A Bayesian Computational Approach to Explore the Optimal Duration of a Cell Proliferation Assay

Cell proliferation assays are routinely used to explore how a low-density monolayer of cells grows with time. For a typical cell line with a doubling time of 12 h (or longer), a standard cell proliferation assay conducted over 24 h provides excellent information about the low-density exponential growth rate, but limited information about crowding effects that occur at higher densities. To explore how we can best detect and quantify crowding effects, we present a suite of in silico proliferation assays where cells proliferate according to a generalised logistic growth model. Using approximate Bayesian computation we show that data from a standard cell proliferation assay cannot reliably distinguish between classical logistic growth and more general non-logistic growth models. We then explore, and quantify, the trade-off between increasing the duration of the experiment and the associated decrease in uncertainty in the crowding mechanism.     

Multi-scale modeling of a wound-healing cell migration assay

A continuum model and a discrete model are developed to capture the population-scale and cell-scale behavior in a wound-healing cell migration assay created from a scrape wound in a confluent cell monolayer. During wound closure, the cell population forms a sustained traveling wave, with close contact between cells behind the wavefront. Cells exhibit contact inhibition of migration and contact-limited proliferation. The continuum model includes the two dominant mechanisms and characteristics of cell migration and proliferation, using a cell diffusivity function that decreases with cell density and a logistic proliferative growth term. The discrete model arises naturally from the continuum model. Individual cells are simulated as continuous-time random walkers with nearest-neighbor transitions, together with a birth/death process. The migration and proliferation parameters are determined by analysing individual mice 3T3 fibroblast cell trajectories obtained during the development of a confluent cell monolayer and in a wound healing assay. The population-scale model successfully predicts the shape and speed of the traveling wave, while the discrete model is also successful in capturing the contact inhibition of migration effects.     

Adipose tissue extract shows potential for wound healing: in vitro proliferation and migration of cell types contributing to wound healing in the presence of adipose tissue preparation and platelet rich plasma

Growth factors are the key elements in wound healing signaling for cell migration, differentiation and proliferation. Platelet-rich plasma (PRP), one of the most studied sources of growth factors, has demonstrated to promote wound healing in vitro and in vivo. Adipose tissue is an alternative source of growth factors. Through a simple lipoaspirate method, adipose derived growth factor-rich preparation (adipose tissue extract; ATE) can be obtained. The authors set out to compare the effects of these two growth factor sources in cell proliferation and migration (scratch) assays of keratinocyte, fibroblast, endothelial and adipose derived stem cells. Growth factors involved in wound healing were measured: keratinocyte growth factor, epidermal growth factor, insulin-like growth factor, interleukin 6, platelet-derived growth factor beta, tumor necrosis factor alfa, transforming growth factor beta and vascular endothelial growth factor. PRP showed higher growth factor concentrations, except for keratinocyte growth factor, that was present in adipose tissue in greater quantities. This was reflected in vitro, where ATE significantly induced proliferation of keratinocytes at day 6 (p < 0.001), compared to plasma and control. Similarly, ATE-treated fibroblast and adipose stem cell cultures showed accelerated migration in scratch assays. Moreover, both sources showed accelerated keratinocyte migration. Adipose tissue preparation has an inductive effect in wound healing by proliferation and migration of cells involved in wound closure. Adipose tissue preparation appears to offer the distinct advantage of containing the adequate quantities of growth factors that induce cell activation, proliferation and migration, particularly in the early phase of wound healing.     

A Growth Curve of Cell Numbers In the Neural Retina of Embryonic Chicks

ABSTRACT The growth of cell numbers in a normal embryonic population of ncural retinal cells is described. the numbers were estimated from a time shortly after the neural retina first becomes recognizable to a time when numbers of retinal cells have become steady. Cell numbers were estimated in preparations of an entire neural retina dispersed into a suspension of single nuclei which were then counted in a Coulter counter. the growth curve of the In numbers of cells has three phases of growth: an exponential phase during which there is steady-state exponential growth, a differentiative phase during which cell proliferation ceases and an end phase when no further change in cell numbers can be detected. the variances of the In numbers of cells were highest during the exponential phase. the variances decreased during the differentiative phase and were at their lowest during the end phase. For variances to decrease requires mechanisms which control the final numbers of cells in the neural retina very precisely. the implications of mechanisms which operate by controlling cell lineages are explored.     

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.     

Low Oxygen Tension Primes Aortic Endothelial Cells to the Reparative Effect of Tissue-Protective Cytokines

Erythropoietin (EPO) has both erythropoietic and tissue-protective properties. The EPO analogues carbamylated EPO (CEPO) and pyroglutamate helix B surface peptide (pHBSP) lack the erythropoietic activity of EPO but retain the tissue-protective properties that are mediated by a heterocomplex of EPO receptor (EPOR) and the β common receptor (βCR). We studied the action of EPO and its analogues in a model of wound healing where a bovine aortic endothelial cells (BAECs) monolayer was scratched and the scratch closure was assessed over 24 h under different oxygen concentrations. We related the effects of EPO and its analogues on repair to their effect on BAECs proliferation and migration (evaluated using a micro-Boyden chamber). EPO, CEPO and pHBSP enhanced scratch closure only at lower oxygen (5%), while their effect at atmospheric oxygen (21%) was not significant. The mRNA expression of EPOR was doubled in 5% compared with 21% oxygen, and this was associated with increased EPOR assessed by immunofluorescence and Western blot. By contrast, βCR mRNA levels were similar in 5% and 21% oxygen. EPO and its analogues increased both BAECs proliferation and migration, suggesting that both may be involved in the reparative process. The priming effect of low oxygen tension on the action of tissue-protective cytokines may be of relevance to vascular disease, including atherogenesis and restenosis.     

Experimental Investigation of HGF Inhibiting Glial Scar In Vitro

To study the inhibitory effect of Hepatocyte growth factor (HGF) on the responsive hyperplasia of damaged astrocytes in vitro. We prepared damaged model of astrocytes to simulate the responsive hyperplasia of damaged astrocytes in vivo by culturing astrocytes in vitro; After the first day of Ad-HGF transfection, astrocytes were scratched, then after the first, the third, and the fifth day of scratch, we detect the expression amount of astrocytes specific glial fibrillary acidic protein (GFAP) and the ratio of S-phase cells with flow cytometry, both of which can reflect the proliferation status of damaged astrocytes; After HGF was added in scratched astrocytes, the activity of SPK and MAPK (P42/44) were detected by autoradiography and immunoblotting test; After adding different concentrations of HGF protein in astrocytes cultured in different serum concentrations and adding diverse concentrations of HGF protein, SPK and SPK inhibitor DMS in scratched astrocytes, we detect cell proliferation with 3H-TDR incorporation. The first day after Ad-HGF transfected astrocytes were scratched, the amount of GFAP secreted by astrocytes were decreased significantly (P < 0.05), and the cells in S phase were declined obviously. HGF has bidirectional regulation on SPK of scratched astrocytes: increases the SPK activity when HGF in low dose, while inhibits when in high dose. In addition, DMS can block the signal passage; HGF had no effects on MAPK (P42/44) of damaged astrocytes cells. In conclusion, after the transfection of Ad-HGF, it can inhibit the responsive hyperplasia of damaged astrocytes by the means of blocking SPK passage.     

Using the scratch assay to study cell migration in an inquiry-based cell biology lab

Cell migration, a fundamental process in development, wound healing, and immune function, is a common topic in undergraduate cell biology courses. We developed laboratory exercises with an inquiry-based learning (IBL) approach in which cell migration could be examined with the scratch assay, adapted from the primary literature. A narrow scratch was created in a confluent monolayer of cells growing on the bottom of a cell culture dish. Migration into the resultant cell-free zone from both sides of the scratch was measured after one day using the scale bar function of a digital camera. The Chinese hamster ovary cell line was used, but any adherent cell type could be examined. Students used the scratch assay to formulate hypotheses and design experiments in which variables affecting cell migration could be investigated. For example, the effect of cytoskeletal disruption was evaluated by adding the microtubule- and microfilament-disrupting drugs, colcemid and phallacidin, respectively, to the growth medium when the scratch was made. Optimal drug concentration parameters were determined for students to reference. Low drug concentrations inhibited cell migration, while higher concentrations killed the cells. This study demonstrated that the scratch assay is an accessible IBL method for studying cell migration.     

In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro

The in vitro scratch assay is an easy, low-cost and well-developed method to measure cell migration in vitro. The basic steps involve creating a "scratch" in a cell monolayer, capturing the images at the beginning and at regular intervals during cell migration to close the scratch, and comparing the images to quantify the migration rate of the cells. Compared to other methods, the in vitro scratch assay is particularly suitable for studies on the effects of cell-matrix and cell-cell interactions on cell migration, mimic cell migration during wound healing in vivo and are compatible with imaging of live cells during migration to monitor intracellular events if desired. Besides monitoring migration of homogenous cell populations, this method has also been adopted to measure migration of individual cells in the leading edge of the scratch. Not taking into account the time for transfection of cells, in vitro scratch assay per se usually takes from several hours to overnight.     

A quantitative, facile, and high-throughput image-based cell migration method is a robust alternative to the scratch assay

Cell migration is a key phenotype for a number of therapeutically important biological responses, including angiogenesis. A commonly used method to assess cell migration is the scratch assay, which measures the movement of cells into a wound made by physically scoring a confluent cell monolayer to create an area devoid of cells. Although this method has been adequate for qualitative characterization of migration inhibitors, it does not provide the highly reproducible results required for quantitative compound structure-activity relationship evaluation because of the inconsistent size and placement of the wound area within the microplate well. The Oris? Cell Migration Assay presents a superior alternative to the scratch assay, permitting formation of precisely placed and homogeneously sized cell-free areas into which migration can occur without releasing factors from wounded or dead cells or damaging the underlying extracellular matrix. Herein the authors compare results from the scratch and Oris? cell migration assays using an endothelial progenitor cell line and the Src kinase inhibitor dasatinib. They find that using the Acumen? Explorer laser microplate cytometer in combination with the Oris? Cell Migration Assay plate provides a robust, efficient, and cost-effective cell migration assay exhibiting excellent signal to noise, plate uniformity, and statistical validation metrics.     

Development,validation and pilot screening of an in vitro multi-cellular three-dimensional cancer spheroid assay for anti-cancer drug testing

It has been demonstrated that two-dimensional (2D) monolayer cancer cell proliferation assay for anti-cancer drug screening is a very artificial model and cannot represent the characteristics of three-dimensional (3D) solid tumors. The multi-cellular in vitro 3D tumor spheroid model is of intermediate complexity, and can provide a bridge to the gap between the complex in vivo tumors and simple in vitro monolayer cell cultures. In this study, a simple and cost-effective cancer 3D spheroid assay suitable for small molecule anti-cancer compound screening was developed, standardized and validated on H292 non-small lung cancer cell line. A pilot screening with this assay was performed utilizing a compound library consisting of 41 anti-cancer agents. The traditional 2D monolayer cell proliferation assay was also performed with the same cell line and compounds. A correlational study based on the IC₅₀ values from the 2D and 3D assays was conducted. There is low correlation with the two sets of biological data, suggesting the two screening methods provide different information regarding the potency of the tested drug candidates.     

Tumour suppressive function of HUWE1 in thyroid cancer

HUWE1 (the HECT, UBA, and WWE domain-containing protein 1) is an ubiquitin E3 ligase which plays an important role in coordinating diverse cellular processes. It has been found to be dysregulated in various cancer type and its functions in tumorigenesis remain controversial. The potential tumour suppressive role of HUWE1 in thyroid cancer development was investigated by knocking down HUWE1 in three authentic thyroid cancer cell lines, WRO, FTC133 and BCPAP, followed by various functional assays, including cell proliferation, scratch wound healing and invasion assays. Xenograft experiment was performed to examine in vivo tumour suppressive properties of HUWE1. Small-interfering RNA mediated knockdown of HUWE1 promoted cell proliferation, cell migration and invasion in thyroid cancer cells. Overexpression of HUWE1 conferred partial sensitivity to chemo drugs interfering with DNA replication in these cells. Moreover, HUWE1 was found to be down-regulated in human thyroid cancer tissues compared with matched normal thyroid tissues. In addition, overexpression of HUWE1 significantly inhibited tumour growth in vivo using xenograft mouse models. Mechanistic investigation revealed that HUWE1 can regulate p53 protein level through its stabilization. HUWE1 functions as a tumour suppressor in thyroid cancer progression, which may represent a novel therapeutic target for prevention or intervention of thyroid cancer.     

Measuring permeability with a whole cell-based biosensor as an alternate assay for angiogenesis: comparison with common in vitro assays

Angiogenesis plays cardinal role in normal developmental processes as well as in numerous pathologies. Multiple cytokines are released and act simultaneously to activate endothelial cells in vivo. The present study investigated the relative ability of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), tumor necrosis factor-alpha (TNF-alpha) in modulating cell monolayer permeability, migration, proliferation and tube formation individually and in combination. While the common methods for assaying angiogenesis were conducted for studying cell migration, proliferation and differentiation, endothelial cell monolayer permeability studies were carried out using a whole cell-based biosensor. The biosensor, consisting of a confluent monolayer of human umbilical vein endothelial cells (HUVECs) on a potassium ion-selective electrode, takes advantage of cell monolayer permeability dysfunction to detect the presence of small quantities of cytokines. When a confluent monolayer of cells was formed on the membrane surface, the response of the electrode toward the marker ion, potassium, was inhibited. The response obtained after exposing this sensor to different cytokines for 1 and 3h, can be attributed to the modulation of monolayer permeability by these cytokines. The present study demonstrated that at the concentrations experimented with, the relative change in permeability assay in the presence of cytokines compared to the control was much higher than that observed in other assays, thereby bolstering the potential of the biosensor to act as a quick screening tool for angiogenesis.     

Crucial role of vinexin for keratinocyte migration in vitro and epidermal wound healing in vivo

In the process of tissue injury and repair, epithelial cells rapidly migrate and form epithelial sheets. Vinexin is a cytoplasmic molecule of the integrin-containing cell adhesion complex localized at focal contacts in vitro. Here, we investigated the roles of vinexin in keratinocyte migration in vitro and wound healing in vivo. Vinexin knockdown using siRNA delayed migration of both HaCaT human keratinocytes and A431 epidermoid carcinoma cells in scratch assay but did not affect cell proliferation. Induction of cell migration by scratching the confluent monolayer culture of these cells activated both EGFR and ERK, and their inhibitors AG1478 and U0126 substantially suppressed scratch-induced keratinocyte migration. Vinexin knockdown in these cells inhibited the scratch-induced activation of EGFR, but not that of ERK, suggesting that vinexin promotes cell migration via activation of EGFR. We further generated vinexin (−/−) mice and isolated their keratinocytes. They similarly showed slow migration in scratch assay. Furthermore, vinexin (−/−) mice exhibited a delay in cutaneous wound healing in both the back skin and tail without affecting the proliferation of keratinocytes. Together, these results strongly suggest a crucial role of vinexin in keratinocyte migration in vitro and cutaneous wound healing in vivo.     

Ethyl isopropyl amiloride inhibits smooth muscle cell proliferation and migration by inducing apoptosis and antagonizing urokinase plasminogen activator activity

Amiloride inhibits activation of the Na(+)-H+ exchanger (NHE), a critical step in smooth muscle cell (SMC) growth. While amiloride treatment reduces SMC proliferation and migration, as well as experimental lesion formation, these effects are not exclusively due to NHE inhibition and remain incompletely understood. The purpose of this study was to examine the mechanisms involved in amiloride-induced attenuation of SMC proliferation and migration, looking specifically at the potential role of apoptosis and urokinase plasminogen activator (uPA) activity in these processes. Rabbit SMCs in tissue culture were exposed to 10-80 microM of the amiloride analogue ethyl isopropyl amiloride (EIPA). Compared with controls, EIPA reduced DNA synthesis, cell number, and mitochondrial respiration, but without toxic effects on quiescent or proliferating cells. In a Boyden chamber assay, EIPA reduced uPA-induced SMC migration. Moreover, in a SMC scratch assay EIPA treatment resulted in a 66% reduction in the number of repopulating cells, a 92% decrease in the number of proliferating cells, and a 37-fold increase in the number of apoptotic cells. SMC apoptosis was frequently localized to the scratch edges, where cell proliferation and bcl-2 expression were absent. Finally, uPA enzymatic activity in the cell culture media was lower for EIPA-treated versus control SMCs. Therefore, EIPA inhibits both SMC proliferation and migration by inducing apoptosis and antagonizing uPA activity, respectively, and requires further study as an agent for reducing vascular lesion formation.     

Adrenaline Increases Cyclic 3′5′-AMP Formation in Hamster Epidermis

CATECHOLAMINES probably influence cell proliferation by delaying cells in the premitotic phase^1,2. Bullough and Laurence found that crude skin extracts contained a tissue-specific protein (chalone) which inhibited epidermal cell proliferation and that the action of this extract was augmented by adrenaline³. They later found that adrenaline alone (0.00025 µg/ml.) reduced epidermal mitotic activity in mouse ears by about 50% in vitro⁴.     

A high-throughput soft agar assay for identification of anticancer compound

A 384-well soft agar assay was developed to identify potential novel anticancer compounds. Normally used to detect cell transformation, the assay is used here to quantitate cell proliferation in a 3-dimensional (3-D) anchorage-independent format. HCC827 cells, which are highly sensitive to epithelial growth factor receptor (EGFR) tyrosine kinase inhibitors, were used to develop the method and a set of 9600 compounds used to validate the assay. Results were compared to a monolayer assay using the same compound set. The assay provides a robust method to discover compounds that could be missed using traditional monolayer formats.     

Space-Limited Mitosis in the Glazier–Graner–Hogeweg Model

The Glazier–Graner–Hogeweg (GGH) model is a cellular automata framework for representing the time evolution of cellular systems, appealing because unlike many other individual-cell-based models it dynamically simulates changes in cell shape and size. Proliferation has seen some implementation into this modelling framework, but without consensus in the literature as to how this behaviour is best represented. Additionally, the majority of published GGH model implementations which feature proliferation do so in order to simulate a certain biological situation where mitosis is important, but without analysis of how these proliferation routines operate on a fundamental level. Here, a method of proliferation for the GGH model which uses separate cell phenotypes to differentiate cells which have entered or just left the mitotic phase of the cell cycle is presented and demonstrated to correctly predict logistic growth on a macroscopic scale (in accordance with experimental evidence). Comparisons between model simulations and the generalised logistic growth model provide an interpretation of the latter’s ‘shape parameter’, and the proliferation routine used here is shown to offer the modeller somewhat predictable control over the proliferation rate, important for ensuring temporal consistency between different cellular behaviours in the model. All results are found to be insensitive to the inclusion of active cell motility. The implications of these simulated proliferation assays towards problems in cell biology are also discussed.     

Growth inhibition of estrogen receptor-positive and aromatase-positive human breast cancer cells in monolayer and spheroid cultures by letrozole, anastrozole, and tamoxifen

Two third-generation aromatase inhibitors, letrozole and anastrozole, and the antiestrogen tamoxifen, were compared for growth-inhibiting activity in two estrogen receptor (ER)-positive aromatase-overexpressing human breast cancer cell lines, MCF-7aro and T-47Daro. Inhibition of hormone (1 nM testosterone)-stimulated proliferation was evaluated in both monolayer cultures and in three-dimensional spheroid cultures. Letrozole and anastrozole were also compared for effectiveness of aromatase inhibition, and relative affinity for aromatase, under both monolayer and spheroid growth conditions. Letrozole was an effective inhibitor of MCF-7aro monolayer cell proliferation, with an estimated 50% inhibitory concentration (IC50) of 50-100 nM, whereas an IC50 was not reached with anastrozole at any concentration tested (100-500 nM). An IC50 of tamoxifen was 1000 nM. Proliferation of T-47Daro monolayer cells was more sensitive to inhibition by all three agents; as with MCF-7aro cells, letrozole was the most effective inhibitor. MCF-7aro spheroids were slightly less sensitive than monolayer cells proliferation-inhibiting effects of letrozole (IC50 about 200 nM), and there was no significant inhibition with 100-200 nM anastrozole or 200-1000 nM tamoxifen. Letrozole and anastrozole significantly inhibited T-47Daro spheroid cell proliferation, at 15-25 and 50 nM, respectively, consistent with the greater sensitivity of T-47Daro monolayer cells to inhibition of proliferation by these agents. Tamoxifen failed to significantly inhibit T-47Daro spheroid cell proliferation over a 100-500 nM concentration range. Determination of aromatase inhibition in monolayers of both cell lines by a direct-access microsomal assay and an intact-cell assay revealed that letrozole was more active than anastrozole in monolayers of both cell lines and in both assays. In MCF-7aro spheroids following cell lysis, only letrozole significantly inhibited aromatase activity, supporting the conclusion that letrozole binds stronger to aromatase than anastrozole does. Our results demonstrate that MCF-7aro and T-47Daro spheroids could be a suitable model for evaluation of growth-inhibitory effects of agents used in hormonal therapy of breast cancer.