1. Submitted by:
Durgesh kr. Jha
I year M. Pharm(Pharmaceutics)
Under the guidance of:
Prof. P. V. Devrajan
Institute of Chemical Technology
Nathalal Parekh Marg, Matunga(East), Mumbai
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2. Overview
 Introduction
 3D vs. 2D cell culture
 Advantages of 3D cell culture
 In vitro tumor microenvironment in 3D system
 Mechanism of formation of spheroids
 3D cell culture techniques for tumor models
 3D in vitro tumor models
 Commercially available 3D cultures
 Recent developments on tumor models
 Applications of 3D tumor models
 References
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3. Introduction
 Tissues are made up of highly complex 3D
arrangement of cells
 Conventional 2D system fails to mimic the complex
tumor microenvironment as in vivo
 But well-defined 3D in vitro tumor model resembles
tumor structures found as in vivo
 They reflect the distinct invasive behavior of human
tumor cells
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4. Introduction
 Recreation of the tumor microenvironment including
tumor-stromal interactions, cell-cell adhesion and
cellular signaling is essential in cancer-related studies
 Most widely used 3D models are spheroid cell
aggregates and scaffold culture systems
 The in vitro 3D tumor model was to closely simulate an
in vivo solid tumor and its microenvironment for
evaluation of anticancer drug delivery systems.
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5. 3D vs. 2D cell culture
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•Physiologic cell-to-cell contact dominates
•Cells 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
VS

6. Advantages of 3D cell culture
 Better mirrors the environment experienced by tumor
cells in the body
 Replicates complex tissue structures and in vivo-like
morphology
 Better reflects normal differentiation, polarization,
cell behavior and intercellular interactions
 More realistic cell biology and function
 More predictive of disease states and drug response
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7. Advantages of 3D cell culture
 More mechanistically accurate modeling of the
target tissue
 Shorter production times relative to similar to
current monolayer culture
 Significant cost saving compared to alternative
approaches
 Less cell numbers required
 Simpler to automate
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8. In vivo tumor microenvironment in 3D system
 The 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 growth
 Infiltrated by inflammatory cells
 Numerous 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
microenvironment
 Stromal 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.
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Typical tumor microenvironment

9. 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 interactions
 Microenvironment influences cellular-differentiation,
proliferation, apoptosis and gene expression
 Hypoxia, necrosis, angiogenesis, invasion, metastasis, cell
adhesion and tumor–immune cell interactions are the
elements of tumor microenvironment
 Heterologous three-dimensional (3D) in vitro model systems
can satisfy these demands reasonably accurately and thus
mimics the in vivo tumor condition
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10. Mechanism of formation of spheroid
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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 via
integrin-ECM binding
(2) a delay period for cadherin
expression & accumulation
(3) formation of compact spheroids
through homophilic cadherin- cadherin
interactions

11. 3D cell culture techniques
3D cell
culture
techniques
Spontaneous
cell
aggregation
Liquid
overlay
cultures
Hanging
drop
method
Spinner
flask
culturesGyratory
shakers &
roller
tubes
Micro-
carrier
beads
Scaffold
based
cultures
Rotary
cell
culture
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12. 3D cell culture techniques
1. Spontaneous cell aggregation
• Based 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 cultures
• The adhesive force between cells is
much higher than the individual cells
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13. 3D cell culture techniques
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• Cells do not adhere to the substratum but grow on it
• Nonadhesive substrates used are: agar/agarose, poly-
hydroxymethyl methaacrylate or Matrigel
3. Hanging drop method

14. 3D cell culture techniques
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4. Spinner flask culture
• Cells suspended in tissue
culture media
• Cells are cultured in high-speed
stirring in ‘ stirred tank bioreactors’
• No attachment to any substrate
• Fluid movement also aid in mass
transport of nutrients to and wastes
from the spheroids

15. 3D cell culture techniques
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5. Gyratory shakers and roller tubes
• Cell suspension is placed in Erlenmeyer flask containing
specific amount of medium
• Flask is then rotated in a gyratory rotation
• Spheroids of required size are produced
6. Microcarrier beads
 This technique uses a microcarrier bead surface to culture
cancer cells
 The primary advantage of microcarrier beads is that they
support the aggregation of attachment dependent cells
and cell lines which do not spontaneously aggregate.

16. 3D cell culture techniques
……Microcarrier 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 (95–210m) and composite material (e.g.
dextran).
 Exihibit cell–cell or cell–substratum interactions.
 Can be used for mass cultivation
 Cell differentiation and maturation,
metabolic studies
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17. 3D cell culture techniques
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7. Scaffold based cultures
• Scaffolds mimics ECMs
• Provide mechanical strength to support cell adhesion
and growth in 3D shape
• It may be of biological( e.g. collagen, laminin, alginate,
chitosan) and synthetic(e.g. poly(lactide-coglycolide),
PEG) polymers
• Recently, 3D biodegradable, pre-engineered scaffolds
shows improved method of simulating ECM
• Cells adhere to the fibers and proliferate into the
interstitial space between fibers

18. 3D cell culture techniques
8. Rotary cell culture system
 Very low shear stress forces
 Minimal contact with vessel wall
 Simulated zero gravity
 Quick production of spheroids
 Culture multiple cell types
 Produce more differentiated complex epithelial shape
 The 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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19. 3D in vitro tumor models
Tumor models Descriptions Advantages Disadvantages
Ex-vivo Thin slices of tumor
tissues are cultured
on a porous
membrane or
embedded in the
ECM matrices
Preserves in vivo
phenotypes,
architectures, and
intracellular
interactions
Requires harvesting
of human or animal
tissues
Hollow-fiber Tumor cells are
cultured in
polyvinylidine
fluoride hollow
fibers
Can be implanted
into animals for in
vivo studies
Fibers may hinder
transport of
nutrients and
macro-biomolecules
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20. 3D in vitro tumor models
Multilayer Tumor cells are
cultured on a
porous
membrane post-
confluence to
form multilayers
Symmetrical
structure mimics
the necrotic core
Inconvenient to
exchange culture
media
Multicellular
spheroid
Tumor cells
aggregate to form a
spheroid
Various methods
are available
Mimics the
physiology of in
vivo solid tumor
Difficult to control
size of spheroids
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21. Commercially available 3D cultures
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22. Recent developments in 3D tumor models
1.Microfluidics system
 Microfluidic 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 environment
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Design of the gel-free 3D microfluidic cell
culture system (3D-mFCCS).

23. Microfluidics..
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• 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 with
a withdrawal flow at the outlet.

24. Recent developments in 3D tumor models
2. Imaging techniques
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Microscopy techniques Uses
Atomic force microscopy To study the effects of
stroma on the tissue
stiffening and tensile
strength in tumors
Light and transmission
electron microscopy
Histological characterization
at the cellular level
Electron microscopy Study of presence of
apoptotic and necrotic cells
Multiphoton confocal
microscopy
Advanced spheroid imaging
Rapid phase contrast
imaging
Evaluation of the effect of
growth-promoting and
growth-suppressing factors
and drugs in spheroids
Two-photon microscopy Tumor invasion and growth

25. Applications of 3D tumor models
1. Study of therapeutic efficacy of anticancer drugs
• 3D cultures are more physiologically related to
microenvironment, proliferation and morphology,
gene expression and cell behavior and drug response
• Thus, an in vitro 3D tumor model is capable of
providing close predictions of in vivo drug efficacy
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26. Case study: MTS model of breast cancer cell
for study of cytotoxic effect of doxorubicin
1.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
scaffold
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27. CASE STUDY…
2. Loading of MTS in microfluidic channel
• simulates 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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28. Applications of 3D tumor models
2. Gene function analysis
• 3D models are employed to investigate genes involved in cell apoptosis,
migration and invasion
3. Model for cell-cell interactions
• Heterotypic spheroid models shows the interaction between tumor
cells and fibroblasts and other stromal components
• Role of stroma derived fibroblasts in tumor progression can be
investigated
4.3D models in nanomedicine research
• Introduction of novel nanomedicine leads to the development in
cancer therapy research
• Nanoparticles can act as drug carriers, delivery vehicles for receptor
ligands
• They can also be used as an agent to improve imaging and targetting
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29. Applications of 3D tumor models
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Schematic representation of representative approaches for using
engineered nanoparticle systems to target, detect, and deliver drugs to
cancer cells in 3D cell culture models

30. REFERENCES
 Whiteside, 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 , 29–34.
 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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31. REFERENCES
 Hamilton, G. (1998). Multicellular spheroids as an in vitro tumor
model. Cancer Lett, 131 , 29–34.
 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): 022001
 Benien, P. S., Archana (2014). "Benien, Parul; Swami, Archana --
3D tumor models- history, advances and future perspectives."
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