3D cell culture techniques for the tumor models and their applications

3D cell culture techniques for tumor models and their applicationsSubmitted by:Durgesh kr. JhaI year M. Pharm(Pharmaceutics)Under the guidance of:Prof. P. V. Devrajan

Institute of Chemical TechnologyNathalal Parekh Marg, Matunga(East), Mumbai

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13d cell culture

Overview Introduction 3D vs. 2D cell cultureAdvantages of 3D cell cultureIn vitro tumor microenvironment in 3D systemMechanism of formation of spheroids3D cell culture techniques for tumor models3D in vitro tumor modelsCommercially available 3D culturesRecent developments on tumor modelsApplications of 3D tumor modelsReferences

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23d cell culture

Introduction Tissues are made up of highly complex 3D arrangement of cellsConventional 2D system fails to mimic the complex tumor microenvironment as in vivoBut well-defined 3D in vitro tumor model resembles tumor structures found as in vivo They reflect the distinct invasive behavior of human tumor cells19/11/20143

Introduction Recreation of the tumor microenvironment including tumor-stromal interactions, cell-cell adhesion and cellular signaling is essential in cancer-related studiesMost widely used 3D models are spheroid cell aggregates and scaffold culture systemsThe in vitro 3D tumor model was to closely simulate an in vivo solid tumor and its microenvironment for evaluation of anticancer drug delivery systems.19/11/20144

3D vs. 2D cell culture

19/11/20145Physiologic cell-to-cell contact dominatesCells interact with extracellular matrix (ECM) Diffusion gradient of drugs, oxygen, nutrients, and waste Co-culture of multiple cell mimics microenvironment Shows resistance to anticancer drug as in vivo tumor Cell-to-cell contact only on edges Cell mostly in contact with plastic Cells contact extracellular matrix mostly on one surface No gradients present Co-culture unable to establish a microenvironment Anticancer drug resistance is not seen

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Advantages of 3D cell cultureBetter mirrors the environment experienced by tumor cells in the bodyReplicates complex tissue structures and in vivo-like morphologyBetter reflects normal differentiation, polarization, cell behavior and intercellular interactionsMore realistic cell biology and functionMore predictive of disease states and drug response19/11/20146

Advantages of 3D cell cultureMore mechanistically accurate modeling of the target tissueShorter production times relative to similar to current monolayer cultureSignificant cost saving compared to alternative approaches Less cell numbers requiredSimpler to automate

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In vivo tumor microenvironment in 3D systemThe tumor microenvironment is created by the tumor and dominated by tumor-induced interactions.Immune cells in the tumor microenvironment fails to exercise antitumor effector functions & are co-opted to promote tumor growthInfiltrated by inflammatory cellsNumerous stromal cells, including endothelial cells of the blood and lymphatic circulation, stromal fibroblasts, and innate and adaptive infiltrating immune cells together comprise the complex tumor microenvironmentStromal ECM contains proteins, such as collagen, elastin and laminin, that give tissues their mechanical properties Help to organize communication between cells embedded within the matrix.19/11/20148

Typical tumor microenvironment

In vivo tumor microenvironment in 3D system Tumor cells interact with those stromal components through growth factor-mediated tumor-stromal cell crosstalk and integrin-mediated tumor-ECM interactionsMicroenvironment influences cellular-differentiation, proliferation, apoptosis and gene expressionHypoxia, necrosis, angiogenesis, invasion, metastasis, cell adhesion and tumorimmune cell interactions are the elements of tumor microenvironmentHeterologous three-dimensional (3D) in vitro model systems can satisfy these demands reasonably accurately and thus mimics the in vivo tumor condition19/11/20149

Mechanism of formation of spheroid19/11/201410

The tumor cells form 3D structures as a result of the interplay of integrin with ECM, leading to cell aggregation and later compaction though cadherin (trans-membrane proteins) interactions

Spheroid formation can be divided into three stages: (1) formation of loose cell aggregates viaintegrin-ECM binding(2) a delay period for cadherin expression & accumulation(3) formation of compact spheroids through homophilic cadherin- cadherin interactions

3D cell culture techniques19/11/201411

3D cell culture techniques1. Spontaneous cell aggregationBased on the fact that the malignant cells has ability to adhere each other(homotypic aggregation) and other cells( heterotypic aggregation)This spontaneous property of malignant cells result in the formation of multicellular aggregates These spheroids are very similar to avascular tumor nodules

2. Liquid overlay culturesThe adhesive force between cells is much higher than the individual cells

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3D cell culture techniques19/11/201413Cells do not adhere to the substratum but grow on itNonadhesive substrates used are: agar/agarose, poly- hydroxymethyl methaacrylate or Matrigel

3. Hanging drop method

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4. Spinner flask cultureCells suspended in tissue culture mediaCells are cultured in high-speed stirring in stirred tank bioreactorsNo attachment to any substrateFluid movement also aid in mass transport of nutrients to and wastes from the spheroids

3D cell culture techniques19/11/201415

5. Gyratory shakers and roller tubesCell suspension is placed in Erlenmeyer flask containing specific amount of mediumFlask is then rotated in a gyratory rotationSpheroids of required size are produced

6. Microcarrier beadsThis technique uses a microcarrier bead surface to culture cancer cellsThe primary advantage of microcarrier beads is that they support the aggregation of attachment dependent cells and cell lines which do not spontaneously aggregate.

3D cell culture techniquesMicrocarrier beads Promote the culture of normally difficult to grow or more sensitive cell lines(e.g.endothelial cells, haemopoietic cells) Co-culturing different cell types is possible Microcarrier beads differs in their coatings (e.g. positivelycharged DEAE, trimethyl-2-hydroxyaminopropyl groups, collagen coated, etc.), sizes (95210m) and composite material (e.g. dextran). Exihibit cellcell or cellsubstratum interactions.Can be used for mass cultivationCell differentiation and maturation, metabolic studies

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3D cell culture techniques19/11/2014177. Scaffold based culturesScaffolds mimics ECMs

Provide mechanical strength to support cell adhesion and growth in 3D shapeIt may be of biological( e.g. collagen, laminin, alginate, chitosan) and synthetic(e.g. poly(lactide-coglycolide), PEG) polymersRecently, 3D biodegradable, pre-engineered scaffolds shows improved method of simulating ECMCells adhere to the fibers and proliferate into the interstitial space between fibers

3D cell culture techniques8. Rotary cell culture systemVery low shear stress forces Minimal contact with vessel wallSimulated zero gravityQuick production of spheroidsCulture multiple cell typesProduce more differentiated complex epithelial shapeThe cylindrical vessel of the bioreactor is completely filled with cell suspension, and is then continuously rotated to maintain cells in a free fall state

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3D in vitro tumor modelsTumor modelsDescriptions Advantages Disadvantages Ex-vivoThin slices of tumor tissues are cultured on a porous membrane or embedded in the ECM matricesPreserves in vivo phenotypes, architectures, and intracellular interactionsRequires harvesting of human or animal tissuesHollow-fiberTumor cells are cultured in polyvinylidine fluoride hollow fibersCan be implanted into animals for in vivo studiesFibers may hinder transport of nutrients and macro-biomolecules

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3D in vitro tumor modelsMultilayer Tumor cells are cultured on a porous membrane post-confluence to form multilayersSymmetrical structure mimics the necrotic coreInconvenient to exchange culture mediaMulticellular spheroidTumor cells aggregate to form a spheroidVarious methods are availableMimics the physiology of in vivo solid tumorDifficult to control size of spheroids

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Commercially available 3D cultures19/11/201421

Recent developments in 3D tumor models1.Microfluidics systemMicrofluidic devices are typically created by bonding a PDMS substrate containing microchannel features created by replica molding with a blank PDMS slab. Various natural and synthetic hydrogels have been incorporated into microfluidic cell culture systems to support cells in 3D This process can be easily automated and is suitable for mass production of tumor spheroids and low volume drug testing. It is advantageous in terms of creating uniform size and composition of spheroids with improved efficiency, in a controlled environment19/11/201422

Design of the gel-free 3D microfluidic cell culture system (3D-mFCCS).

Microfluidics..19/11/201423

In situ formation and immobilization of 3D multi-cellular aggregates in the microchannel. Cells were suspended in cell culture medium with dissolved inter-cellular linker and seeded into the microfluidic channel witha withdrawal flow at the outlet.

Recent developments in 3D tumor models2. Imaging techniques

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Microscopy techniquesUsesAtomic force microscopyTo study the effects of stroma on the tissue stiffening and tensile strength in tumorsLight and transmission electron microscopyHistological characterization at the cellular levelElectron microscopyStudy of presence of apoptotic and necrotic cellsMultiphoton confocal microscopyAdvanced spheroid imagingRapid phase contrast imagingEvaluation of the effect of growth-promoting and growth-suppressing factors and drugs in spheroidsTwo-photon microscopyTumor invasion and growth

Applications of 3D tumor models1. Study of therapeutic efficacy of anticancer drugs3D cultures are more physiologically related to microenvironment, proliferation and morphology, gene expression and cell behavior and drug responseThus, an in vitro 3D tumor model is capable of providing close predictions of in vivo drug efficacy

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Case study: MTS model of breast cancer cell for study of cytotoxic effect of doxorubicin1.Culture of uniformly sized MTS in a hydrogel scaffold containing microwells(A) A master template was pressed into the gelatin solution mixed with HCl and glutaraldehyde followed by warming at 60 C to allow cross-linking. Upon removal of the master template, the hydrogel scaffold containing microwells was rinsed prior to cell seeding. (B) MCF-7 cells suspended in 50% Matrigel solution were transferred onto the hydrogel scaffold. After MTS were formed,Matrigel encapsulating MTS were released from the hydrogel scaffold19/11/201426

CASE STUDY2. Loading of MTS in microfluidic channelsimulates dynamic fluidic movement of in vivo microenvironment.

3. DOX Treated MTS in the Microfluidic Channel.Fluorescence images of the tumor models were acquired to observe drug accumulation or distribution.The concentration of DOX that is cytocidal on MCF-7 monolayers shows no significant cell death in the 3D model whether in static or dynamic condition Confirms the 3D tumor model was less sensitive to DOX than the monolayer

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Applications of 3D tumor models2. Gene function analysis3D models are employed to investigate genes involved in cell apoptosis, migration and invasion

3. Model for cell-cell interactionsHeterotypic spheroid models shows the interaction between tumor cells and fibroblasts and other stromal componentsRole of stroma derived fibroblasts in tumor progression can be investigated

4.3D models in nanomedicine researchIntroduction of novel nanomedicine leads to the development in cancer therapy researchNanoparticles can act as drug carriers, delivery vehicles for receptor ligandsThey can also be used as an agent to improve imaging and targetting

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Applications of 3D tumor models19/11/201429

Schematic representation of representative approaches for using engineered nanoparticle systems to target, detect, and deliver drugs to cancer cells in 3D cell culture models

REFERENCESWhiteside, T. L. (2008). "The tumor microenvironment and its role in promoting tumor growth." Oncogene 27(45): 5904-5912.Ong, S. M., C. Zhang, Y. C. Toh, S. H. Kim, H. L. Foo, C. H. Tan, D. van Noort, S. Park and H. Yu (2008). "A gel-free 3D microfluidic cell culture system." Biomaterials 29(22): 3237-3244.Shin, C. S., B. Kwak, B. Han, K. Park and A. Panitch (2013). "3D cancer tumor models for evaluating chemotherapeutic efficacy." 445-460.Hamilton, G. (1998). Multicellular spheroids as an in vitro tumor model. Cancer Lett, 131 , 2934.Francesco Pampaloni, E. G. R. a. E. H. K. S.(2007) "Nature Reviews Molecular Cell Biology Volume 8 issue 10 " The third dimension bridges the gap between cell culture and liv.Huh, D., G. A. Hamilton and D. E. Ingber (2011). "From 3D cell culture to organs-on-chips." Trends Cell Biol 21(12): 745-754.

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REFERENCESHamilton, G. (1998). Multicellular spheroids as an in vitro tumor model. Cancer Lett, 131 , 2934. Kim, J. B. (2005). "Three-dimensional tissue culture models in cancer biology." Semin Cancer Biol 15(5): 365-377.Leong, D. T. and K. W. Ng (2014). "Probing the relevance of 3D cancer models in nanomedicine research." Adv Drug Deliv Rev.Shin, C. S., B. Kwak, B. Han and K. Park (2013). "Development of an in vitro 3D tumor model to study therapeutic efficiency of an anticancer drug." Mol Pharm 10(6): 2167-2175.Wang, C., Z. Tang, Y. Zhao, R. Yao, L. Li and W. Sun (2014). "Three-dimensional in vitro cancer models: a short review." Biofabrication 6(2): 022001Benien, P. S., Archana (2014). "Benien, Parul; Swami, Archana -- 3D tumor models- history, advances and future perspectives."

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