This document discusses 3D cell culture techniques. It defines 3D cell culture as permitting biological cells to grow and interact in all three dimensions, mimicking their natural environment. This is an improvement over 2D cultures where cells grow unnaturally. The document describes various 3D culture methods including scaffold-based techniques using polymeric scaffolds or biological scaffolds, and non-scaffold methods like hanging drop plates, spheroid plates, microfluidics, and gels. It also discusses bioreactors and lists applications of 3D cultures.

1. 3D CELL CULTURES
By:
Ajisafe Victor Ayobami
OSMANIA UNIVERSITY,
HYDERABAD , INDIA

2. 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.

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

4. 2D vs 3D Cell Cultures

5. 3D Cell Culture Techniques
a) 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
c) Bioreactors
a) Gels
3D Cell culture Bioreactor
3D Cell Culture Gels
Non Scaffold Based
Scaffold Based

6. 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

7. 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

8. 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
biology analysis at the
molecular level
Round Square Slit pattern
Scaffold Based

9. 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
or buffer volumes
• Suitable for use as primary cell-
based tissue models and tumor
models that include
immortalized cancer cell lines
Top view Bottom view
Non Scaffold Based

10. 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

11. 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
Non Scaffold Based

12. 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,

13. 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.

14. Applications of 3D Cell Cultures
Applications

15. Consideration in 3D Cell Cultures
• 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.
CONCLUSSION