Validation of a semi automatic device to standardize quantification of Colony-Forming Unit (CFU) on hematopoietic stem cell products.

Accurate characterization of hematopoietic stem cells (HSC) products is needed to better anticipate the hematopoietic reconstitution and the outcome in patients. Although CD34+ viable cells enumeration is a key predictor of time to correction of aplasia, it does not fully inform about functionality of cells contained in the graft. CFU assay is the gold standard in vitro potency assay to assess clonogenicity of HSC and consists on the count and identification of colonies several days after culture in a semi solid media. Manual count of colonies with optic microscope is the most commonly used method but its important variability and subjectivity hinders the universal implementation of this potency assay. The aim of this study is to validate a standardized method using the STEMvision™ system, the first semi-automated instrument for imaging and scoring hematopoietic colonies, according to French and European recommendations. Results obtained highlight better performance criteria with STEMvision™ system than the manual method. This semi-automatic device tends to reduce the coefficients of variation of repeatability, inter-operator variability and intermediate precision. This newly available platform could represent an interesting option, significantly improving performances of CFU assays used for the characterization of hematopoietic progenitors.

Use of an imaging station for rapid colony counting in radiobiology studies.

Colony counting by eye is time consuming and subjective. Here comparison between the measurements of proliferative growth inhibition in plates of radiation-treated cells by an imaging station correlated highly significantly with counts determined by eye. This would suggest that an imaging station could be a viable alternative for colony counting for doses over 200KBq.

In Vitro Approaches for Assessing the Genotoxicity of Nanomaterials.

Genotoxicity is associated with serious health effects and includes different types of DNA lesions, gene mutations, structural chromosome aberrations involving breakage and/or rearrangements of chromosomes (referred to as clastogenicity) and numerical chromosome aberrations (referred to as aneuploidy). Assessing the potential genotoxic properties of chemicals, including nanomaterials (NMs), is a key element in regulatory safety assessment. State-of-the-art genotoxicity testing includes a battery of assays covering gene mutations, structural and numerical chromosome aberrations. Typically various in vitro assays are performed in the first tier. It is not very likely that NMs may induce as yet unknown types of genotoxic damage beyond what is already known for chemicals. Thus, principles of genotoxicity testing as established for chemicals should be applicable to NMs as well. However, established test guidelines (i.e., OECD TG) may require adaptations for NM testing, as currently under discussion at the OECD. This chapter gives an overview of genotoxicity testing of NMs in vitro based on experiences from various research projects. We recommend a combination of a mammalian gene mutation assay (at either Tk or HPRT locus), the in vitro comet assay, and the cytokinesis-block micronucleus assay, which are discussed in detail here. In addition we also include the Cell Transformation Assay (CTA) as a promising novel test for predicting NM-induced cell transformation in vitro.

Clonogenic Culture of Mouse Thymic Epithelial Cells.

The thymus plays an essential role in the development and selection of T cells by providing a unique microenvironment that is mainly composed of thymic epithelial cells (TECs). We previously identified stem cells of medullary TECs (mTECs) that are crucial for central tolerance induction using a novel clonogenic culture system. We also found that medullary thymic epithelial stem cells (mTESCs) maintain life-long mTECs regeneration and central T cell self-tolerance in mouse models. The clonogenic efficiency of TECs in vitro is highly correlated to the TEC reconstitution activity in vivo. Here, we describe the clonogenic culture system to evaluate the self-renewing activity of TESCs. The colonies are derived from TESCs, are visualized and quantified by rhodamine-B staining on a feeder layer, and can be passaged in vitro. Thus, our system enables quantitative evaluation of TESC activity and is useful for dissecting the mechanisms that regulate TESC activity in physiological aging as well as in various clinical settings.

A new preclinical 3-dimensional agarose colony formation assay.

The evaluation of new drug treatments and combination treatments for gliomas and other cancers requires a robust means to interrogate wide dose ranges and varying times of drug exposure without stain-inactivation of the cells (colonies). To this end, we developed a 3-dimensional (3D) colony formation assay that makes use of GelCount technology, a new cell colony counter for gels and soft agars. We used U251MG, SNB19, and LNZ308 glioma cell lines and MiaPaCa pancreas adenocarcinoma and SW480 colon adenocarcinoma cell lines. Colonies were grown in a two-tiered agarose that had 0.7% agarose on the bottom and 0.3% agarose on top. We then studied the effects of DFMO, carboplatin, and SAHA over a 3-log dose range and over multiple days of drug exposure. Using GelCount we approximated the area under the curve (AUC) of colony volumes as the sum of colony volumes (microm2xOD) in each plate to calculate IC50 values. Adenocarcinoma colonies were recognized by GelCount scanning at 3-4 days, while it took 6-7 days to detect glioma colonies. The growth rate of MiaPaCa and SW480 cells was rapid, with 100 colonies counted in 5-6 days; glioma cells grew more slowly, with 100 colonies counted in 9-10 days. Reliable log dose versus AUC curves were observed for all drugs studied. In conclusion, the GelCount method that we describe is more quantitative than traditional colony assays and allows precise study of drug effects with respect to both dose and time of exposure using fewer culture plates.

Determination of cell colony formation in a high-content screening assay.

The use of cell colony formation assays for research and clinical applications to assess the functional integrity of cells after in vitro manipulations is extensive. Key areas include hematopoietic stem cell research, cell transformation studies, and predicting the response of tumors to chemotherapeutic agents. Traditionally, enumeration of colonies has involved laborious and subjective counting by hand using a microscope. Here, laser scanning microplate cytometry has been used to provide an automated high-content readout of the effects of cytostatic agents on colony formation. This approach determines colony number through the application of a volume algorithm. Such an approach permits the differentiation of cytostatic effects where the number of colonies and size remains constant, and cytotoxic effects where the size and number may be reduced. Application of microplate cytometry thus offers significant benefits over alternative analytical methods in the search for novel chemotherapeutic agents.

Multiple lineage colony growth from human marrow in plasma clot diffusion chambers.

Marrow cells from ten healthy adult donors were cultured in plasma clot diffusion chambers implanted intraperitoneally into mice. Host animals were conditioned by two injections of phenylhydrazine and 600 cGy of x-rays. Cultures (5 X 10(4) cells/chamber) were continued for between 2 and 40 days and the chambers were retransplanted into new host animals every 5 days. Following termination of cultures, plasma clots were stained with benzidine-hematoxylin and analyzed microscopically. Erythroid, neutrophil, monocyte, eosinophil, megakaryocyte, mixed, undifferentiated, and fibroblastoid colonies were grown with neutrophil, erythroid, monocyte, and eosinophil colonies being the most frequent. A total of between 25 and 60 colonies was observed per chamber at any time point.

Improved soft-agar colony assay in a fluid processing apparatus.

The standard method for quantitating bone marrow precursor cells has been to count the number of colony-forming units that form in semisolid (0.3%) agar. Recently we adapted this assay for use in hardware, the Fluid Processing Apparatus, that is flown in standard payload lockers of the space shuttle. When mouse or rat macrophage colony-forming units were measured with this hardware in ground-based assays, we found significantly more colony growth than that seen in standard plate assays. The improved growth correlates with increased agar thickness but also appears to be due to properties inherent to the Fluid Processing Apparatus. This paper describes an improved method for determining bone marrow macrophage precursor numbers in semisolid agar.

Emergent properties of nanosensor arrays: applications for monitoring IgG affinity distributions, weakly affined hypermannosylation, and colony selection for biomanufacturing.

It is widely recognized that an array of addressable sensors can be multiplexed for the label-free detection of a library of analytes. However, such arrays have useful properties that emerge from the ensemble, even when monofunctionalized. As examples, we show that an array of nanosensors can estimate the mean and variance of the observed dissociation constant (KD), using three different examples of binding IgG with Protein A as the recognition site, including polyclonal human IgG (KD µ = 19 µM, σ(2) = 1000 mM(2)), murine IgG (KD µ = 4.3 nM, σ(2) = 3 µM(2)), and human IgG from CHO cells (KD µ = 2.5 nM, σ(2) = 0.01 µM(2)). Second, we show that an array of nanosensors can uniquely monitor weakly affined analyte interactions via the increased number of observed interactions. One application involves monitoring the metabolically induced hypermannosylation of human IgG from CHO using PSA-lectin conjugated sensor arrays where temporal glycosylation patterns are measured and compared. Finally, the array of sensors can also spatially map the local production of an analyte from cellular biosynthesis. As an example, we rank productivity of IgG-producing HEK colonies cultured directly on the array of nanosensors itself.

A method of test substance removal in agar colony assays using glass capillaries.

A technique has been developed to remove test substances, after defined incubation periods, from clonogenic in vitro assays using agar-containing glass capillaries. Following removal from the capillaries, the entire agar gels were washed in petri dishes and redrawn into new capillaries. Using 8 radioactive biochemicals of molecular masses ranging from 150 to 1300 dalton the kinetics of diffusion between 1 and 20 min were determined. Using a wash solution-to-assay volume ratio of 20:1, a single washing for 10 min yielded between 90% and 99% removal by diffusion of test substances. By incorporating myelopoietic stem cells it was demonstrated that the cells to be assayed can be quantitatively transferred, without loss or stress, out of and back into capillaries. Thus the reversibility of test substance action can examined under defined conditions avoiding technical problems of previous methods.

Sistema automático para contagem de colônias em placas de Petri / Automatic system for colonies counting in Petri dishes

Esse trabalho apresenta o projeto e os testes de um protótipo de um instrumento simples, compacto e de baixo custo, para a contagem de Unidades Formadoras de Colônias (UFCs) em placas de petri. O protótipo é composto por um sistema de iluminação, uma câmera CCD, uma placa de aquisição de vídeo e um microcomputador IBM/PC ou compatível. O sistema de iluminação com LEDs é inovador e possibilita a aquisição de imagens das placas de petri com excelente contraste entre as colônias e o background. Essa característica possibilitou o desenvolvimento de um algoritmo de processamento digital de imagens simples, rápido e eficaz. O programa adquire e calcula a média de vinte imagens de uma placa de petri e utiliza o método background subtraction para separar as colônias de bactérias do restante da imagem e atenuar pequenas diferenças de iluminação. A imagem é então limiarizada e as UFCs contadas com um algoritmo recursivo capaz de localizar e determinar a dimensão, em pixels, de cada uma das colônias. Os resultados dos vários testes realizados durante o desenvolvimento demonstraram que o instrumento é capaz de contar as UFCs com área superior a 0,8mm2, sendo que o coeficiente de correlação entre os resultados obtidos com o método de contagem convencional e a contagem feita pelo protótipo é superior a 0,99