Conventional Monolayer Culture Research Articles

Enhanced Differentiation Potential of Primary Human Endometrial Cells Cultured on 3D Scaffolds.

Novel approaches for culturing primary human cells in vitro are increasingly needed to study cell and tissue physiology and to grow replacement tissue for regenerative medicine. Conventional 2D monolayer cultures of endometrial epithelial and stromal cells fail to replicate the complex 3D architecture of tissue. A fully synthetic scaffold that mimics the microenvironment of the human endometrium can ultimately provide a robust platform for investigating tissue physiology and, hence, take significant steps toward tackling female infertility and IVF failure. In this work, emulsion-templated porous polymers (known as polyHIPEs) were investigated as scaffolds for the culture of primary human endometrial epithelial and stromal cells (HEECs and HESCs). Infiltration of HEECs and HESCs into cell-seeded polyHIPE scaffolds was assessed by histological studies, and phenotype was confirmed by immunostaining. Confocal microscopy revealed that the morphology of HEECs and HESCs is representative of that found in vivo. RNA sequencing was used to investigate transcriptome differences between cells grown on polyHIPE scaffolds and in monolayer cultures. The differentiation status of HEECs and HESCs grown in polyHIPE scaffolds and in monolayer cultures was further evaluated by monitoring the expression of endometrial marker genes. Our observations suggest that a 3D cell culture model that could approximate native human endometrial architecture and function can be developed using tailored polyHIPE scaffolds.

Defined Geldrop Cultures Maintain Neural Precursor Cells

Distinct micro-environmental properties have been reported to be essential for maintenance of neural precursor cells (NPCs) within the adult brain. Due to high complexity and technical limitations, the natural niche can barely be studied systematically in vivo. By reconstituting selected environmental properties (adhesiveness, proteolytic degradability, and elasticity) in geldrop cultures, we show that NPCs can be maintained stably at high density over an extended period of time (up to 8 days). In both conventional systems, neurospheres and monolayer cultures, they would expand and (in the case of neurospheres) differentiate rapidly. Further, we report a critical dualism between matrix adhesiveness and degradability. Only if both features are functional NPCs stay proliferative. Lastly, Rho-associated protein kinase was identified as part of a pivotal intracellular signaling cascade controlling cell morphology in response to environmental cues inside geldrop cultures. Our findings demonstrate that simple manipulations of the microenvironment in vitro result in an important preservation of stemness features in the cultured precursor cells.

Treatment of Bone Defects by Transplantation of Genetically Modified Mesenchymal Stem Cell Spheroids

Cell transplantation is promising for regenerative medicine. A combination of a three-dimensional spheroid culture system with gene transfection was developed to enhance the therapeutic effects of mesenchymal stem cell (MSC) transplantation. The spheroid cell culture system is based on micropatterned substrates composed of a regular array of 100-μm-diameter cell-adhesion areas coated with a temperature-responsive polymer, poly (N-isopropylacrylamide-co-methacrylic acid), which allows for spheroid detachment by simply cooling the plates. In this study, MSC spheroids were transfected with plasmid DNA encoding runt-related transcription factor 2 (Runx2) and tested for their ability to enhance bone regeneration. In vitro analyses revealed that osteogenic differentiation of the MSCs was enhanced by forming spheroids and was further promoted by Runx2 expression. The enhanced osteogenic differentiation was maintained under pathological conditions, such as hypoxia and inflammation. Transplanting Runx2-transfected MSC spheroids into bone defects on rat femurs induced significantly faster bone regeneration compared with nontransfected MSC spheroids or genetically modified MSCs from conventional monolayer culture. MSC migration into the bone defect area was enhanced by upregulation of cell-migration-related genes. In conclusion, genetically modified MSC spheroids are effective for enhancing bone regeneration, providing a promising option for cell transplantation therapy in the fields of regenerative medicine.

Impact of RUNX2 gene silencing on the gemcitabine sensitivity of p53‑mutated pancreatic cancer MiaPaCa‑2 spheres.

Recently, it has been well‑recognized that the response toward anticancer drugs differs between two‑ and three‑dimensional (2D and 3D) invitro cancer cell growth models. In the present study, we have demonstrated that, similar to the conventional 2D monolayer culture systems which often lack invivo physiological insights, RUNX2 gene silencing increases the gemcitabine (GEM) sensitivity of the 3D spheres generated from p53‑mutated pancreatic cancer MiaPaCa‑2 cells. According to our results, MiaPaCa‑2 cells, but not p53‑wild‑type pancreatic cancer SW1990 cells efficiently formed sphere structures in serum‑free sphere‑forming medium. Although GEM treatment caused a marked induction of TAp73/TAp63 in MiaPaCa‑2 spheres accompanied by the transcriptional activation of p53 family‑target genes such as p21WAF1 and NOXA, only 20% of cells underwent cell death. Under these experimental conditions, mutant p53 expression level was increased in response to GEM and RUNX2 remained unchanged at the protein level regardless of GEM exposure, which may suppress the pro‑apoptotic activity of TAp73/TAp63. Notably, RUNX2 gene silencing markedly augmented GEM‑mediated cell death of MiaPaCa‑2 spheres compared to that of non‑depleted ones. Expression analyses revealed that forced depletion of RUNX2 further stimulated GEM‑induced upregulation of TAp63 as well as its downstream target genes such as p21WAF1 and NOXA. In summary, our observations strongly indicated that, similarly to 2D monolayer culture, RUNX2 gene silencing increased GEM sensitivity of MiaPaCa‑2 spheres and highlighted the therapeutic potential of RUNX2 in pancreatic cancer with p53 mutation.

Evaluation of cartilage specific matrix synthesis of human articular chondrocytes after extended propagation on microcarriers by image analysis.

Cell-based technologies for the repair of cartilage defects usually rely on the expansion of low numbers of chondrocytes isolated from biopsies of healthy cartilage. Proliferating chondrocytes are known to undergo dedifferentiation characterized by downregulation of collagen type II and proteoglycan production, and by upregulation of collagen type I synthesis. Re-expression of cartilage specific matrix components by expanded chondrocytes is therefore critical for successful cartilage repair. Human articular chondrocytes were expanded on microcarriers Cytodex 3. The growth area was increased by adding empty microcarriers. Added microcarriers were colonized by bead-to-bead transfer of the cells. The chondrocytes were harvested from the microcarriers and characterized by their ability to synthesize collagen type II when cultivated in alginate beads using chondrogenic growth factors. A semi-automatic image analysis technique was developed to determine the fractions of collagen type II and type I positive cells. The expansion of human articular chondrocytes on microcarriers yielded high cell numbers and propagation rates compared to chondrocytes expanded in flask culture for one passage. The proportion of collagen type II positive cells compared to collagen type I synthesizing cells was increased compared to chondrocytes expanded using conventional methods. The matrix synthesis upon treatment with chondrogenic factors IGF-I and BMP-7 was enhanced whereas TGF-ss had an inhibitory effect on microcarrier expanded chondrocytes. Expanding human articular chondrocytes on microcarriers omitting subcultivation steps leads to superior ratios of collagen type II to type I forming cells compared to the expansion in conventional monolayer culture.

A comprehensive analysis of human dental pulp cell spheroids in a three-dimensional pellet culture system

ObjectiveThree-dimensional (3D) cell culture methods are of high importance to studies of biological processes. This is particularly the case with spheroid cultures, which create 3D cell aggregates without the use of exogenous materials. Compared to conventional monolayer cultures, cellular spheroid cultures have been demonstrated to improve multilineage potential and extracellular matrix production. To address this issue in depth, we present a more comprehensive analysis of 3D human dental pulp cell (hDPC) spheroids. DesignhDPC spheroids were fabricated by the pellet culture method and were cultured without adding any reagent to induce differentiation. The gene-expression profiles of the 3D and two-dimensional (2D) cultured hDPCs were compared by complementary DNA microarray analysis. Odontoblastic and osteoblastic differentiation marker gene expression was evaluated by quantitative real-time PCR (RT-qPCR). Hematoxylin-eosin (HE) staining and transmission electron microscopy (TEM) were applied to examine the morphology of hDPC spheroids and extracellular matrix components. ResultsCompared with 2D monolayer culture, microarray analysis identified 405 genes and 279 genes with twofold or greater differential expression after 3 days and 28 days of 3D culture, respectively. In 3D hDPC spheroids, gene ontology analysis revealed upregulation of extracellular matrix-related genes and downregulation of cell growth-related genes. RT-qPCR analysis showed higher expression levels of osteocalcin, dentin sialophosphoprotein, and alkaline phosphatase. TEM revealed the morphological characteristics of the fibrillar collagen-rich matrix and cell-cell interactions. ConclusionsThe present findings provide clues to understanding the mechanisms of pellet-cultured hDPCs and contribute to future research in the comparative studies of different 3D culture methods.

Abstract PD4-13: Estrogen receptor-negative breast adenomyoepitheliomas are driven by co-occurring HRAS hotspot and PI3K pathway gene mutations: A genetic and functional analysis

Abstract Introduction:Adenomyoepithelioma (AME) of the breast is a rare biphasic tumor, characterized by epithelial and myoepithelial differentiation. Although AMEs have an indolent clinical course, a subset may progress to carcinoma and metastasize. We sought to define the mutational landscape of AMEs and investigate the functional impact of recurrent pathogenic mutations identified in these tumors. Methods: Thirty-one AMEs were subjected to whole-exome sequencing (WES, n=8) or massively parallel sequencing targeting all coding regions of 410 key cancer genes and intronic and regulatory regions of selected genes (n=23). Somatic genetic alterations were defined using state-of-the-art bioinformatics algorithms. In an additional set of 12 AMEs, Sanger sequencing analysis of HRAS, PIK3CA and AKT1 was performed. Non-tumorigenic estrogen receptor (ER)-negative mammary epithelial cells (i.e. MCF10A, MCF10A with a PIK3CA H1047R mutation knock-in and MCF12A) were utilized for functional studies using both conventional monolayer and three-dimensional (3D) culture assays. Results: 27 (63%) and 16 (37%) AMEs were ER-positive and ER-negative, respectively. ER-negativity was significantly associated with histologic features predictive of a more aggressive behavior, with a higher number of mutations and copy number alterations, and with a distinct mutational profile as compared to ER-positive AMEs. Of the 27 ER-positive AMEs, 12 cases (44%) harbored PIK3CA hotspot mutations, and 5 PIK3CA wild-type cases displayed E17K AKT1 hotspot mutations. By contrast, of the 16 ER-negative AMEs, 9 (56%), 9 (56%) and 3 (19%) harbored HRAS, PIK3CA (mostly E545K and H1047R hotspots) and PIK3R1 mutations, respectively. Strikingly, all HRAS mutations were restricted to ER-negative AMEs, affected the hotspot codon Q61 (Q61R/K), and all but one co-occurred with PIK3CA or PIK3R1 mutations. In addition, HRAS Q61 hotspot mutations were significantly associated with necrosis (p=0.01) and high mitotic rates (p=0.03). CDKN2A homozygous deletions were also detected only in ER-negative AMEs (19%) and found to be significantly associated with progression to carcinoma (p=0.001). Forced expression of HRAS Q61R in MCF10A and MCF12A cells resulted in i) increased proliferation and transformation, ii) an irregular growth pattern in 3D organotypic cell cultures, iii) partial loss of the epithelial phenotype, and iv) acquisition of myoepithelial differentiation, which was more overt in PIK3CA-mutant MCF10A cells. HRAS Q61Rinduced hyperactivation of the PI3K pathway, but both PI3K and MAPK pathways likely contributed to the RAS-mediated proliferation, which was completely arrested by combined AKT and MEK inhibition. Conclusion: AMEs are phenotypically and genetically heterogeneous. Whilst pathogenic mutations in PI3K pathway-related genes occur across the spectrum of lesions, HRAS Q61 hotspot mutations are restricted to ER-negative AMEs. Our genomic and functional analyses indicate that HRAS Q61 mutations are driver events in the pathogenesis of ER-negative AMEs and, in conjunction with mutant PIK3CA, may lead to the acquisition of myoepithelial differentiation in breast epithelial cells. Citation Format: Geyer FC, Li A, Papanastasiou AD, Smith A, Selenica P, Burke KA, Edelweiss M, Wen H-C, Piscuoglio S, Schultheis AM, Martelotto LG, Pareja F, Kumar R, Brandes A, Lozada J, Macedo GS, Muenst S, Terracciano LM, Jungbluth A, Foschini MP, Wen HY, Brogi E, Palazzo J, Rubin BP, Ng CKY, Norton L, Varga Z, Ellis IO, Rakha E, Chandarlapatty S, Weigelt B, Reis-Filho JS. Estrogen receptor-negative breast adenomyoepitheliomas are driven by co-occurring HRAS hotspot and PI3K pathway gene mutations: A genetic and functional analysis [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD4-13.

Engineered Models of Metastasis with Application to Study Cancer Biomechanics.

Three-dimensional complex biomechanical interactions occur from the initial steps of tumor formation to the later phases of cancer metastasis. Conventional monolayer cultures cannot recapitulate the complex microenvironment and chemical and mechanical cues that tumor cells experience during their metastatic journey, nor the complexity of their interactions with other, noncancerous cells. As alternative approaches, various engineered models have been developed to recapitulate specific features of each step of metastasis with tunable microenvironments to test a variety of mechanistic hypotheses. Here the main recent advances in the technologies that provide deeper insight into the process of cancer dissemination are discussed, with an emphasis on three-dimensional and mechanical factors as well as interactions between multiple cell types.

A long-term culture system based on a collagen vitrigel membrane chamber that supports liver-specific functions of hepatocytes isolated from mice with humanized livers.

During drug discovery, in vitro models are used to predict the in vivo pharmacokinetic and toxicological properties of drug candidates in humans. However, the conventional method of culturing human hepatocytes as monolayers does not necessarily replicate biologic reactions and does not support liver-specific functions, such as cytochrome P450 (CYP) activities, for prolonged periods. To remedy these problems and thus increase and prolong hepatic functions, we developed a culture system comprising a collagen vitrigel membrane (CVM) chamber and PXB-cells®, fresh hepatocytes isolated from liver-humanized chimeric mice (PXB-mice®). To quantitatively assess our new system, we evaluated the activities of 5 major CYP isoforms (CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A), albumin secretion, and urea synthesis. First, between Days 14 and 21, the activities of all CYP isoforms tested in vitrigel culture were equal to or higher than in conventional monolayer culture system. Second, the activities of CYP3A, CYP2C9, and CYP2C19 during Days 10 through 17 were higher in vitrigel culture than in suspended PXB-cells prepared on Day 0 (suspension assay). Third, albumin secretion and urea synthesis were higher in vitrigel culture than in conventional monolayer culture. Fourth, the vitrigel-cultured PXB-cells showed the characteristic morphology of parenchymal hepatocytes and were almost all alive in monolayer. These results indicate that our vitrigel culture method is superior to the conventional monolayer method in terms of diverse liver-specific functions, including CYP activity. Our findings suggest that the vitrigel culture method could be a powerful in vitro tool for predicting the pharmacokinetic and toxicological properties of drug candidates in humans.

Involvement of mTOR-autophagy in the selection of primitive mesenchymal stem cells in chitosan film 3-dimensional culture

Mesenchymal stem cells (MSCs) in conventional monolayer culture are heterogeneous and contain a significant portion of senescent cells. MSCs cultured on chitosan film form 3-dimenional spheres, increase in stemness and differentiation capability; however, the underlying mechanisms remain elusive. We first demonstrate chitosan film culture induces apoptosis at 2 days, with specificity in late senescent cells. Especially in senescent cells, chitosan film culture activates mTOR, which activates S6K/S6/4E-BP1 to enhance fibronection synthesis and peripheral dead cell attachment, and phosphorylates ULK1 at S757 to further inactivate ULK1, LC3A and autophagy, thereby inducing apoptosis. Combination of chitosan film culture with mTOR inhibition prevents peripheral dead cell attachment, thereby further increasing pluripotent gene expression, in vitro osteogenesis and in vivo bone formation. These data successfully figure out the role of mTOR signaling in chitosan film culture and develop a method by combination of rapamycin treatment for promoting stemness and differentiation capability in MSCs.

Proteomic Characterization of Primary Mouse Hepatocytes in Collagen Monolayer and Sandwich Culture.

Dedifferentiation of primary hepatocytes in vitro makes their application in long-term studies difficult. Embedding hepatocytes in a sandwich of extracellular matrix is reported to delay the dedifferentiation process to some extent. In this study, we compared the intracellular proteome of primary mouse hepatocytes (PMH) in conventional monolayer cultures (ML) to collagen sandwich culture (SW) after 1 day and 5 days of cultivation. Quantitative proteome analysis of PMH showed no differences between collagen SW and ML cultures after 1 day. Glycolysis and gluconeogenesis were strongly affected by long-term cultivation in both ML and SW cultures. Interestingly, culture conditions had no effect on cellular lipid metabolism. After 5 days, PMH in collagen SW and ML cultures exhibit characteristic indications of oxidative stress. However, in the SW culture the defense system against oxidative stress is significantly up-regulated to deal with this, whereas in the ML culture a down-regulation of these important enzymes takes place. Regarding the multiple effects of ROS and oxidative stress in cells, we conclude that the down-regulation of these enzymes seem to play a role in the loss of hepatic function observed in the ML cultivation. In addition, enzymes of the urea cycle were clearly down-regulated in ML culture. Proteomics confirms lack in oxidative stress defense mechanisms as the major characteristic of hepatocytes in monolayer cultures compared to sandwich cultures. J. Cell. Biochem. 119: 447-454, 2018. © 2017 Wiley Periodicals, Inc.

Hydrogel Environment Supports Cell Culture Expansion of a Grade IV Astrocytoma.

Malignant astrocytomas are aggressive cancers of glial origin that can develop into invasive brain tumors. The disease has poor prognosis and high recurrence rate. Astrocytoma cell lines of human origin are an important tool in the experimental pathway from bench to bedside because they afford a convenient intermediate system for in vitro analysis of brain cancer pathogenesis and treatment options. We undertook the current study to determine whether hydrogel culture methods could be adapted to support the growth of astrocytoma cell lines, thereby facilitating a system that may be biologically more similar to in vivo tumor tissue. Our experimental protocols enabled maintenance of Grade IV astrocytoma cell lines in conventional monolayer culture and in the extracellular matrix hydrogel, Geltrex™. Light and fluorescence microscopy showed that hydrogel environments promoted cellular reorganization from dispersed cells into multilayered aggregates. Transmission electron microscopy revealed the prevalence of autophagy and nuclear membrane distortions in both culture systems. Analysis of microarray Gene Expression Omnibus (GEO) DataSets highlighted expression of genes implicated in pathways for cancer progression and autophagy. A pilot quantitative polymerase chain reaction (qPCR) analysis of the autophagic biomarkers, Beclin 1 (BECN1) and microtubule-associated proteins 1A/1B light chain 3B (MAP1LC3B), with two reference genes (beta actin, ACTB; glyceraldehyde 3-phosphate dehydrogenase, GAPDH), uncovered a relative increase of BECN1 and LC3B in hydrogel cultures of astrocytoma as compared to the monolayer. Taken together, results establish that ultrastructural and molecular characteristics of autophagy are features of this astrocytoma cell line, and that hydrogel culture systems can afford novel opportunities for in vitro studies of glioma.

An oxygen-permeable spheroid culture chip (Oxy chip) promotes osteoblastic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are being clinically used for bone and cartilage regeneration. However, the preparation of MSCs for implantation is still costly and time consuming, and controlling the differentiation of stem cells remains a challenge. Although much attention has been paid to three-dimensional cultures in the fields of tissue engineering and regenerative medicine, adequate oxygen supply remains a challenge for growing thicker and larger cellular constructs. To solve this problem, we have developed an oxygen-permeable spheroid culture device (Oxy chip) that enables direct oxygen supply to the cells. The aim of this study was to examine the effect of a three-dimensional culture and oxygenation to the cells on the differentiation of mouse MSC strain D1 cells. Our data demonstrated that MSCs grown in the Oxy chip differentiated into osteoblasts more quickly and efficiently than those grown in the conventional non-oxygen permeable chip and monolayer culture. DNA array and energy metabolism analyses revealed that the Oxy chip facilitated osteoblastic differentiation and aerobic glycolysis, rather than chondrogenic differentiation and anaerobic glycolysis. Together, we revealed for the first time that the oxygenation by the Oxy chip was effective on the osteoblastic differentiation and survival of three-dimensional cultured MSCs. This chip is useful for preparing differentiated cells and controlling the direction of differentiation of MSCs. Moreover, this approach may be useful for transitioning spheroid cultures as a therapy in regenerative medicine.

Development of size-customized hepatocarcinoma spheroids as a potential drug testing platform using a sacrificial gelatin microsphere system

Sacrificial gelatin microspheres can be developed as a cell delivery vehicle for non-anchorage dependent cells – its incorporation into a macroscopic scaffold system not only allows the cells to be cultured in suspension within cavities left behind by the sacrificial material, it also allows scaffold-free tissue development to be confined within the cavities. In this study, dense and highly viable hepatocarcinoma spheroids were developed by means of encapsulation in sacrificial gelatin microspheres produced via a simple water-in-oil emulsion technique. By initial selection of microsphere size and distribution, spheroid size can be controlled for various applications such as uniform tumor spheroids as a reproducible three-dimensional drug screening and testing platform that better mimics the in vivo nature of tumors (instead of conventional monolayer culture), as this study has suggested as a proof-of-concept with chemotherapy drug Doxorubicin.

Neuronal-glial populations form functional networks in a biocompatible 3D scaffold

Monolayers of neurons and glia have been employed for decades as tools for the study of cellular physiology and as the basis for a variety of standard toxicological assays. A variety of three dimensional (3D) culture techniques have been developed with the aim to produce cultures that recapitulate desirable features of intact. In this study, we investigated the effect of preparing primary mouse mixed neuron and glial cultures in the inert 3D scaffold, Alvetex. Using planar multielectrode arrays, we compared the spontaneous bioelectrical activity exhibited by neuroglial networks grown in the scaffold with that seen in the same cells prepared as conventional monolayer cultures. Two dimensional (monolayer; 2D) cultures exhibited a significantly higher spike firing rate than that seen in 3D cultures although no difference was seen in total signal power (<50Hz) while pharmacological responsiveness of each culture type to antagonism of GABAAR, NMDAR and AMPAR was highly comparable. Interestingly, correlation of burst events, spike firing and total signal power (<50Hz) revealed that local field potential events were associated with action potential driven bursts as was the case for 2D cultures. Moreover, glial morphology was more physiologically normal in 3D cultures. These results show that 3D culture in inert scaffolds represents a more physiologically normal preparation which has advantages for physiological, pharmacological, toxicological and drug development studies, particularly given the extensive use of such preparations in high throughput and high content systems.

Human hepatoma cell lines on gas foaming templated alginate scaffolds for in vitro drug-drug interaction and metabolism studies

Liver in vitro systems that allow reliable prediction of major human in vivo metabolic pathways have a significant impact in drug screening and drug metabolism research. In the present study, a novel porous scaffold composed of alginate was prepared by employing a gas-in-liquid foaming approach. Galactose residues were introduced on scaffold surfaces to promote cell adhesion and to enhance liver specific functions of the entrapped HepG2/C3A cells. Hepatoma cells in the gal-alginate scaffold showed higher levels of liver specific products (albumin and urea) and were more responsive to specific inducers (e.g. dexamethasone) and inhibitors (e.g. ketoconazole) of the CYP3A4 system than in conventional monolayer culture. HepG2/C3A cells were also more efficient in terms of rapid elimination of testosterone, used as a model substance, at rates comparable to those of in vivo excretion. In addition, an improvement in metabolism of testosterone, in terms of phase II metabolite formation, was also observed when the more differentiated HepaRG cells were used. Together the data suggest that hepatocyte/gas templated alginate-systems provide an innovative high throughput platform for in vitro drug metabolism and drug–drug interaction studies, with broad fields of application, and might provide a valid tool for minimizing animal use in preclinical testing of human relevance.

Inhibition of Src family kinases overcomes anoikis resistance induced by spheroid formation and facilitates cisplatin-induced apoptosis in human mesothelioma cells.

Malignant mesothelioma is an aggressive tumor arising from mesothelial cells of serous membranes, and forms spheroid-like cell aggregates in pleural and peritoneal effusions. We examined the levels of anoikis, apoptosis induced by the detachment of cells from the extracellur matrix, in suspension culture in the human mesothelioma cell line NCI-H2052. NCI-H2052 cells were adherent in conventional monolayer cultures, but were found to form spheroids in suspension cultures using dishes with ultra-low cell binding capacity. NCI-H2052 cells proliferated in both cultures, but the proliferation rate was markedly lower in suspension cultures than in monolayer cultures. In addition, NCI-H2052 cells in suspension cultures showed little apoptosis, suggesting that the suspension culture induces anoikis resistance. Western blot analysis revealed that suspension cultures induced activation of Src family kinases (SFK) after spheroid formation. Dasatinib, an inhibitor of multi-tyrosine kinases including SFK, abolished anoikis resistance in suspension cultures, indicating that SFK activated by spheroid formation are responsible for anoikis resistance. Cisplatin induced apoptosis in NCI-H2052 cells, but the apoptotic rate was significantly lower in suspension cultures than in monolayer cultures, suggesting that spheroid formation is involved in cisplatin resistance. Furthermore, a combination of dasatinib and cisplatin induced apoptosis more significantly than either alone in suspension cultures. These results suggest that spheroid formation induces resistance to anoikis and to cisplatin through SFK activation and that dasatinib facilitates cisplatin-induced apoptosis in human mesothelioma cells.

Flow perfusion effects on three-dimensional culture and drug sensitivity of Ewing sarcoma

Three-dimensional tumor models accurately describe different aspects of the tumor microenvironment and are readily available for mechanistic studies of tumor biology and for drug screening. Nevertheless, these systems often overlook biomechanical stimulation, another fundamental driver of tumor progression. To address this issue, we cultured Ewing sarcoma (ES) cells on electrospun poly(ε-caprolactone) 3D scaffolds within a flow perfusion bioreactor. Flow-derived shear stress provided a physiologically relevant mechanical stimulation that significantly promoted insulin-like growth factor-1 (IGF1) production and elicited a superadditive release in the presence of exogenous IGF1. This finding is particularly relevant, given the central role of the IGF1/IGF-1 receptor (IGF-1R) pathway in ES tumorigenesis and as a promising clinical target. Additionally, flow perfusion enhanced in a rate-dependent manner the sensitivity of ES cells to IGF-1R inhibitor dalotuzumab (MK-0646) and showed shear stress-dependent resistance to the IGF-1R blockade. This study demonstrates shear stress-dependent ES cell sensitivity to dalotuzumab, highlighting the importance of biomechanical stimulation on ES-acquired drug resistance to IGF-1R inhibition. Furthermore, flow perfusion increased nutrient supply throughout the scaffold, enriching ES culture over static conditions. Our use of a tissue-engineered model, rather than human tumors or xenografts, enabled precise control of the forces experienced by ES cells, and therefore provided at least one explanation for the remarkable antineoplastic effects observed by some ES tumor patients from IGF-1R targeted therapies, in contrast to the lackluster effect observed in cells grown in conventional monolayer culture.

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells.

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Core-shell hydrogel beads with extracellular matrix for tumor spheroid formation.

Creating multicellular tumor spheroids is critical for characterizing anticancer treatments since they may provide a better model of the tumor than conventional monolayer culture. Moreover, tumor cell interaction with the extracellular matrix can determine cell organization and behavior. In this work, a microfluidic system was used to form cell-laden core-shell beads which incorporate elements of the extracellular matrix and support the formation of multicellular spheroids. The bead core (comprising a mixture of alginate, collagen, and reconstituted basement membrane, with gelation by temperature control) and shell (comprising alginate hydrogel, with gelation by ionic crosslinking) were simultaneously formed through flow focusing using a cooled flow path into the microfluidic chip. During droplet gelation, the alginate acts as a fast-gelling shell which aids in preventing droplet coalescence and in maintaining spherical droplet geometry during the slower gelation of the collagen and reconstituted basement membrane components as the beads warm up. After droplet gelation, the encapsulated MCF-7 cells proliferated to form uniform spheroids when the beads contained all three components: alginate, collagen, and reconstituted basement membrane. The dose-dependent response of the MCF-7 cell tumor spheroids to two anticancer drugs, docetaxel and tamoxifen, was compared to conventional monolayer culture.