A comparison of different methods to determine the end of exponential growth in CHO cell cultures for optimization of scale-up

Maximizing cell growth rate and cell yield are among the most important features of a successful mammalian cell culture production process. To minimize time and resources needed to scale up cell mass it is important to maintain the cultures in exponential growth at every scale. Here we report results comparing viable cell counts, packed cell volume, intracellular nucleotide ratios, cell cycle analysis, and on-line oxygen uptake rates (OUR) and optical density for the determination of the end of exponential growth to optimize transfer times during scale-up of CHO cell cultures. Viable cell concentration, packed cell volume, and relative abundance of cells in S-phase were not very reliable at determining the end of exponential growth during the process. In contrast, on-line determination of OUR and off-line determination of intracellular nucleotide ratios (U-ratio) were very sensitive to changes in growth rate, enabling clear determination of the end of exponential growth within a short time. Although on-line OUR was found to be the most convenient and fastest method, it is restricted to instrumented and continuously monitored cultures. In contrast the nucleotide method can be applied with any culture scale and condition but needs the availability of an operator running an HPLC system and takes about an hour from sampling to result. Optical density showed an inflection along with OUR and U-ratio but was less sensitive in determining the end of exponential growth.     

Surface:volume relationship in cardiac myocytes studied with confocal microscopy and membrane capacitance measurements: species-dependence and developmental effects.

The quantitative analysis of the contribution of ion fluxes through membrane channels to changes of intracellular ion concentrations would benefit from the exact knowledge of the cell volume. It would allow direct correlation of ionic current measurements with simultaneous measurements of ion concentrations in individual cells. Because of various limitations of conventional light microscopy a simple method for accurate cell volume determination is lacking. We have combined the optical sectioning capabilities of fluorescence laser scanning confocal microscopy and the whole-cell patch-clamp technique to study the correlation between cell volume and membrane capacitance. Single cardiac myocytes loaded with the fluorescent dye calcein were optically sectioned to produce a series of confocal images. The volume of cardiac myocytes of three different mammalian species was determined by three-dimensional volume rendering of the confocal images. The calculated cell volumes were 30.4 +/- 7.3 pl (mean +/- SD) in rabbits (n = 28), 30.9 +/- 9.0 pl in ferrets (n = 23), and 34.4 +/- 7.0 pl in rats (n = 21), respectively. There was a positive linear correlation between membrane capacitance and cell volume in each animal species. The capacitance-volume ratios were significantly different among species (4.58 +/- 0.45 pF/pl in rabbit, 5.39 +/- 0.57 pF/pl in ferret, and 8.44 +/- 1.35 pF/pl in rat). Furthermore, the capacitance-volume ratio was dependent on the developmental stage (8.88 +/- 1.14 pF/pl in 6-month-old rats versus 6.76 +/- 0.62 pF/pl in 3-month-old rats). The data suggest that the ratio of surface area:volume of cardiac myocytes undergoes significant developmental changes and differs among mammalian species. We further established that the easily measurable parameters of cell membrane capacitance or the product of cell length and width provide reliable but species-dependent estimates for the volume of individual cells.     

Multiplexing Spheroid Volume,Resazurin and Acid Phosphatase Viability Assays for High-Throughput Screening of Tumour Spheroids and Stem Cell Neurospheres

Three-dimensional cell culture has many advantages over monolayer cultures, and spheroids have been hailed as the best current representation of small avascular tumours in vitro. However their adoption in regular screening programs has been hindered by uneven culture growth, poor reproducibility and lack of high-throughput analysis methods for 3D. The objective of this study was to develop a method for a quick and reliable anticancer drug screen in 3D for tumour and human foetal brain tissue in order to investigate drug effectiveness and selective cytotoxic effects. Commercially available ultra-low attachment 96-well round-bottom plates were employed to culture spheroids in a rapid, reproducible manner amenable to automation. A set of three mechanistically different methods for spheroid health assessment (Spheroid volume, metabolic activity and acid phosphatase enzyme activity) were validated against cell numbers in healthy and drug-treated spheroids. An automated open-source ImageJ macro was developed to enable high-throughput volume measurements. Although spheroid volume determination was superior to the other assays, multiplexing it with resazurin reduction and phosphatase activity produced a richer picture of spheroid condition. The ability to distinguish between effects on malignant and the proliferating component of normal brain was tested using etoposide on UW228-3 medulloblastoma cell line and human neural stem cells. At levels below 10 µM etoposide exhibited higher toxicity towards proliferating stem cells, whereas at concentrations above 10 µM the tumour spheroids were affected to a greater extent. The high-throughput assay procedures use ready-made plates, open-source software and are compatible with standard plate readers, therefore offering high predictive power with substantial savings in time and money.     

Methods for the quantification ofFrankia cell biomass

Six methods for the estimation of microbial biomass were compared for determination ofFrankia cell concentrations. Six strains ofFrankia were cultivated in stationary culture, harvested by centrifugation, washed with saline buffer and diluted to five standardized concentrations. These cell suspensions were then used to assess reliability of each of the biomass determination methods. The destructive total protein determination methods were the most sensitive and reliable. Two non-destructive methods, packed cell volume and turbidity measurement, were also accurate, and because of their simplicity hold advantage for routine growth measurements and inoculum dilutions. Dry weight determinations were inconsistent for the small cell masses used in this study. An ELISA procedure demonstrated reliability but little sensitivity.     

Three-dimensional hydrogel culture conditions promote the differentiation of human induced pluripotent stem cells into hepatocytes

Background aims

Human induced pluripotent stem cells (hiPSCs) are becoming increasingly popular in research endeavors due to their potential for clinical application; however, such application is challenging due to limitations such as inferior function and low induction efficiency. In this study, we aimed to establish a three-dimensional (3D) culture condition to mimic the environment in which hepatogenesis occurs in vivo to enhance the differentiation of hiPSCs for large-scale culture and high throughput BAL application.

Measurement of biophysical properties of red blood cells by resistive pulse spectroscopy: volume, shape, surface area, and deformability

This paper presents a simple, new approach to the determination of size, shape, surface area, and deformability information for cells, notably red blood cells. The results are obtained by combining experimental measurements from resistive pulse spectroscopy (an extension of electronic cell-sizing methodology) with theoretical calculations for model cell systems. Assuming constancy of surface area and approximating red cell shapes by both prolate and oblate ellipsoids of revolution, values are determined for cell shape factor and volume under a variety of conditions. For red blood cells under low-stress conditions, shape factor, volume, and surface area results are found to be consistent with those available from the literature, when the oblate model is used. The applicability of this approach for determination of red cell properties under altered conditions is demonstrated by results for cell volume, at varying osmotic pressure and mechanical shear (tensile) stress. By quantitating the change in cell shape with stress, a new numerical scale for measuring cell deformability is also obtained, and data are presented on its variation for red cells at different osmolalities, over the range of 140 to 500 mOsm.     

A computational model for populations of dividing cells.

A mathematical model of the cell movements due to cell division is presented. In the model we assume that every cell is a computational object with a given volume, and that the cell pushes the neighbouring cells in order to acquire the space for this volume. The Force that each cell exerts over the other cells is derived from a harmonic arbitrary Potential. The main parameter of the model is the average distance among the cells, that checks if the system is in spatial equilibrium or not. We show that just changing the physical constraints we can model two different systems, a two-dimensional culture on a plate and a three-dimensional early embryo. In both cases the patterns of the cell populations we obtain are similar to the real ones.     

Mapping three-dimensional stress and strain fields within a soft hydrogel using a fluorescence microscope

Three-dimensional cell culture is becoming mainstream as it is recognized that many animal cell types require the biophysical and biochemical cues within the extracellular matrices to perform truly physiologically realistic functions. However, tools for characterizing cellular mechanical environment are largely limited to cell culture plated on a two-dimensional substrate. We present a three-dimensional traction microscopy that is capable of mapping three-dimensional stress and strain within a soft and transparent extracellular matrix using a fluorescence microscope and a simple forward data analysis algorithm. We validated this technique by mapping the strain and stress field within the bulk of a thin polyacrylamide gel layer indented by a millimeter-size glass ball, together with a finite-element analysis. The experimentally measured stress and strain fields are in excellent agreements with results of the finite-element simulation. The unique contributions of the presented three-dimensional traction microscopy technique are: 1), the use of a fluorescence microscope in contrast with the confocal microscope that is required for the current three-dimensional traction microscopes in the literature; 2), the determination of the pressure field of an incompressible gel from strains; and 3), the simple forward-data-analysis algorithm. Future application of this technique for mapping animal cell traction in three-dimensional nonlinear biological gels is discussed.     

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.     

Application of fluorimetric analysis of plant esterases to study of programmed cell death and effects of cadmium(II) ions

Esterases (EC 3.1.1.x) represent a diverse group of hydrolases catalyzing the cleavage and formation of carboxyl ester bonds. Their connection with development has made them a suitable marker of development in plants. In the present work, we focused on the fluorimetric determination of the plant esterases in plant cell cultures (tobacco BY-2 cells and early somatic embryos of Norway spruce, clone 2/32) with respect to application the method for the study of programmed cell death and the influence of cadmium(II) ions on the plant cells. The programmed cell death has been triggered by sodium nitroprusside and glucose oxidase. The determination of the esterase activity by the proposed technique in a cell extract determined very small difference in enzyme activity, which was a reliable marker of metabolic changes. In addition, the esterase activity of spruce somatic embryos decreased with the increase in medium Cd concentration.     

Modelling tissues in 3D: the next future of pharmaco-toxicology and food research?

The development and validation of reliable in vitro methods alternative to conventional in vivo studies in experimental animals is a well-recognised priority in the fields of pharmaco-toxicology and food research. Conventional studies based on two-dimensional (2-D) cell monolayers have demonstrated their significant limitations: the chemically and spatially defined three-dimensional (3-D) network of extracellular matrix components, cell-to-cell and cell-to-matrix interactions that governs differentiation, proliferation and function of cells in vivo is, in fact, lost under the simplified 2-D condition. Being able to reproduce specific tissue-like structures and to mimic functions and responses of real tissues in a way that is more physiologically relevant than what can be achieved through traditional 2-D cell monolayers, 3-D cell culture represents a potential bridge to cover the gap between animal models and human studies. This article addresses the significance and the potential of 3-D in vitro systems to improve the predictive value of cell-based assays for safety and risk assessment studies and for new drugs development and testing. The crucial role of tissue engineering and of the new microscale technologies for improving and optimising these models, as well as the necessity of developing new protocols and analytical methods for their full exploitation, will be also discussed.     

Early cell death detection with digital holographic microscopy

Background

Digital holography provides a non-invasive measurement of the quantitative phase shifts induced by cells in culture, which can be related to cell volume changes. It has been shown previously that regulation of cell volume, in particular as it relates to ionic homeostasis, is crucially involved in the activation/inactivation of the cell death processes. We thus present here an application of digital holographic microscopy (DHM) dedicated to early and label-free detection of cell death.

Methods and Findings

We provide quantitative measurements of phase signal obtained on mouse cortical neurons, and caused by early neuronal cell volume regulation triggered by excitotoxic concentrations of L-glutamate. We show that the efficiency of this early regulation of cell volume detected by DHM, is correlated with the occurrence of subsequent neuronal death assessed with the widely accepted trypan blue method for detection of cell viability.

Conclusions

The determination of the phase signal by DHM provides a simple and rapid optical method for the early detection of cell death.     

Respiratory activity of avian blood cells

The respiratory activity of avian blood cells was determined with samples of whole blood from individual male and female chickens. this oxygen consumption represents only that of the cells since no measurable activity was found in the plasma samples. the precision of determining respiratory activity was examined statistically and found to be approximately that obtained with a blood cell count but much less precise than the packed cell volume determination. the variability of cell count and mean corpuscular volume indicates that neither is a good means for expressing oxygen consumption – the most meaningful basis is oxygen consumption per milliliter of cells. the relationship between blood cell respiration and temperature is described.     

Fluorescence correlation spectroscopy in small cytosolic compartments depends critically on the diffusion model used

Fluorescence correlation spectroscopy (FCS) is a powerful technique for measuring low concentrations of fluorescent molecules and their diffusion constants. In the standard case, fluorescence fluctuations are measured in an open detection volume defined by the confocal optics. However, if FCS measurements are carried out in cellular processes that confine the detection volume, the standard FCS model leads to erroneous results. In this paper, we derive a modified FCS model that takes into account the confinement of the detection volume. Using this model, we have carried out the first FCS measurements in dendrites of cultured neurons. We further derive, for the case of confined diffusion, the limits within which the standard two- and three-dimensional diffusion models give reliable results.     

RAPID ALDOSTERONE-INDUCED CELL VOLUME INCREASE OF ENDOTHELIAL CELLS MEASURED BY THE ATOMIC FORCE MICROSCOPE

Atomic force microscopy (AFM) is a useful technique for imaging the surface of living cells in three dimensions. The authors applied AFM to obtain morphological information of individual cultured endothelial cells of bovine aorta under stationary and strain conditions and to simultaneously measure changes in cell volume in response to aldosterone. This mineralocorticoid hormone is known to have acute, non-genomic effects on intracellular pH, intracellular electrolytes and inositol-1,4,5-triphosphate production. In this study whether endothelial cells under tension change their volume in response to aldosterone was tested. Such changes were already shown in human leukocytes measured by Coulter counter. In contrast to leukocytes that are more or less spherical and live in suspension, endothelial cells exhibit a complex morphology and adhere to a substrate. Thus, measurements of discrete cell volume changes in endothelial cells under physiological condition is only feasible with more sophisticated techniques. By using AFM we could precisely measure the absolute cell volume of individual living endothelial cells. Before the addition of aldosterone the cell volume of mechanically stressed endothelial cells mimicking arterial blood pressure was 1827±172fl. Cell volume was found to increase by 28% 5min after hormone exposure. Twenty-five minutes later cell volume was back to normal despite the continuous presence of aldosterone in the medium. Amiloride, a blocker of the plasma membrane Na⁺/H⁺exchanger prevented the initial aldosterone-induced volume increase. Taken together, AFM disclosed a transient swelling of endothelial cells induced by the activation of an aldosterone sensitive plasma membrane Na⁺/H⁺exchanger.     

Generation of renal tubules at the interface of an artificial interstitium.

During kidney development a multitude of tubular portions is formed. Little knowledge is available by which cellbiological mechanism a cluster of embryonic cells is able to generate the three-dimensional structure of a tubule. However, this know-how is most important in tissue engineering approaches such as the generation of an artificial kidney module or for the therapy of renal diseases using stem cells. To obtain cellbiological insights in parenchyme development we elaborate a new technique to generate under in vitro conditions renal tubules derived from the embryonic cortex of neonatal rabbits. The aim of the experiments is to establish a specific extracellular environment allowing optimal three-dimensional development of renal tubules under serum-free culture conditions. In the present paper we demonstrate features of the renal stem cell niche and show their isolation as intact microcompartments for advanced tissue culture. Perfusion culture in containers exhibiting a big dead fluid volume results in the development of a flat collecting duct (CD) epithelium at the surface of the tissue explant. In contrast, by fine-tuning the dead fluid volume within a perfusion culture container by an artificial interstitium made of a polyester fleece shows the generation of tubules. It is an up to date unknown morphogenetic information which tells the cells to form tubular structures.     

Cell-to-Cell Diversity in a Synchronized Chlamydomonas Culture As Revealed by Single-Cell Analyses

In a synchronized photoautotrophic culture of Chlamydomonas reinhardtii, cell size, cell number, and the averaged starch content were determined throughout the light-dark cycle. For single-cell analyses, the relative cellular starch was quantified by measuring the second harmonic generation (SHG). In destained cells, amylopectin essentially represents the only biophotonic structure. As revealed by various validation procedures, SHG signal intensities are a reliable relative measure of the cellular starch content. During photosynthesis-driven starch biosynthesis, synchronized Chlamydomonas cells possess an unexpected cell-to-cell diversity both in size and starch content, but the starch-related heterogeneity largely exceeds that of size. The cellular volume, starch content, and amount of starch/cell volume obey lognormal distributions. Starch degradation was initiated by inhibiting the photosynthetic electron transport in illuminated cells or by darkening. Under both conditions, the averaged rate of starch degradation is almost constant, but it is higher in illuminated than in darkened cells. At the single-cell level, rates of starch degradation largely differ but are unrelated to the initial cellular starch content. A rate equation describing the cellular starch degradation is presented. SHG-based three-dimensional reconstructions of Chlamydomonas cells containing starch granules are shown.     

Cell kinetic analysis of a murine macrophage cell line

For the present study, which was performed to find a reliable method suitable for determination of the cell kinetic parameters of a continuous cell line, use was made of the macrophage cell line J774.1. The doubling time of the cell population was approximately 27 h. The continuous labeling curve showed that all the cells divide and almost no quiescent cells occur. The cell-cycle time as determined from the curve of the labeled cells in mitosis, the course of the stathmokinetic index, and time-lapse videorecordings, was about 19 h. The discrepancy between the population doubling time and the cell-cycle time must be due to death and disintegration of cells during culture in vitro. The results indicate that the doubling time of a cell population is not a reliable parameter to determine the kinetics of a population of continuously proliferating cells and that determination of the course of the stathmokinetic index offers a rapid and simple method to establish the cell-cycle time reliably.     

Invasiveness of malignant ST/A mouse lung cells in vitro

ST/A mouse lung cells underwent apparently spontaneous malignant alteration in tissue culture. We have compared the capacity of these cells to form malignant tumours in syngeneic animals with their behaviour in vitro. ST-L1, ST-L22, and ST-L104 cells were malignant, whilst ST-L108 and ST-L109 cells were not. ST-L1 and ST-L22 cells showed anchorage-dependence of growth, whilst ST-L104, ST-L108 and ST-L109 cells did not. ST-L22, ST-L104, ST-L108 and ST-L109 performed directional migration from a spheroid explanted on glass. This capacity was lost in ST-L1 cells, which produced so-called round-cell transformants. All but ST-L108 cells produced type C viral particles. The tumorigenic cell lines ST-L1, ST-L22 and ST-L104 invaded fragments of embryonic chick heart in three-dimensional culture, whereas the non-tumorigenic ST-L108 and ST-L109 cells did not. Furthermore, the histology of ST-L cells invading in three-dimensional culture resembled that of the invasive sarcomas which they produced in vivo. The present observations with ST-L cells confirm that invasiveness in three-dimensional culture is a reliable criterion for malignant of tissue culture lines.