Assuring Clonality on the Beacon Digital Cell Line Development Platform

During biomanufacturing cell lines development, the generation and screening for single‐cell derived subclones using methods that enable assurance of clonal derivation can be resource‐ and time‐intensive. High‐throughput miniaturization, automation, and analytic strategies are often employed to reduce such bottlenecks. The Beacon platform from Berkeley Lights offers a strategy to eliminate these limitations through culturing, manipulating, and characterizing cells on custom nanofluidic chips via software‐controlled operations. However, explicit demonstration of this technology to provide high assurance of a single cell progenitor has not been reported. Here, a methodology that utilizes the Beacon instrument to ensure high levels of clonality is described. It is demonstrated that the Beacon platform can efficiently generate production cell lines with a superior clonality data package, detailed tracking, and minimal resources. A stringent in‐process quality control strategy is established to enable rapid verification of clonal origin, and the workflow is validated using representative Chinese hamster ovary‐derived cell lines stably expressing either green or red fluorescence protein. Under these conditions, a >99% assurance of clonal origin is achieved, which is comparable to existing imaging‐coupled fluorescence‐activated cell sorting seeding methods.     

Use of DNA-specific anthraquinone dyes to directly reveal cytoplasmic and nuclear boundaries in live and fixed cells

Image-based, high-content screening assays demand solutions for image segmentation and cellular compartment encoding to track critical events — for example those reported by GFP fusions within mitosis, signalling pathways and protein translocations. To meet this need, a series of nuclear/cytoplasmic discriminating probes have been developed: DRAQ5™ and CyTRAK Orange™. These are spectrally compatible with GFP reporters offering new solutions in imaging and cytometry. At their most fundamental they provide a convenient fluorescent emission signature which is spectrally separated from the commonly used reporter proteins (e.g. eGFP, YFP, mRFP) and fluorescent tags such as Alexafluor 488, fluorescein and Cy2. Additionally, they do not excite in the UV and thus avoid the complications of compound UV-autofluorescence in drug discovery whilst limiting the impact of background sample autofluorescence. They provide a convenient means of stoichiometrically labelling cell nuclei in live cells without the aid of DMSO and can equally be used for fixed cells. Further developments have permitted the simultaneous and differential labelling of both nuclear and cytoplasmic compartments in live and fixed cells to clearly render the precise location of cell boundaries which may be beneficial for quantitative expression measurements, cell-cell interactions and most recently compound in vitro toxicology testing.     

Analysis of the paired TCR α- and β-chains of single human T cells

Analysis of the paired i.e. matching TCR α- and β-chain rearrangements of single human T cells is required for a precise investigation of clonal diversity, tissue distribution and specificity of protective and pathologic T-cell mediated immune responses. Here we describe a multiplex RT-PCR based technology, which for the first time allows for an unbiased analysis of the complete sequences of both α- and β-chains of TCR from single T cells. We validated our technology by the analysis of the pathologic T-cell infiltrates from tissue lesions of two T-cell mediated autoimmune diseases, psoriasis vulgaris (PV) and multiple sclerosis (MS). In both disorders we could detect various T cell clones as defined by multiple T cells with identical α- and β-chain rearrangements distributed across the tissue lesions. In PV, single cell TCR analysis of lesional T cells identified clonal CD8(+) T cell expansions that predominated in the epidermis of psoriatic plaques. An MS brain lesion contained two dominant CD8(+) T-cell clones that extended over the white and grey matter and meninges. In both diseases several clonally expanded T cells carried dual TCRs composed of one Vβ and two different Vα-chain rearrangements. These results show that our technology is an efficient instrument to analyse αβ-T cell responses with single cell resolution in man. It should facilitate essential new insights into the mechanisms of protective and pathologic immunity in many human T-cell mediated conditions and allow for resurrecting functional TCRs from any αβ-T cell of choice that can be used for investigating their specificity.     

A single‐step FACS sorting strategy in conjunction with fluorescent vital dye imaging efficiently assures clonality of biopharmaceutical production cell lines

The development of biopharmaceutical production cell lines typically starts with generation of heterogeneous populations of cells, from which then single cell clones are established. Several regulatory guidelines require that production cell lines are clonal, and the actual demonstration of clonality has been increasingly demanded by regulatory authorities over the last years. Here, the authors describe the relative contribution of flow cytometry mediated deposition of single cells in multiwell plates and subsequent imaging to assurance of clonality in a state of the art approach to single cell generation. Within the flow cytometry step, two unit operations are evaluated separately, doublet discrimination during event selection for deposition and droplet deposition accuracy. The imaging procedure is evaluated for the accuracy of detection of non‐clonal populations. By employing mixing experiments of cell populations, the authors demonstrate that doublet discrimination is highly efficient, and that an appropriately set up flow cytometry system already can generate >99.5% true single cell clones. The efficiency of the described imaging process depends on several factors, reaching an optimal detection rate of non‐clonal wells of about 99.8%. Our results demonstrate that one well characterized cloning step generate biopharmaceutical production cell lines with a probability of clonality of >99.99%.     

Examination of CD8+ T cell function in humans using MHC class I tetramers: similar cytotoxicity but variable proliferation and cytokine production among different clonal CD8+ T cells specific to a single viral epitope

Following infection by human T cell lymphotrophic virus-I (HTLV-I), high frequencies of polyclonal Tax11-19-reactive CD8(+) T cells can be detected in the peripheral blood. To investigate whether there are differences in the effector functions of these cells, we generated a panel of Tax11-19-reactive T cell clones by single cell sorting of HLA-A2/Tax11-19 tetramer binding CD8(+) T cells followed by repeated stimulation with PHA and IL-2. Examination of the TCRs revealed 17 different T cell clones with unique clonal origins. Nine representative CD8(+) T cell clones showed a similar cytotoxic dose-response activity against Ag-pulsed target cells, even though they express different TCRs. This cytotoxic effector function was not influenced by the engagement of either CD28 or CD2 costimulatory molecules. In contrast to the cytotoxic activity, qualitatively different degrees of proliferative response and cytokine secretion were observed among T cell clones of different clonal origin. The induction of proliferation and cytokine secretion required the engagement of costimulatory molecules, particularly CD2-LFA-3 interaction. These results indicate that functionally diverse, polyclonal CTL populations can be activated specific to a single immunodominant viral epitope; they can manifest virtually identical cytotoxic effector function but have marked differences in proliferation and cytokine secretion.     

Skeletal muscle cell populations

Summary Cell suspensions from the breast muscles of 10-day old chicken embryos were separated into non-myogenic, fibroblast-like cell fractions and a mononucleated, myogenic cell fraction by Percoll^TM density centrifugation. lsolated populations were characterized by their morphology in both mass cultures and individual macroscopic clones and by the immunocytochemical detection of skeletal muscle-and smooth muscle-specific proteins in individual cells. Cell populations were also characterized by their protein patterns using sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The less dense, non-myogenic cells comprised 16% of the cells. In culture they were predominantly flatiened, stellate cells and gave rise to clones lacking myotubes. These fibroblast-like cells were negative for skeletal muscle myosin or muscle type creatine phosphokinase. Less than 0.1% of these cells demonstrated strong fluorescence when stained with anti-desmin or anti-smooth muscle specific actin. This observation suggested that the vast majority of these cells were not related to vascular smooth muscle cells. Also, over 99% of the non-myogenic cells did not display characteristic properties of endothelial cells. The denser myogenic cell fraction comprised over 80% of the cells and in clonal cultures gave rise to about 70% myogenic clones. An additional 30% of clones from this fraction were non-myogenic indicating heterogeneity in this population. We conclude that Percoll centrifugation can be employed for the isolation of myogenic and non-myogenic cell populations directly from the embryonic muscle. Moreover, this procedure allows the direct analysis of cell-specific proteins (e.g., by gel electrophoresis) without the need for cell culturing. The results thus obtained closely reflect the status of the cells in the intact muscle.     

Extreme clonality in lymphoblastoid cell lines with implications for allele specific expression analyses

Lymphoblastoid cell lines (LCL) are being actively and extensively used to examine the expression of specific genes and genome-wide expression profiles, including allele specific expression assays. However, it has recently been shown that approximately 10% of human genes exhibit random patterns of monoallelic expression within single clones of LCLs. Consequently allelic imbalance studies could be significantly compromised if bulk populations of donor cells are clonal, or near clonal. Here, using X chromosome inactivation as a readout, we confirm and quantify widespread near monoclonality in two independent sets of cell lines. Consequently, we recommend where possible the use of bulk, non cell line, ex vivo cells for allele specific expression assays.     

Broadening cell selection criteria with micropallet arrays of adherent cells.

A host of technologies exists for the separation of living, nonadherent cells, with separation decisions typically based on fluorescence or immunolabeling of cells. Methods to separate adherent cells as well as to broaden the range of possible sorting criteria would be of high value and complementary to existing strategies. Cells were cultured on arrays of releasable pallets. The arrays were screened and individual cell(s)/pallets were released and collected. Conventional fluorescence and immunolabeling of cells were compatible with the pallet arrays, as were separations based on gene expression. By varying the size of the pallet and the number of cells cultured on the array, single cells or clonal colonies of cells were isolated from a heterogeneous population. Since cells remained adherent throughout the isolation process, separations based on morphologic characteristics, for example cell shape, were feasible. Repeated measurements of each cell in an array were performed permitting the selection of cells based on their temporal behavior, e.g. growth rate. The pallet array system provides the flexibility to select and collect adherent cells based on phenotypic and temporal criteria and other characteristics not accessible by alternative methods.     

Cell behaviour of Drosophila fat cadherin mutations in wing development

We have studied several cell behaviour parameters of mutant alleles of fat (ft) in Drosophila imaginal wing disc development. Mutant imaginal discs continue growing in larvae delayed in pupariation and can reach sizes of several times those of wild-type. Their growth is, however, basically allometric. Homozygous ft cells grow faster than their twin cells in clones and generate larger territories, albeit delimited by normal clonal restrictions. Moreover, ft cells in clones tend to grow towards wing proximal regions. These behaviours can be related with failures in cell adhesiveness and cell recognition. Double mutant combinations with alleles of other genes, e.g. of the Epidermal growth factor receptor (DER) pathway, modify ft clonal phenotypes, indicating that adhesiveness is modulated by intercellular signalling. Mutant ft cells show, in addition, smaller cell sizes during proliferation and abnormal cuticular differentiation, which reflect cell membrane and cytoskeleton anomalies, which are not modulated by the DER pathway.     

Efficiency of Plasmocin™ on various mammalian cell lines infected by mollicutes in comparison with commonly used antibiotics in cell culture: a local experience

Mycoplasma contamination is a deleterious event for cell culture laboratories. Plasmocin™ is used to prevent and eradicate mycoplasma infections from cell. In this study, 80 different mammalian cell lines from various sources; human, monkey, mice, hamster and rat were used to study and evaluate plasmocin™ efficiency and compare it to commonly used antibiotics such as BM-cyclin, ciprofloxacin and mycoplasma removal agent (MRA). It was shown that mycoplasma infections were eradicated by plasmocin™, BM-cyclin, ciprofloxacin and MRA in 65%, 66.25%, 20%, and 31.25%, respectively, of infected cell cultures. However, re-infection with mycoplasmas after the period of 4 months occurred in 10–80% of the studied cell lines. Cell cytotoxicity and culture death was observed in 25, 17.5 and 10% of the treated cells, for plasmocin™, BM-cyclin and MRA, respectively. In this study, Plasmocin™ showed strong ability to eradicate mollicutes from our cell lines with minimal percentage of regrowth. However, due to its high cell cytotoxicity it should be used with caution especially when dealing with expensive or hard-to-obtain cell lines. Amongst the antibiotics tested, BM-cyclin was shown to remove mycoplasma with the highest efficiency.     

Growth advantage (“clonal dominance”) of metastatically competent tumor cell variants expressed under selective two- or three-dimensional tissue culture conditions

Summary In previous experiments it was shown that injection into syngeneic CBA/J mice of cell mixtures containing an excess of non-metastatic SP1 mouse mammary carcinoma cells with aras transfected metastatic variant of SP1 called C1, always resulted in the eventual dominance of the C1 subpopulation at the site of inoculation. This occurred despite the growth rates of the two cell populations being identical in vivo when grown separately. The means by which the C1 subpopulation achieved “clonal dominance” is thought to involve its responsiveness to stimulatory paracrine growth factors liberated by the non-metastatic SP1 population. The clonal dominance process, however, could not be recapitulated in conventional monolayer tissue culture conditions in which SP1 and C1 cells were grown together in high concentrations of serum, i.e. under non-limiting culture conditions. We now show that clonal dominance of C1 cells can be observed when the cell mixture is maintained in tissue culture for extended periods, or when the cells are grown under selective, limiting conditions, some of which may mimic growth conditions in vivo more accurately. These conditions were a) growth in low (limiting) serum concentrations; and b) growth as three-dimensional multicellular aggregates, i.e. as “tumor spheroids”. Under all of these conditions dominance of the C1 subpopulation always took place, but with an efficiency 6- to 40-fold less than generally observed in vivo. C1 cells were also able to form more stable (compact) spheroids compared to SP1 cells. Entrapment of the latter in mixed C1/SP1 spheroids increased the recovery of the SP1 cells suggesting some kind of “rescue” mechanism in which cells are protected from physical forces by three-dimensional structure. The relevance of these in vitro interactions for clonal dominance in primary tumors and metastasis in vivo are discussed.     

Estimating the number of hematopoietic or lymphoid stem cells giving rise to clonal chromosome aberrations in blood T lymphocytes

Quantifying the proliferative capacity of long-term hematopoietic stem cells in humans is important for bone marrow transplantation and gene therapy. Obtaining appropriate data is difficult, however, because the experimental tools are limited. We hypothesized that tracking clonal descendants originating from hematopoietic stem cells would be possible if we used clonal chromosome aberrations as unique tags of individual hematopoietic stem cells in vivo. Using FISH, we screened 500 blood T lymphocytes from each of 513 atomic bomb survivors and detected 96 clones composed of at least three cells with identical aberrations. The number of clones was inversely related to their population size, which we interpreted to mean that the progenitor cells were heterogeneous in the number of progeny that they could produce. The absolute number of progenitor cells contributing to the formation of the observed clones was estimated as about two in an unexposed individual. Further, scrutiny of ten clones revealed that lymphocyte clones could originate roughly equally from hematopoietic stem cells or from mature T lymphocytes, thereby suggesting that the estimated two progenitor cells are shared as one hematopoietic stem cell and one mature T cell. Our model predicts that one out of ten people bears a non- aberrant clone comprising >10% of the total lymphocytes, which indicates that clonal expansions are common and probably are not health-threatening.     

Isolation and characterization of clonal vascular smooth muscle cell lines from spontaneously hypertensive and normotensive rat aortas

Summary Vascular smooth muscle cells were isolated from the aortas of spontaneously hypertensive rats and normotensive Wistar-Kyoto rats by use of the explant method on collagen gels. Clonal cell lines derived from these enriched populations possessed ultrastructural characteristics of vascular smooth muscle cells in culture; they grew in hill and valley configuration, immunostained with the muscle actin antibody HHF35, and failed to react with von Willebrand Factor VIII antibody. Fourteen clonal cell lines were characterized for growth and ligand binding characteristics. Large variations in growth rate and cell density at saturation were exhibited by clones of both strains. Similar variability was noted for specific binding of endothelial 1 and Sar¹,Ile⁸-angiotensin II to their receptors, indicating considerable phenotypic heterogeneity among the clonal cell lines. Six selected clones were further characterized for angiotensin II receptor linkage to G proteins. Cells of both strains exhibited comparable affinity shifts in the presence of GTPγS. These clonal cell lines should be useful for a variety of analyses of the comparative biology of aortic cells. It is possible that the diversity of phenotypic traits exhibited by these clones reflects the heterogeneity of vascular smooth muscle tissue found in vivo.     

Skeletal muscle cell populations. Separation and partial characterization of fibroblast-like cells from embryonic tissue using density centrifugation

Cell suspensions from the breast muscles of 10-day old chicken embryos were separated into non-myogenic, fibroblast-like cell fractions and a mononucleated, myogenic cell fraction by Percoll density centrifugation. Isolated populations were characterized by their morphology in both mass cultures and individual macroscopic clones and by the immunocytochemical detection of skeletal muscle- and smooth muscle-specific proteins in individual cells. Cell populations were also characterized by their protein patterns using sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The less dense, non-myogenic cells comprised 16% of the cells. In culture they were predominantly flattened, stellate cells and gave rise to clones lacking myotubes. These fibroblast-like cells were negative for skeletal muscle myosin or muscle type creatine phosphokinase. Less than 0.1% of these cells demonstrated strong fluorescence when stained with anti-desmin or anti-smooth muscle specific actin. This observation suggested that the vast majority of these cells were not related to vascular smooth muscle cells. Also, over 99% of the non-myogenic cells did not display characteristic properties of endothelial cells. The denser myogenic cell fraction comprised over 80% of the cells and in clonal cultures gave rise to about 70% myogenic clones. An additional 30% of clones from this fraction were non-myogenic indicating heterogeneity in this population. We conclude that Percoll centrifugation can be employed for the isolation of myogenic and non-myogenic cell populations directly from the embryonic muscle. Moreover, this procedure allows the direct analysis of cell-specific proteins (e.g., by gel electrophoresis) without the need for cell culturing. The results thus obtained closely reflect the status of the cells in the intact muscle.     

All beta cells contribute equally to islet growth and maintenance

In healthy adult mice, the beta cell population is not maintained by stem cells but instead by the replication of differentiated beta cells. It is not known, however, whether all beta cells contribute equally to growth and maintenance, as it may be that some cells replicate while others do not. Understanding precisely which cells are responsible for beta cell replication will inform attempts to expand beta cells in vitro, a potential source for cell replacement therapy to treat diabetes. Two experiments were performed to address this issue. First, the level of fluorescence generated by a pulse of histone 2B-green fluorescent protein (H2BGFP) expression was followed over time to determine how this marker is diluted with cell division; a uniform loss of label across the entire beta cell population was observed. Second, clonal analysis of dividing beta cells was completed; all clones were of comparable size. These results support the conclusion that the beta cell pool is homogeneous with respect to replicative capacity and suggest that all beta cells are candidates for in vitro expansion. Given similar observations in the hepatocyte population, we speculate that for tissues lacking an adult stem cell, they are replenished equally by replication of all differentiated cells.     

Isolation and characterization of epithelial cells from bovine pancreatic duct

Summary Epithelial cells derived from bovine pancreatic duct have been grown continuously in culture for 30 weeks (approximately 90 doublings of the cell population). The cells were grown in Eagle's minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 0.1 mM nonessential amino acids, and antibiotics. In confluent cultures, the cells are multilayered and form circular structures. When tested at various passages, the cells neither formed colonies in soft agar nor produced tumors after inoculation into athymic, nude mice. Hydrocortisone (1 and 5 μg per ml) and insulin (1,5 and 10 μg per ml) had no effect on the growth of the cells. β-Retinyl acetate inhibited growth rate and cell yield at a concentration of 5 μg per ml but was not growth-inhibitory at lower concentrations. By electron microscopy the cells have numerous mitochondria, Golgi and microvilli. Mucous droplets were observed in a small proportion of the cells. Desmosome-like structures and occluding junctions were observed more frequently between cells that had been transferred as aggregates than between cells transferred as single cells. Cytochemical studies indicated that some cells produce PAS positive granules that were not removed after treatment of the cultures with diastase. Eleven cell clones were isolated from the mass culture. The growth rates of the clones are different as well as the period of time in which the clones can be propagated in vitro. This work was supported in part by Y01 CP 60204 and N01 CP 43237.     

Clonal In Vitro Analysis of Neurotrophin Receptor p75-Immunofluorescent Cells Reveals Phenotypic Plasticity of Primary Rat Olfactory Ensheathing Cells

Clonal in vitro analysis represents a powerful tool for studying cellular differentiation. In the present study, microscope-assisted single cell transfer was combined with immunofluorescence to establish clonal cultures of identified primary rat olfactory ensheathing cells (OECs). During development, OECs originate from the neural crest, a transient population of multipotent cells. Since only neural crest cells have been analyzed at clonal density, it remained unclear whether OECs may retain multipotent features. Neurotrophin receptor p75 (p75^NTR)-immunolabelled rat OECs were seeded at clonal density under visual control using a semiautomated cell selection and transfer device (Quixell?) and emerging clones were analyzed with regard to proliferation and antigenic expression. We demonstrate that OECs from neonatal (P1) and 7 day-old (P7) but not from adult rats formed clones in the presence of OEC- and astrocyte-conditioned media (OEC-CM, A-CM). Cloning efficiency but not in vitro growth of OECs was independent of age but increased upon treatment with OEC-CM. Interestingly, about 75 % of P1 compared to 27 % of P7 OEC clones lost p75^NTR expression during 2 weeks in vitro and acquired immunoreactivity for Thy-1. The observation that primary OECs from P1 lost expression of p75^NTR at clonal density and initiated expression of the fibroblast marker Thy-1 may suggest that their developmental potential is greater than previously anticipated. Since microscope-assisted selection of immunofluorescent cells combined with semiautomated transfer guarantees monoclonality in a single step and affords selection of cells according to fluorescent label and/or morphological criteria it may be relevant for a variety of other cell types.     

Cell lineage dependent and independent control of Purkinje cell number in the mammalian CNS: further quantitative studies of lurcher chimeric mice

Recent quantitative studies of lurcher chimeric mice have shown that the adult population of cerebellar Purkinje cells can properly be described as a small number of developmental clones of cells. The clones are not seen as patches of contiguous neurons; rather, the cells of any one clone distribute throughout the half-cerebellum that contains them, intermingling extensively with the Purkinje cells of other linkages. Lurcher----wild-type chimeras were analyzed using the cell autonomous Purkinje-cell-lethal mutant, lurcher (+/Lc), as a cell marker. Cell counts from these chimeras revealed that the number of surviving Purkinje cells was always an integral multiple of a unit clone size. These numerical quanta are the evidence for the existence of Purkinje cell developmental clones. When two different inbred strains of mouse were compared (C3H/HeJ and C57BL/6), the resulting clonal analysis showed that the unit clone size (i.e., the number of Purkinje cells in one quantum) is an autonomous property of the lineage and hence, presumably, intrinsic to the progenitor cell that founded it. The current study uses the lurcher chimeric mouse system to examine the cell lineage relationships among the Purkinje cells of a third inbred strain of mouse, AKR/J. The data both support and extend our previous studies. Quantitative analysis reveals that the Purkinje cells of this strain also exist in clones, and the size of these clones is also strain-specific. The number of cells in a single clone (7850), however, is different from either C3H/HeJ (10,200) or C57BL/6 (9200). The fact that this value is so highly polymorphic among the inbred strains of mouse makes it likely that, rather than being a function of different alleles at a single genetic locus, clone size may well represent a multifactorial (but still cell-autonomous) property of developing Purkinje cells. Additional results from a single chimeric animal suggest strongly that clone number (i.e., the number of progenitors selected to found the population) is not strain-specific but results instead from cell:cell interactions during early nervous system formation.     

A nanoparticle-based immobilization assay for prion-kinetics study

A technique for permanently capturing a replica impression of biological cells has been developed to facilitate analysis using nanometer resolution imaging tools, namely the atomic force microscope (AFM). The method, termed Bioimprint™, creates a permanent cell 'footprint' in a non-biohazardous Poly (dimethylsiloxane) (PDMS) polymer composite. The transfer of nanometer scale biological information is presented as an alternative imaging technique at a resolution beyond that of optical microscopy. By transferring cell topology into a rigid medium more suited for AFM imaging, many of the limitations associated with scanning of biological specimens can be overcome. Potential for this technique is demonstrated by analyzing Bioimprint™ replicas created from human endometrial cancer cells. The high resolution transfer of this process is further detailed by imaging membrane morphological structures consistent with exocytosis. The integration of soft lithography to replicate biological materials presents an enhanced method for the study of biological systems at the nanoscale.