Multilayered Culture Research Articles

A High-Throughput Screening Model of the Tumor Microenvironment for Ovarian Cancer Cell Growth

The tumor microenvironment plays an important role in the processes of tumor growth, metastasis, and drug resistance. We have used a multilayered 3D primary cell culture model that reproduces the human ovarian cancer metastatic microenvironment to study the effect of the microenvironment on the pharmacological responses of different classes of drugs on cancer cell proliferation. A collection of oncology drugs was screened to identify compounds that inhibited the proliferation of ovarian cancer cells growing as monolayers or forming spheroids, on plastic and on a 3D microenvironment culture model of the omentum metastatic site, and also cells already in preformed spheroids. Target-based analysis of the pharmacological responses revealed that several classes of targets were more efficacious in cancer cells growing in the absence of the metastatic microenvironment, and other target classes were less efficacious in cancer cells in preformed spheres compared to forming spheroid cultures. These findings show that both the cellular context of the tumor microenvironment and cell adhesion mode have an essential role in cancer cell drug resistance. Therefore, it is important to perform screens for new drugs using model systems that more faithfully recapitulate the tissue composition at the site of tumor growth and metastasis.

多層灌流共培養のための2層コラーゲンマイクロチューブデバイス

多層灌流共培養のための2層コラーゲンマイクロチューブデバイス

Construction and characterization of a multilayered gingival keratinocyte culture model: the TURK-U model.

In construction of epithelial cells as multilayers, the cells are grown submerged to confluence on fibroblast-embedded collagen gels and, then, lifted to air to promote their stratification. We recently demonstrated that gingival epithelial cells form uniform monolayers on semi-permeable nitrocellulose membranes, supported with a semi-solid growth medium, which allows the cells to grow at an air-liquid-solid interface from the beginning of the culturing protocol. In this study, the aim was to further develop our previous model to form a multilayered gingival epithelial culture model. Two different epithelial cell lines (HaCaT from skin and HMK from gingiva) were used in all experiments. Both cell lines were grown first as monolayers for 3days. After that, keratinocytes were trypsinized, counted and seeded on a sterile semi-permeable nitrocellulose membrane placed on the top of a semi-solid growth medium, forming an air-liquid-solid interface for the cells to grow. At days 1, 4, and 7, epithelial cells were fixed, embedded in paraffin, and sectioned for routine Hematoxylin-Eosin staining and immunohistochemistry for cytokeratin (Ck). At day 1, HMK cells grew as monolayers, while HaCaT cells stratified forming an epithelium with two to three layers. At day 4, a stratified epithelium in the HMK model had four to five layers and its proliferation continued up to day 7. HaCaT cells formed a dense and weakly proliferating epithelium with three to four layers of stratification at day 4 but the proliferation disappeared at day 7. At all days, both models were strongly positive for Ck5, Ck7, and Ck 19, and weakly positive for Ck10. Gingival epithelial cells stratify successfully on semi-permeable nitrocellulose membranes, supported with a semi-solid growth medium. This technique allows researchers to construct uniform gingival epithelial cell multilayers at an air-liquid-solid interface, without using collagen gels, resulting in a more reproducible method.

Erratum: Quantitative high throughput screening using a primary human three-dimensional organotypic culture predicts in vivo efficacy

The tumour microenvironment contributes to cancer metastasis and drug resistance. However, most high throughput screening (HTS) assays for drug discovery use cancer cells grown in monolayers. Here we show that a multilayered culture containing primary human fibroblasts, mesothelial cells and extracellular matrix can be adapted into a reliable 384- and 1,536-multi-well HTS assay that reproduces the human ovarian cancer (OvCa) metastatic microenvironment. We validate the identified inhibitors in secondary in vitro and in vivo biological assays using three OvCa cell lines: HeyA8, SKOV3ip1 and Tyk-nu. The active compounds directly inhibit at least two of the three OvCa functions: adhesion, invasion and growth. In vivo, these compounds prevent OvCa adhesion, invasion and metastasis, and improve survival in mouse models. Collectively, these data indicate that a complex three-dimensional culture of the tumour microenvironment can be adapted for quantitative HTS and may improve the disease relevance of assays used for drug screening.

Development of low-cost on demand multilayer cell culture platforms

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Canine Adipose-Derived Stem Cell Aggregates as a Viable Substitute to Actual Canine Dermal Papillae

Abstract Hair loss is a major dermatological disease in veterinary and human medicine. Active studies on hair regeneration with mesenchymal stem cells have been performed in an effort to solve the limitations of conservative treatments in human medicine. Our understanding of the canine hair follicle (HF), considering a useful model for the study of the human alopecia, is limited. This study was designed to broaden our understanding of canine dermal papilla (DP), and to reconstruct dermal papilla-like tissue (DPLT) using canine adipose-derived mesenchymal stem cells (AD-MSCs), as an alternative to actual DP. We cultured canine DPs, observed their culture patterns and compared their expression level of DP-related genes and proteins with those of DPLTs by performing RT-PCR analysis and Western blotting. Canine dermal papilla cells (DPCs) showed multilayer culture patterns with pseudo-papillae. Reconstruction of DPLTs was performed successfully. Not only were they morphologically similar to actual DPs, but we also observed similarities between DPCs and DPLTs in molecular characteristics. These findings suggested that DPLT was a viable substitute for DP. This study will not only be helpful for understanding the morphological and molecular characteristics of canine DPCs, but may also serve as a basis for understanding human hair follicle biology and potential therapeutic strategies for alopecia.

In silico modelling and analysis of the electrical and mechanical properties of in vitro cardiac cultures with different fiber architectures.

Today, in vitro cardiac cultures are widely exploited to investigate several aspects of the electromechanical behavior of the cardiac tissue. Thus, new forecasts may derive from modelling their properties. In particular, in this paper, we focus on the fiber architecture of cultures, i.e. on the way cellular sarcomeres are locally oriented, when they are designed to be cardiac patches. We employ a three-dimensional model to simulate the bioelectrical activity and the biomechanics of a multilayered culture made of ventricular cells and with four possible architectures consisting of: i) random fibers in all cells; ii) randomly rotating fibers among layers; iii) structurally rotating fibers from the bottom layer to the top one; iv) parallel fibers among layers. Our results suggest that the best configuration for a patch may be the architecture with structurally rotating fibers, which is the one that most approaches the anisotropic structure of the in vivo heart, thanks to its better electrical and mechanical performances.

Quantitative high throughput screening using a primary human three-dimensional organotypic culture predicts in vivo efficacy.

The tumour microenvironment contributes to cancer metastasis and drug resistance. However, most high throughput screening (HTS) assays for drug discovery use cancer cells grown in monolayers. Here we show that a multilayered culture containing primary human fibroblasts, mesothelial cells and extracellular matrix can be adapted into a reliable 384- and 1,536-multi-well HTS assay that reproduces the human ovarian cancer (OvCa) metastatic microenvironment. We validate the identified inhibitors in secondary in vitro and in vivo biological assays using three OvCa cell lines: HeyA8, SKOV3ip1 and Tyk-nu. The active compounds directly inhibit at least two of the three OvCa functions: adhesion, invasion and growth. In vivo, these compounds prevent OvCa adhesion, invasion and metastasis, and improve survival in mouse models. Collectively, these data indicate that a complex three-dimensional culture of the tumour microenvironment can be adapted for quantitative HTS and may improve the disease relevance of assays used for drug screening.

Elucidating the Uptake and Distribution of Nanoparticles in Solid Tumors via a Multilayered Cell Culture Model.

Multicellular layers (MCLs) have previously been used to determine the pharmacokinetics of a variety of different cancer drugs including paclitaxel, doxorubicin, methotrexate, and 5-fluorouracil across a number of cell lines. It is not known how nanoparticles (NPs) navigate through the tumor microenvironment once they leave the tumor blood vessel. In this study, we used the MCL model to study the uptake and penetration dynamics of NPs. Gold nanoparticles (GNPs) were used as a model system to map the NP distribution within tissue-like structures. Our results show that NP uptake and transport are dependent on the tumor cell type. MDA-MB-231 tissue showed deeper penetration of GNPs as compared to MCF-7 one. Intracellular and extracellular distributions of NPs were mapped using CytoViva imaging. The ability of MCLs to mimic tumor tissue characteristics makes them a useful tool in assessing the efficacy of particle distribution in solid tumors.Electronic supplementary materialThe online version of this article (doi:10.1007/s40820-014-0025-1) contains supplementary material, which is available to authorized users.

A multilayered cell culture model for transport study in solid tumors: Evaluation of tissue penetration of polyethyleneimine based cationic micelles

Limited drug distribution is partially responsible for the efficacy gap between preclinical and clinical studies of nano-sized drug carriers for cancer therapy. In this study, we examined the transport behavior of cationic micelles formed from a triblock copolymer of poly(d,l-lactide-co-glycolide)-block-branched polyethyleneimine-block-poly(d,l-lactide-co-glycolide) using a unique in vitro tumor model composed of a multilayered cell culture (MCC) and an Ussing chamber system. The Cy3-labeled cationic micelles showed remarkable Cy3 distribution in the MCC whereas charge-shielded micelles with a poly(ethylene glycol) surface accumulated on the surface of the MCC. Penetration occurred against convectional flow caused by a hydraulic pressure gradient. The study using fluorescence resonance energy transfer (FRET) showed that the cationic micelles dissociate at the interface between the culture media and the MCC or possibly inside of the first-layer cells and penetrates into the MCC as unimers. The penetration and distribution were energy-dependent and suppressed by various endocytic inhibitors. These suggest that cationic unimers mainly utilized clathrin-mediated endocytosis and macropinocytosis for cellular entry and a significant fraction were exocytosed by an unknown mechanism.

Multilayer three-dimensional filter paper constructs for the culture and analysis of aortic valvular interstitial cells

Culturing aortic valvular interstitial cells in an environment that models the aortic valve is an essential step towards understanding the progression of calcific aortic valve disease. Here the adaption of a three-dimensional (3-D) stacked paper-based culture system is presented for analyzing valve cells in a thick collagen gel matrix. Filter paper layers, modeled after a 96-well plate design, were printed with a wax well-plate template and then seeded with valve cell and collagen mixtures that quickly gelled into 3-D cultures. Stacking these layers permitted extensive customization of culture thickness and cell density profiles to model the full thickness of native valve tissue. Aortic valvular interstitial cells seeded into the paper-based constructs consistently demonstrated high survival up to 14days of culture with significant increases in cell number through the first 3days of culture. After 4days following seeding, valve cells in single layer cultures showed reduced smooth muscle α-actin expression with a stabilized cell density, suggesting a transition from an activated phenotype to a more quiescent state. Valve cells in multilayer cultures demonstrated the ability to migrate from layer to layer and had the highest smooth muscle α-actin expression in areas with predicted low oxygen tensions. These results establish the filter-paper-based method as a viable culture system for analyzing valve cells in an in vitro 3-D model of the aortic valve.

Cultivated Vaginal Microbiomes Alter HIV-1 Infection and Antiretroviral Efficacy in Colonized Epithelial Multilayer Cultures

There is a pressing need for modeling of the symbiotic and at times dysbiotic relationship established between bacterial microbiomes and human mucosal surfaces. In particular clinical studies have indicated that the complex vaginal microbiome (VMB) contributes to the protection against sexually-transmitted pathogens including the life-threatening human immunodeficiency virus (HIV-1). The human microbiome project has substantially increased our understanding of the complex bacterial communities in the vagina however, as is the case for most microbiomes, very few of the community member species have been successfully cultivated in the laboratory limiting the types of studies that can be completed. A genetically controlled ex vivo model system is critically needed to study the complex interactions and associated molecular dialog. We present the first vaginal mucosal culture model that supports colonization by both healthy and dysbiotic VMB from vaginal swabs collected from routine gynecological patients. The immortalized vaginal epithelial cells used in the model and VMB cryopreservation methods provide the opportunity to reproducibly create replicates for lab-based evaluations of this important mucosal/bacterial community interface. The culture system also contains HIV-1 susceptible cells allowing us to study the impact of representative microbiomes on replication. Our results show that our culture system supports stable and reproducible colonization by VMB representing distinct community state types and that the selected representatives have significantly different effects on the replication of HIV-1. Further, we show the utility of the system to predict unwanted alterations in efficacy or bacterial community profiles following topical application of a front line antiretroviral.

Ultrastructure of blood and lymphatic vascular networks in three-dimensional cultured tissues fabricated by extracellular matrix nanofilm-based cell accumulation technique

Cell accumulation technique is an extracellular matrix (ECM) nanofilm-based tissue-constructing method that enables formation of multilayered hybrid culture tissues. In this method, ECM-nanofilm is constructed using layer-by-layer assembly of fibronectin and gelatin on culture cells. The ECM-nanofilm promotes cell-to-cell interaction; then the three-dimensional (3D) multilayered tissue can be constructed with morphological change of the cells mimicking living tissues. By using this method, we have successfully produced tubular networks of human umbilical venous endothelial cells (HUVECs) and human dermal lymphatic endothelial cells (HDLECs) in 3D multilayered normal human dermal fibroblasts (NHDFs). This study demonstrated morphological characteristics of the vascular networks in the engineered tissues by using light and electron microscopy. In light microscopy, HUVECs and HDLECs formed luminal structures such as native blood and lymphatic capillaries, respectively. Electron microscopy showed distinct ultrastructural aspects of the vasculature of HUVECs or HDLECs with intermediated NHDFs and abundant ECM. The vasculature constructed by HUVECs exhibited structures similar to native blood capillaries, involving overlapping endothelial connections with adherens junctions, abundant vesicles in the endothelial cells and basement membrane-like structure. The detection of laminin around HUVEC-constructed vessels supported the presence of perivascular basal lamina. The vasculature constructed by HDLECs showed some ultrastructural characteristics similar to those of native lymphatic capillaries such as irregular vascular shape, loose adhesive connection and gap formation between endothelial cells. In conclusion, our novel vascular network models fabricated by the cell accumulation technique provide highly organized blood and lymphatic capillary networks mimicking the vasculatures in vivo.

Two-Stage Transplantation of Cell-Engineered Autologous Auricular Chondrocytes to Regenerate Chondrofat Composite Tissue

The authors have developed a unique multilayered culture method that expands to large volumes elastic chondrocytes from a small piece of human auricular cartilage. In this study, the authors applied the two-stage transplantation method for cultured auricular chondrocytes to difficult cases of nasal/chin reconstruction where subcutaneous tissue is thin or scarred. Auricular chondrocytes were cultured and expanded to sufficiently large volumes, and then, in a two-stage transplantation process, injection-transplanted into a patient's lower abdomen, where they were regenerated into larger chondrofat composite tissue in 6 months and used as a material for nasal/chin reconstruction. The authors then performed histologic and electron microscopic analysis of serial cross-sections and magnetic resonance imaging analysis of the chondrofat composite tissue. The cultured auricular chondrocytes consistency regenerated intraabdominally to a larger, stable neocartilage, with adherent fat tissue within 6 months. Eighteen patients (nose, n = 14; chin, n = 4) underwent this procedure, and the chondrofat composite tissue was stable after 1 to 5 years' postoperative follow-up. The chondrofat composite tissue maintained good shape, with no major complications. Magnetic resonance imaging showed that the chondrofat composite tissue was regenerated and vascularized in the abdomen in all 18 cases (100 percent). Infection and total absorption were not seen. Only partial absorption was noted (5.6 percent). The chondrofat composite tissue was found to be a new innovative graft material in which neocartilage is regenerated to be continuous with fat tissue by means of the neoperichondrium. It has thereby become possible to perform the previously impossible simultaneous reconstruction of cartilage and fat tissue.

P. gingivalis Modulates Keratinocytes through FOXO Transcription Factors

P. gingivalis is a prominent periodontal pathogen that has potent effects on host cells. In this study we challenged gingival epithelial cells with P. gingivalis with the aim of assessing how mRNA levels of key target genes were modulated by P. gingivalis via the transcription factors FOXO1 and FOXO3. Primary mono- and multi-layer cultures of gingival epithelial cells were challenged and barrier function was examined by fluorescent dextran and apoptosis was measured by cytoplasmic histone associated DNA. Gene expression levels were measured by real-time PCR with and without FOXO1 and FOXO3 siRNA compared to scrambled siRNA. P. gingivalis induced a loss of barrier function and stimulated gingival epithelial cell apoptosis in multilayer cultures that was in part gingipain dependent. P. gingivalis stimulated an increase in FOXO1 and FOXO3 mRNA, enhanced mRNA levels of genes associated with differentiated keratinocyte function (keratin-1, -10, -14, and involucrin), increased mRNA levels of apoptotic genes (BID and TRADD), reduced mRNA levels of genes that regulate inflammation (TLR-2 and -4) and reduced those associated with barrier function (integrin beta-1, -3 and -6). The ability of P. gingivalis to modulate these genes was predominantly FOXO1 and FOXO3 dependent. The results indicate that P. gingivalis has pronounced effects on gingival keratinocytes and modulates mRNA levels of genes that affect host response, differentiation, apoptosis and barrier function. Moreover, this modulation is dependent upon the transcription factors FOXO1 or FOXO3. In addition, a new function for FOXO1 was identified, that of suppressing TLR-2 and TLR-4 and maintaining integrin beta -1, beta -3 and beta -6 basal mRNA levels in keratinocytes.

Optimal Differentiation of In Vitro Keratinocytes Requires Multifactorial External Control

For almost 30 years, keratinocyte differentiation has been studied in numerous cell models including keratinocyte primary culture with various supplemented culture media. In this respect, it has become quite difficult to draw comparisons between studies using such a variety of culture conditions. Serum-free condition with low calcium has been used to culture basal proliferating cells, though differentiation is induced by various procedures. These latter include the addition of calcium at mM concentration and a concomitant addition of serum and calcium. Lowering the incubation temperature of cells has also been reported to induce a premature differentiation of keratinocytes in organotypic skin culture. This effect of temperature on keratinocyte differentiation has been poorly depicted, although average human skin temperature has been shown to be about 32°C. However, studying differentiation and quantifying shifts in the differentiation rate of a cell population implies to precisely know i) the proportion of differentiated cells in the whole population, and ii) to which extent and to which level of expression, the induction of a gene or a protein might be considered as a marker of differentiation. This lack has rarely been taken into consideration and has surely led to over-interpretations of single protein induction and to consequent extrapolations to real differentiation processes. By means of paralleled analyses with immunocytofluorescence, flow cytometry, and with multiple differentiation markers quantify by qPCR and western-blot, we studied the paradoxical connection between calcium, serum, multilayer culture and incubation temperature on the differentiation of in vitro keratinocytes. Conversely to previous reports, we have shown that calcium switch is indeed a potent model for inducing calcium-dependent genes, but is not an efficient procedure when one wishes to assess the keratinocyte differentiation rate. Moreover, we have demonstrated that a synergic stimulation by calcium, serum, confluence and lower incubation temperature amplified the differentiation rate.

Polymer-based mesh as supports for multi-layered 3D cell culture and assays

Three-dimensional (3D) culture systems can mimic certain aspects of the cellular microenvironment found in vivo, but generation, analysis and imaging of current model systems for 3D cellular constructs and tissues remain challenging. This work demonstrates a 3D culture system-Cells-in-Gels-in-Mesh (CiGiM)-that uses stacked sheets of polymer-based mesh to support cells embedded in gels to form tissue-like constructs; the stacked sheets can be disassembled by peeling the sheets apart to analyze cultured cells-layer-by-layer-within the construct. The mesh sheets leave openings large enough for light to pass through with minimal scattering, and thus allowing multiple options for analysis-(i) using straightforward analysis by optical light microscopy, (ii) by high-resolution analysis with fluorescence microscopy, or (iii) with a fluorescence gel scanner. The sheets can be patterned into separate zones with paraffin film-based decals, in order to conduct multiple experiments in parallel; the paraffin-based decal films also block lateral diffusion of oxygen effectively. CiGiM simplifies the generation and analysis of 3D culture without compromising throughput, and quality of the data collected: it is especially useful in experiments that require control of oxygen levels, and isolation of adjacent wells in a multi-zone format.

Osmoprotective effects of supplemental epidermal growth factor in an ex vivo multilayered human conjunctival model under hyperosmotic stress

To analyze the effects of supplemental epidermal growth factor (EGF) and the roles of inflammatory cytokines (interleukin [IL]-6) in an ex vivo dry-eye model under hyperosmotic stress using a multilayered culture of human conjunctival epithelial cells (HCECs). Multilayered cultures of HCECs were exposed to hyperosmotic stress (400 mOsm/L) for 24 h in addition to 0.5 ng/mL EGF (low-EGF group) or 25 ng/mL EGF (high-EGF group). Apoptosis was analyzed using the TUNEL assay. Cell proliferation was measured using the [3H]-thymidine incorporation assay. The expression of IL-6, EGF, EGF receptor (EGFR), and phosphorylated extracellular signal-regulated kinase (p-ERK) was measured by western blot analysis. The secretion of IL-6 was measured using ELISA. Western blot analysis was also performed using antibodies against cleaved caspase-3. The percentage of apoptotic cells was lower in the high-EGF group (6.7%) than in the low-EGF group (10.3%). The high-EGF group demonstrated increased proliferation (323.7 counts/min in the low-EGF group vs 649.1 counts/min in the high-EGF group). EGF induced higher phosphor-EGFR expression and upregulated p-ERK in HCECs. In addition, EGF significantly decreased the secretion of IL-6 and cleaved caspase-3 in HCECs. The level of IL-6 was increased in the ex vivo HCEC dry-eye model that was under hyperosmotic stress. Supplemental EGF reduces the level of IL-6, decreases apoptosis, and increases proliferation. These findings indicate that EGF has potential as a therapeutic agent for the treatment of dry eyes.

Monitoring MSCs during expansion in a stirred tank bioreactor

Human mesenchymal stromal/stem cells (hMSCs) are an attractive target for clinical study as therapeutic agents. However, multilayer flatbed culture expansion paradigms are cumbersome, time consuming and typically limited in the ability to monitor cell characteristics during the growth process. We described a new expansion paradigm that uses a scalable, single use, stirred tank bioreactor with microcarrier scaffold for hMSC expansion. In addition to ease of handling improvements and lower medium volume requirements, the bioreactor system enables sampling of the hMSCs at any time during the expansion campaign. The cells can then be assessed for a variety of markers that denote desired identity and purity, and for expected cell health status. Beyond the obvious cell viability assessment, apoptosis and cell cycle provide a toolbox of assays for cell health. Changes in the protein markers or cell health status can be used during process development to identify optimal conditions or as a process quality assessment following expansion. In this work, we demonstrate that the combination of cell health assays and protein markers for identity and purity are key assessment tools to support and characterize the expansion of hMSCs in a scalable bioreactor system.

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and organs(1,2,6,7). During development, toxic chemicals or genetic alterations can cause an increase in PCD or change PCD patterns resulting in developmental abnormalities and birth defects(3-5). To understand the etiology of these defects, the study of embryos can be complemented with in vitro assays that use differentiating embryonic stem (ES) cells. Apoptosis is a well-studied form of PCD that involves both intrinsic and extrinsic signaling to activate the caspase enzyme cascade. Characteristic cell changes include membrane blebbing, nuclear shrinking, and DNA fragmentation. Other forms of PCD do not involve caspase activation and may be the end-result of prolonged autophagy. Regardless of the PCD pathway, dying cells need to be removed. In adults, the immune cells perform this function, while in embryos, where the immune system has not yet developed, removal occurs by an alternative mechanism. This mechanism involves neighboring cells (called "non-professional phagocytes") taking on a phagocytic role-they recognize the 'eat me' signal on the surface of the dying cell and engulf it(8-10). After engulfment, the debris is brought to the lysosome for degradation. Thus regardless of PCD mechanism, an increase in lysosomal activity can be correlated with increased cell death. To study PCD, a simple assay to visualize lysosomes in thick tissues and multilayer differentiating cultures can be useful. LysoTracker dye is a highly soluble small molecule that is retained in acidic subcellular compartments such as the lysosome(11-13). The dye is taken up by diffusion and through the circulation. Since penetration is not a hindrance, visualization of PCD in thick tissues and multi-layer cultures is possible(12,13). In contrast, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) analysis(14), is limited to small samples, histological sections, and monolayer cultures because the procedure requires the entry/permeability of a terminal transferase. In contrast to Aniline blue, which diffuses and is dissolved by solvents, LysoTracker Red DND-99 is fixable, bright, and stable. Staining can be visualized with standard fluorescent or confocal microscopy in whole-mount or section using aqueous or solvent-based mounting media(12,13). Here we describe protocols using this dye to look at PCD in normal and sonic hedgehog null mouse embryos. In addition, we demonstrate analysis of PCD in differentiating ES cell cultures and present a simple quantification method. In summary, LysoTracker staining can be a great complement to other methods of detecting PCD.