and carcinogenic factors. To control therapy, it is necessary to find a reliable and general method to track and identify implanted cells in the recipient. This is especially challenging for autologous transplantations, as standard fingerprinting methods cannot be applied. An optimal technique for in vivo cell monitoring does not yet exist, and its development holds several challenges: small numbers of transplanted cells, possibility of cell number quantification, minimal transfer of the contrast agent to non-transplanted cells, and no genetic modification. This review discusses most of the proposed solutions, including magnetic resonance imaging, magnetic particle imaging, positron emission tomography, single-photon emission computed tomography, and optical imaging methods. Additionally, the recent research on cell labeling for stem cell monitoring after transplantation including in vitro, ex vivo, and in vivo imaging studies is described. Promising future imaging modalities for stem cell monitoring after transplantation are shown.

Fluorescence imaging for in situ detection of cell surface sialic acid by competitive binding of 3-(dansylamino)phenylboronic acid

Sialic acid (SA) usually locates at the terminal position of the sugar chains on cell membranes, and its expression level is closely associated with cancer. Here polysialic acid (PSA) embedded gold nanoparticles (AuNPs) were prepared and functionalized with fluorescent 3-(dansylamino)phenylboronic acid (DAPB) for in situ imaging and detection of cell surface SA. The fluorescence resonance energy transfer (FRET) from DAPB to AuNPs quenched the fluorescence of DAPB. In the presence of additional SA or SA-abundant cells, the competitive binding of DAPB with SA and PSA led to the release of the assembled DAPB from the surface of PSA-embedded AuNPs, resulting in fluorescence of DAPB on SA-abundant cell surface. The proposed methods realized the in situ imaging and monitoring of cell surface SA, and could also be applied to the quantification of cell number and the amounts of cell surface SA. This work not only proposed a convenient visualization method for the analysis of SA on cell membranes, but also provided a potential tool for accelerating the elucidation of the basic role of SA in various biological processes and development of anti-cancer therapies.

Abstract 2617: SUMOylation inhibition attenuates the cancer stem cell population in glioblastoma and basal breast cancer

Abstract INTRO: TFAP2A and mouse homolog Tcfap2a are transcription factors that play a critical role in development and cancer. We have recently shown that SUMOylation of TFAP2A is a key posttranslational modification that maintains the basal breast cancer phenotype. Treatment with anacardic acid (AA), a SUMOylation inhibitor, led to depletion of the CD44+/hi/CD24-/lo cancer stem cell population. Interestingly, glioblastoma has also been shown to express TFAP2A and demonstrates an expanded cancer stem cell population. METHODS: Mouse 4T1 basal mammary cancer, IOWA-1T human basal breast cancer and UG118 glioblastoma cell lines were analyzed. MTT was used to measure cell viability. Coulter Counter and hemocytometer were used for cell quantification. Matrigel invasion and western blot were used to further characterize the cell lines. RESULTS: Treatment of 4T1 and UG118 cells with AA in vitro resulted in a 67% and 65% decrease in cell viability by MTT assay, respectively (p &amp;lt; 0.05 for both). Quantification of cell number confirmed that AA treatment inhibited cell proliferation. At 48 hours, 4T1 cells demonstrated a decrease of 73-77% (p &amp;lt; 0.01), and UG 118 cells decreased by 97 to 98% (p &amp;lt; 0.001). Cell invasiveness assessed by Matrigel invasion assay was attenuated in 4T1 and UG118 cells after treatment (84%, p &amp;lt; 0.01 and 28%, p &amp;lt; 0.05, respectively). Western blot demonstrated a dose-dependent decrease in expression of the cancer stem cell marker CD44 or the mouse homolog CD49f following AA treatment of UG118 and 4T1 cells, respectively. Western blot confirmed that while total TFAP2A levels were unchanged, SUMO conjugated TFAP2A was decreased after AA treatment of 4T1 cells. To further establish relevance to human cancer, an aggressive chemotherapeutic resistant patient-derived xenograft, IOWA-1T, similarly demonstrated a 41% drop (p &amp;lt; 0.007) in cell invasiveness and a concomitant decrease in SUMOylated TFAP2A with AA treatment. CONCLUSION: Inhibition of the SUMO pathway attenuates the cancer stem cell population in basal breast cancer and glioblastoma. While further investigation is needed, SUMOylation may be a common biological posttranslational modification that defines cancer stem cells. Furthermore, basal breast cancer, brain cancer, and other cancers that express TFAP2A may be amenable to SUMOylation inhibitors such as AA. Citation Format: Jung M. Park, Tong Wu, Sarah Van Dorin, Ronald Weigel. SUMOylation inhibition attenuates the cancer stem cell population in glioblastoma and basal breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2617. doi:10.1158/1538-7445.AM2015-2617

PLGA-encapsulated perfluorocarbon nanoparticles for simultaneous visualization of distinct cell populations by 19F MRI.

In vivo imaging using (19)F MRI is advantageous, due to its ability to quantify cell numbers, but is limited for a lack of suitable labels. Here, we formulate two stable and clinically applicable labels for tracking two populations of primary human dendritic cells (DCs) simultaneously. Plasmacytoid and myeloid DCs are able to take up sufficient nanoparticles (200 nm) for imaging (10(12 19)F's per cell), despite being relatively nonphagocytic. Clinically relevant numbers of labeled DCs could be imaged in about 10 min, even on a clinical scanner. We demonstrate the use of perfluorocarbon nanoparticles for simultaneous (19)F MRI of distinct cell populations in a clinical setting, without spectroscopic imaging.

In vivo Quantification of the Effects of Radiation and Presence of Hair Follicle Pores on the Proliferation of Fibroblasts in an Acellular Human Dermis in a Dorsal Skinfold Chamber: Relevance for Tissue Reconstruction following Neoadjuvant Therapy.

IntroductionIn neoadjuvant therapy, irradiation has a deleterious effect on neoangiogenesis. The aim of this study was to examine the post-implantation effects of neoadjuvant irradiation on the survival and proliferation of autologous cells seeded onto an acellular human dermis (hAD; Epiflex). Additionally, we examined the influence of dermal hair follicle pores on viability and proliferation. We used dorsal skinfold chambers implanted in rats and in-situ microscopy to quantify cell numbers over 9 days.Methods24 rats received a skinfold chamber and were divided into 2 main groups; irradiated and unirradiated. In the irradiated groups 20Gy were applied epicutaneously at the dorsum. Epiflex pieces were cut to size 5x5mm such that each piece had either one or more visible hair follicle pores, or no such visible pores. Fibroblasts were transduced lentiviral with a fluorescent protein for cell tracking. Matrices were seeded statically with 2.5x104 fluorescent fibroblasts and implanted into the chambers. In each of the two main groups, half of the rats received Epiflex with hair follicle pores and half received Epiflex without pores. Scaffolds were examined in-situ at 0, 3, 6 and 9 days after transplantation. Visible cells on the surface were quantified using ImageJ.ResultsIn all groups cell numbers were decreased on day 3. A treatment-dependent increase in cell numbers was observed at subsequent time points. Irradiation had an adverse effect on cell survival and proliferation. The number of cells detected in both irradiated and non-irradiated subjects was increased in those subjects that received transplants with hair follicle pores.DiscussionThis in-vivo study confirms that radiation negatively affects the survival and proliferation of fibroblasts seeded onto a human dermis transplant. The presence of hair follicle pores in the dermis transplants is shown to have a positive effect on cell survival and proliferation even in irradiated subjects.

Improved Osteoblast and Chondrocyte Adhesion and Viability by Surface-Modified Ti6Al4V Alloy with Anodized TiO₂ Nanotubes Using a Super-Oxidative Solution.

Titanium (Ti) and its alloys are amongst the most commonly-used biomaterials in orthopedic and dental applications. The Ti-aluminum-vanadium alloy (Ti6Al4V) is widely used as a biomaterial for these applications by virtue of its favorable properties, such as high tensile strength, good biocompatibility and excellent corrosion resistance. TiO2 nanotube (NTs) layers formed by anodization on Ti6Al4V alloy have been shown to improve osteoblast adhesion and function when compared to non-anodized material. In his study, NTs were grown on a Ti6Al4V alloy by anodic oxidation for 5 min using a super-oxidative aqueous solution, and their in vitro biocompatibility was investigated in pig periosteal osteoblasts and cartilage chondrocytes. Scanning electron microscopy (SEM), energy dispersion X-ray analysis (EDX) and atomic force microscopy (AFM) were used to characterize the materials. Cell morphology was analyzed by SEM and AFM. Cell viability was examined by fluorescence microscopy. Cell adhesion was evaluated by nuclei staining and cell number quantification by fluorescence microscopy. The average diameter of the NTs was 80 nm. The results demonstrate improved cell adhesion and viability at Day 1 and Day 3 of cell growth on the nanostructured material as compared to the non-anodized alloy. In conclusion, this study evidences the suitability of NTs grown on Ti6Al4V alloy using a super-oxidative water and a short anodization process to enhance the adhesion and viability of osteoblasts and chondrocytes. The results warrant further investigation for its use as medical implant materials.

Anchoring dipalmitoyl phosphoethanolamine to nanoparticles boosts cellular uptake and fluorine-19 magnetic resonance signal.

Magnetic resonance (MR) methods to detect and quantify fluorine (19F) nuclei provide the opportunity to study the fate of cellular transplants in vivo. Cells are typically labeled with 19F nanoparticles, introduced into living organisms and tracked by 19F MR methods. Background-free imaging and quantification of cell numbers are amongst the strengths of 19F MR-based cell tracking but challenges pertaining to signal sensitivity and cell detection exist. In this study we aimed to overcome these limitations by manipulating the aminophospholipid composition of 19F nanoparticles in order to promote their uptake by dendritic cells (DCs). As critical components of biological membranes, phosphatidylethanolamines (PE) were studied. Both microscopy and MR spectroscopy methods revealed a striking (at least one order of magnitude) increase in cytoplasmic uptake of 19F nanoparticles in DCs following enrichment with 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE). The impact of enriching 19F nanoparticles with PE on DC migration was also investigated. By manipulating the nanoparticle composition and as a result the cellular uptake we provide here one way of boosting 19F signal per cell in order to overcome some of the limitations related to 19F MR signal sensitivity. The boost in signal is ultimately necessary to detect and track cells in vivo.

Cytotoxic and genotoxic effects of high concentrations of the immunosuppressive drugs cyclosporine and tacrolimus in MRC-5 cells

Immunosuppressive drugs are used to suppress immune system activity in transplant patients and reduce the risk of organ rejection. The present study evaluated the potential cytotoxic, genotoxic and mutagenic of the immunosuppressive drugs cyclosporine (CsA) and tacrolimus (FK-506) on normal human fibroblasts (MRC-5 cells). Based on plasma concentrations of the immunosuppressive drugs, which were obtained from the records of kidney transplant patients at the Kidney Institute of Londrina, Brazil, 11 concentrations of each immunosuppressive were chosen to evaluate cell viability using the MTT assay. From these results, CsA and FK-506 concentrations of 135, 300, 675, and 1520ng/ml and 8, 16, 24, and 32ng/ml, respectively, were evaluated using (i) the comet assay, (ii) the nuclear division index (NDI), (iii) the micronucleus test (CBMN) and (iv) cell proliferation curves generated by quantifying cell numbers and protein levels. In this study, 1520 to 3420ng/ml CsA decreased cell viability after 48h of exposure. Genotoxic effects were observed only with a concentration of 1520ng/ml after 3h of exposure and with concentrations of 675 and 1520ng/ml after 24h of exposure. Mutagenic effects were observed only for the concentration of 1520ng/ml. FK-506 decreased cell viability after 72h of exposure for concentrations up to 20ng/ml; genotoxic effects were observed with concentrations up to 8ng/ml for both treatment times (3 and 24h) and mutagenic effects were observed with concentrations of 24 and 32ng/ml after 24h of treatment. The cell proliferation curves demonstrated the absence of cytostatic effects of these drugs, and these data were confirmed by the NDI analysis. Our results suggest that concentrations lower than 300ng/ml of CsA and 16ng/ml of FK-506 are safe for use, as they did not induce genotoxic and mutagenic damage or affect MRC-5 cell viability and proliferation.

Abstract 68: Tumor operational signaling pathways in an Nf1 mutant mouse model system

Abstract Purpose/Objectives: NF1 is recognized as a tumor suppressor, via its role as a Ras GTPase activating protein. However, the precise mechanisms critical to tumor development in NF1 patients remain unclear. NF1 heterozygosity confers susceptibility to tumorigenesis, promoted by genotoxins such as radiation. We mutagenized Nf1 heterozygous mice using radiation to generate a diverse panel of Nf1 null tumors. Using these novel reagents we interrogated signaling pathways essential for tumor development and propagation towards identification of potential targets for therapeutic approaches. Materials/Methodology: Cell lines were derived from solid tumors developing after focal irradiation of Nf1 heterozygous mice. Radiation-induced tumors included mammary carcinomas, squamous carcinomas, and sarcomas. We employed a high-throughput toxicity screen of 94 conventional and targeted FDA-approved chemotherapeutics directed across diverse core cellular signaling pathways. Drug sensitivity profiles were determined by quantifying cell numbers after a 72 hour drug exposure, and tumor cell lines, non-transformed wild-type and Nf1 null MEFs, and mutant Ras-transformed cell lines (added as controls) were compared. Validation of identified drug sensitivities was performed by enzyme activity assays, phosphoprotein analysis, and soft-agar colony formation. Results: Nf1 mutant tumors failed to demonstrate a common drug sensitivity signature, but instead revealed a heterogeneous pattern of differential sensitivities, with subgroups clustering independent of tumor histology. However, radiation-induced Nf1 null tumor lines exhibited increased sensitivity to inhibition of the Ras-Erk and PI3K-mTOR axis as compared to non-transformed controls. Enhanced sensitivity to agents targeting cell cycle and survival pathways was also observed selectively within the Nf1 null tumor cell lines. Conclusions: These studies indicate that Nf1 null tumors, even those of the same histology, do not intrinsically share a signature sensitivity profile to pharmacologic inhibition. This suggests that therapeutic targeting of signaling pathways in Nf1 mutant tumors is not likely to be defined strictly by Nf1 loss alone. Notably, these Nf1 null tumors evidence varied operational dependence upon PI3K-mTOR and Raf-MEK activity. Current efforts are directed towards further resolving essential mediators, with the goal of more precisely defining signaling pathways which drive progression of the malignant state. Citation Format: Steve Braunstein, Rana Mroue, Brian Huang, Sourav Bandyopadhyay, Jean Nakamura. Tumor operational signaling pathways in an Nf1 mutant mouse model system. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 68. doi:10.1158/1538-7445.AM2014-68

Model-based cell number quantification using online single-oxygen sensor data for tissue engineering perfusion bioreactors.

Online and non-invasive quantification of critical tissue engineering (TE) construct quality attributes in TE bioreactors is indispensable for the cost-effective up-scaling and automation of cellular construct manufacturing. However, appropriate monitoring techniques for cellular constructs in bioreactors are still lacking. This study presents a generic and robust approach to determine cell number and metabolic activity of cell-based TE constructs in perfusion bioreactors based on single oxygen sensor data in dynamic perfusion conditions. A data-based mechanistic modeling technique was used that is able to correlate the number of cells within the scaffold (R(2) = 0.80) and the metabolic activity of the cells (R(2) = 0.82) to the dynamics of the oxygen response to step changes in the perfusion rate. This generic non-destructive measurement technique is effective for a large range of cells, from as low as 1.0 × 10(5) cells to potentially multiple millions of cells, and can open-up new possibilities for effective bioprocess monitoring.

Microscale acoustic disruption of mammalian cells for intracellular product release

High-throughput microscale models for cell culture are critical for biopharmaceutical process development and drug discovery compound screening. While analytical methods are readily available for quantifying cell number and secreted product concentration, the recovery and measurement of intracellular products are significantly affected by the method of cell disruption. For example, the detergents often used in product extraction are incompatible with lipid-enveloped viruses. To provide an alternative to detergent-mediated disruption of mammalian cells, we have developed an effective yet gentle mechanical method compatible with 96-well plates using adaptive focused acoustics technology. This method was adapted for the release of Varicella–Zoster virus from MRC-5 cells and then applied to investigate infectious virus yield as a function of the cell density at infection. This microscale, high-throughput mechanical cell disruption method may be applicable to a variety of mammalian cell culture systems and intracellular products, thus expanding the scope of high-throughput screening.

A rapid and sensitive automated image-based approach for in vitro and in vivo characterization of cell morphology and quantification of cell number and neurite architecture.

Stereological methods for tissue cell counting, specifically for neuron quantification, decrease systematic error and sampling bias; however, they are tedious, labor intensive, and time consuming. Approaches for cell (neuron) quantification in vitro are not accurate, sensitive, or subsequently reproducible. Neuronal phenotype is related to alterations in cell morphology and neurite pattern. The techniques currently available for quantification of these features present several limitations. In this unit, we provide validated automated procedures for in vivo and in vitro quantification of cell number, morphological cell changes, and neurite morphometry in a fast, simple, and reliable manner. Our method counts up to 8 times as many neurons in less than 5% to 10% of the time required for stereological analysis (optical fractionator). In summary, this technology offers an unparalleled opportunity to examine features of cells at high resolution in a complex three-dimensional environment. These techniques provide an exceptional in vivo and in vitro system for neurotoxicity studies, disease modeling, and drug discovery.

Quantification of Protoporphyrin IX Accumulation in Glioblastoma Cells: A New Technique.

Introduction. 5-Aminolevulinic Acid (5-ALA) is a precursor of heme synthesis. A metabolite, protoporphyrin IX (PpIX), selectively accumulates in neoplastic tissue including glioblastoma. Presurgical administration of 5-ALA forms the basis of fluorescence-guided resection (FGR) of glioblastoma (GBM) tumors. However, not all gliomas accumulate sufficient quantities of PpIX to fluoresce, thus limiting the utility of FGR. We therefore developed an assay to determine cellular and pharmacological factors that impact PpIX fluorescence in GBM. This assay takes advantage of a GBM cell line engineered to express yellow fluorescent protein. Methods. The human GBM cell line U87MG was transfected with a YFP expression vector. After treatment with a series of 5-ALA doses, both PpIX and YFP fluorescence were measured. The ratio of PpIX to YFP fluorescence was calculated. Results. YFP fluorescence permitted the quantification of cell numbers and did not interfere with 5-ALA metabolism. The PpIX/YFP fluorescence ratio provided accurate relative PpIX levels, allowing for the assessment of PpIX accumulation in tissue. Conclusion. Constitutive YFP expression strongly correlates with cell number and permits PpIX quantification. Absolute PpIX fluorescence alone does not provide information regarding PpIX accumulation within the cells. Our research indicates that our PpIX/YFP ratio assay may be a promising model for in vitro 5-ALA testing and its interactions with other compounds during FGR surgery.

Review on Impedance Detection of Cellular Responses in Micro/Nano Environment

In general, cell culture-based assays, investigations of cell number, viability, and metabolic activities during culture periods, are commonly performed to study the cellular responses under various culture conditions explored. Quantification of cell numbers can provide the information of cell proliferation. Cell viability study can understand the percentage of cell death under a specific tested substance. Monitoring of the metabolic activities is an important index for the study of cell physiology. Based on the development of microfluidic technology, microfluidic systems incorporated with impedance measurement technique, have been reported as a new analytical approach for cell culture-based assays. The aim of this article is to review recent developments on the impedance detection of cellular responses in micro/nano environment. These techniques provide an effective and efficient technique for cell culture-based assays.

Silibinin promotes osteoblast differentiation of human bone marrow stromal cells via bone morphogenetic protein signaling

Silibinin is the major active constituent of the natural compound silymarin; several studies suggest that silibinin possesses antihepatotoxic properties and anticancer effects against carcinoma cells. However, no study has yet investigated the effect of silibinin on osteogenic differentiation of human bone marrow stem cells (hBMSCs). The aim of this study was to evaluate the effect of silibinin on osteogenic differentiation of hBMSCs. In this study, the hBMSCs were cultured in an osteogenic medium with 0, 1, 10 or 20μmol/l silibinin respectively. hBMSCs viability was analyzed by cell number quantification assay and cells osteogenic differentiation was evaluated by alkaline phosphatas (ALP) activity assay, Von Kossa staining and real time-polymerase chain reaction (RT-PCR). We found that silibinin promoted ALP activity in hBMSCs without affecting their proliferation. The mineralization of hBMSCs was enhanced by treatment with silibinin. Silibinin also increased the mRNA expressions of Collagen type I (COL-I), ALP, Osteocalcin (OCN), Osterix, bone morphogenetic protein-2 (BMP-2) and Runt-related transcription factor 2 (RUNX2). The BMP antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated silibinin-promoted ALP activity. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by silibinin treatment. These results indicate that silibinin enhances osteoblast differentiation probably by inducing the expressions of BMPs and activating BMP and RUNX2 pathways. Thus, silibinin may play an important therapeutic role in osteoporosis patients by improving osteogenic differentiation of BMSCs.

Uncertainty analysis of cell counting by metabolic assays

Cell counting is a fundamental procedure in living cell culture-based experiments and protocols in which the cell number quantification is required. The number of cells is one of the parameters necessary to investigate several cell culture features requiring to be monitored as function of time, such as cell viability, proliferation, growth, fitness and metabolism. Aim of this paper is contributing to declare a comprehensive uncertainty budget for cell counting through metabolic assays according to the EURACHEM/CITAC Guide Quantifying Uncertainty in Analytical Measurement.