Bioprinting: Chapter 6

2-dimensional (2D) static cell culture TOOLS

• cell culture flasks

• multiwall plates

• petri dishes

2D cell culture

• Cells grow as a flat layer on plastic/glass surfaces.

• Requires frequent media changes for nutrients and waste removal.

• Does not mimic the 3D structure of real tissues.

2D cell culture limitations

• Over-simplifies the natural environment of cells.

• Cells behave differently in flat 2D culture compared to 3D tissues in the body.

• (eg: does not replicate blood circulation and complex tissue structure)

Animal model limiations

• ethical

• time consuming

• animal immunity is not representative of humans

5 different types of 3d cell culture models

• scaffold-based

• scaffold-free spheroid

• gel

• bioreactor

• microchip

(some say girls bully men)

Scaffold-based 3D cell culture

• temporary, artificial ECM to accomodate cells and support tissue regeneration

Hydrogel scaffolds

• mimics natural ECM

• ex:

  • alginate (cell transplantation, drug delivery)

  • PEG/HA (cancer cells, stem cells)

  • Macroporous hydrogel (3d cancer cell cultures)

Scaffold-free multicellular spheroids

• 3D structures made from cells naturally coming together to form a cluster

• cells organize themselves and therefore do not need a scaffold

Spheroids as other In Vitro Tissue Model Examples

• liver tissue models

• bone tissue models

• smooth muscle tissues

• skeletal muscle tissues

fabrication techniques for spheroids

• rotating-wall vessels

• U-shaped low attachment microwell plates

• hanging-drop method

rotating-wall vessels

• Cells + ECM-coated beads are placed in a rotating vessel.

• Rotation keeps cells suspended and allows 3D tissue formation.

• Gas-permeable membrane provides oxygen exchange.

U-shaped, low-attachment microwell

• curved bottom glass flask (of sometime)

• cells are suspended, floating around

• they condense to from a spheroid (because of the slope of the glass)

• cells do not adhere to the walls because of some kind of coating

Challenges of spheroids for 3D cell cultures

• lack of control over size and structure/shape

• lack of nutrient and oxygen supply

• not similar to natural tissues

bioreactor

• device where biochemical/biological processes develop under a controlled and monitored environment

Types of Bioreactors

1. spinner flask bioreactor

2. rotating wall bioreactor

3. compression bioreactor

4. strain bioreactor

5. flow perfusion bioreactor

(some rats can’t stay fat)

Spinner flask bioreactor

1. Cells are placed on a scaffold inside a flask containing nutrient-rich culture medium.

2. A spinner (stirrer) mixes the medium, creating fluid movement around the scaffold.

3. This movement improves nutrient and oxygen delivery to cells through convection (fluid flow), rather than only diffusion.

4. Cells grow and produce tissue on the scaffold (commonly used for bone tissue engineering).

5. Gas exchange occurs through porous covers on the flask.

Spinner flask bioreactor limitation

• Nutrients do not reach all areas equally → more cells grow on the outside of the scaffold than the centre.

Rotating wall bioreactor

• Scaffolds + cells are placed in culture medium between two cylinders.

• The outer cylinder rotates, keeping scaffolds suspended and moving.

• This creates a low-gravity (microgravity-like) environment.

• Cells experience gentle movement and can form more uniform 3D tissues.

Compression bioreactor

• A mechanical system compresses the scaffold containing cells.

• Compression can be static (constant) or dynamic (repeated).

• The pressure pushes fluid through the scaffold, improving nutrient and oxygen delivery.

• Helps cells grow and form tissue.

• (FLUID FLOWS THROUGH SCAFFOLD)

Strain bioreactor

• Cells are grown on a scaffold that is clamped in place.

• The scaffold is stretched with cyclic tensile strain (repeated pulling).

• Mechanical strain stimulates cells to differentiate and form specific tissues.

• Used for tendon, bone, ligament, cartilage, and cardiovascular tissue.

Flow perfusion bioreactor

• A pump pushes culture medium through a scaffold containing cells.

• Continuous flow provides nutrients and oxygen throughout the scaffold.

Bioreactor advantages & disadvantages

advantages:

• high volume cell production

• customizable for different physical signals and process

disadvantages:

• high cost

• engineering and fabrication challenges

Microchips

• Miniature devices that mimic human organs/tissues on a chip using human cells.

• Recreate tissue–tissue and organ–organ interactions to study diseases and test treatments.

• Used as an alternative to animal testing.

Advantages & Disadvantages

Advantages:

• More accurately represents human biology than animal models.

• Can study diseases that cannot be replicated well in animals.

• Reduces the need for animal testing.

• Allows drug testing in a controlled environment.

Disadvantages:

• Complex and expensive to develop.

• Difficult to fully replicate the complexity of a whole human body.

• Limited adoption due to technical challenges and standardisation issues