3D Cell Cultures: Ajisafe Victor Ayobami

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3D CELL CULTURES

By:

Ajisafe Victor Ayobami


OSMANIA UNIVERSITY,
HYDERABAD , INDIA
What is 3D Cell Culture?
• An artificially-created
environment in which
biological cells are permitted to
grow or interact with their
surroundings in all three
dimensions.

• An improvement over the


previous method of
growing cells in 2D.

• Accurately mimics the cell


natural environment.
Why 3D Cell Culture?
Before (2D) Now (3D)
• Cells are grown on flat dishes and • Cells attach to one another and
cellular attachment is unnatural. form cell-to-cell attachments
• Cellular communication and synthesized by the cells.
signaling is minimal. • Cellular communication and
• Grown cells are simple with signaling is maximized.
divergent functions from native • Grown cells have complex
tissues. phenotype and functions that
• Not valid for targeting and testing closely mimics native tissues
new drug discoveries. • More valid for targeting and
• Monolayer of cells are formed testing new drug discoveries.
• Multi layers of cells are formed.
2D vs 3D Cell Cultures
3D Cell Culture Techniques
a) Scaffold Based Scaffold Based
 Polymeric Hard Scaffolds
 Biologic Scaffolds
 Micropatterned Surface
Microplates

b) Non Scaffold Based


 Hanging Drop Microplates
 Spheroid Microplates
containing Ultra-Low
Attachment (ULA) coating
 Microfluidic 3D cell culture Non Scaffold Based

3D Cell culture Bioreactor


c) Bioreactors

a) Gels
3D Cell Culture Gels
Scaffold Based
Polymeric Hard Scaffold
• Typical size is 150 - 200µm
• Lactide and glycolide copolymer
may be used as materials
• In regenerative medicine, cells
are grown on this scaffold for
transplantation purposes to
replace defective tissue
• Widely used for engineering
bone, cartilage, ligament, skin,
vascular, neural and skeletal
muscle tissues.
• In preclinical in-vitro testing, cells
are grown to model tumors in the
lab.
Scaffold Based
Biological Scaffolds
• Produced from natural
components of biological origin
e.g proteins such as fibronectin,
collagen, laminin and gelatin.
• Cells attach and reorganize into
desired organs’ 3D structure.
• Provides suitable
microenvironment for the
anatomical and physiological
development of cells.
• Widely used in regenerative
medicine as well as tissue
engineering
Scaffold Based
Micropatterned Surface Microplates
• Each plate contains
micrometer sized
compartments arrayed on the
bottom.
• Usually coated to create a low
adhesion surface.
• Bottom is thin and transparent
for microscopic imaging.
• Used in drug testing, cell Round Square Slit pattern
biology analysis at the
molecular level
Non Scaffold Based
Hanging Drop Microplates
• Cells can self assemble into 3D
spheroid structure in the
absence of a scaffold.
• Spheroid size is controlled by
the number of cells dispensed
into each drop.
• Suitable for long term culturing
by transfer of culture onto
another plate of larger media Top view Bottom view
or buffer volumes
• Suitable for use as primary cell-
based tissue models and tumor
models that include
immortalized cancer cell lines
Non Scaffold Based
Spheroid Microplate
• Used to create round multi
cell tissue or tumor models.

• Wells are shaped to a depth


of SBS 96 – 384-well
microplate.

• Widely used for experimental


purposes.

• ULA is used to coat the


bottom to minimize cell
adherence for spheroid
formation
Non Scaffold Based
Microfluidic
• Used to create 3D architecture of in-vivo
tissues and tumors
• To create heterogeneous models.
• Permits the continuously supply necessary
nutrients and growth requirements as well
as waste removal through culture medium.
• Contains physical barriers that are made up
of glass or silicon, polymers such as
polycarbonate (PC), polystyrene (PS), and
chromatographic or filter paper.
• Cells may be combined with matrix to
encourage cell-ECM interaction for the
assembly of 3D structures.
• Suitable for stem cells, primary and cancer
cells
Bioreactor
• Ideal for mass cell production and ex vivo
tissue engineering applications.
• Suited for cell expansion applications or
scaled production of cellular products, such
as antibodies.
• Bioreactors for 3D cell culture fall into 4
main categories:
– The spinner flask
– Low-shear-stress rotating wall vessels
– Perfusion bioreactors
– Combination of bioreactors with
scaffolds,
Gels
• Have soft-tissue like stiffness that
mimic the ECM.
• Can be made from ECM mixtures
of natural sources such as
collagen and alginate.
• Most widely used is Matrigel
• Synthetic gels include
polyethylene glycol (PEG) – Based
Hydrogel.
• Difficult to use due to inability to
maintain viscosity, and may be
used in combination with other
methods.
Applications of 3D Cell Cultures
Applications
Consideration in 3D Cell Cultures
CONCLUSSION
• There are many methods
available to create in-vivo
cellular environments
• Some are amenable for
re-creating in-vitro barrier
models.
• others are more suitable
for mimicking cancerous
tumors or tissues.

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