8 - TE Basics and Scaffolds

Question 1

What is tissue engineering?

Answer 1

Replacing diseased or damaged living tissue with living tissue designed and constructed for the needs of the individual

Question 2

What is the basic TE workflow?

Answer 2

1. Cell isolation and enrichment for the cells we want to use therapeutically
2. Expansion of cell number in culture
3. Seeding cells onto a suitable scaffold to give the tissue its 3D nature whilst promoting growth and differentiation
4. Tissue maturation on the scaffold in a bioreactor until functionality achieved
5. Implantation of the engineered tissue back into the patent the cells were first isolated from

Question 3

What are the 2 main sources of cells for TE?

Answer 3

1. Autologous - self
2. Allogeneic - non self

Question 4

Give me some examples of methods used to isolate/enrich cell populations and what they select for?

Answer 4

1. Differential adhesion - based on how well cells adhere to different surfaces
2. Density centrifugation - based on size
3. Fluorescence-activated cell sorting - based on size, granularity and surface markers
4. Magnetic-activated cell sorting - based on surface markers

Question 5

Give me some issues that arise when trying to expand your enriched cell population

Answer 5

• Population will still be heterogeneous and cells grow at different rates
• Culture conditions directly impact the cellular growth and function so need to be carefully made
• Ensuring all cells reach final phenotype is key as any remaining SC’s pose a teratoma risk

Question 6

Give me some modern ways to expand cell populations enough for TE

Answer 6

1. Multilayered flasks - cell hotels
2. Bioprocessing - bioreactors

Question 7

How does the stirred flask bioreactor work to expand cell populations?

Answer 7

• Spherical stirrer = high SA:V for cells to grow on
• Stirred = efficient flow of nutrients to and waste away

Question 8

How does the fluidised bed bioreactor work to expand cell populations?

Answer 8

• Culture medium flows through column
• In the column is cells and microcarriers for the cells to adhere to (high SA:V)
• Fluid flow fluidises the particles, keeping them suspended to promote their adhesion to microcarriers not the column walls
  ALSO means nutrients flow to, waste away

Question 9

How do hollow fibre bioreactors work to expand cell populations?

Answer 9

Same principle as fluidised bed but has many straws = very high SA:V that you can then all bundle together to form a larger tissue
can grow cells on the inside and outside = ideal for blood vessels with internal endo and external smooth muscle

Question 10

What is the principle of using a scaffold in TE?

Answer 10

Acts as 3D support for cell growth by directing the tissue to grow in a way that looks like the tissue in vivo, making it more functional

Question 11

What are the key parameters that scaffolds should have and why are they important?

Answer 11

1. Biocompatible - no immune response once transplanted
2. Biodegradeable - as tissue matures it will replace it with its own ECM
3. Cytocompatible - works well with the cells you’re wanting to grow on it
4. Porous - properly populate the scaffold densly with cells and efficient nutrient and waste flow
5. Mechanically appropriate - grown under same mechanical conditions tissue is subjected to in vivo to make it functional
6. Architecturally appropriate - Look like the tissue in vivo
7. Growth promoting - otherwise cells wont actually grow on it

Question 12

What are the 2 main categories of natural scaffold materials and give me some examples of them

Answer 12

1. Polypeptides - collagen, fibronectin, laminin, silk protein, zein
2. Polysaccharides - Hyaluronic acid, alginate, chitosan

Question 13

What are the main issues surrounding using natural scaffolds for TE?

Answer 13

1. High batch to batch variation
2. Some of them aren’t scalable

Question 14

What are the main pros of using natural scaffold materials?

Answer 14

1. Already biocompatible and cytocompatible
2. Already architecturally appropriate

Question 15

Give me three examples of synthetic polymers used for scaffolds

Answer 15

1. Poly-caprolactone
2. Poly-lactic acid
3. Poly-glycolic acid

Question 16

Give me some pros of using synthetic polymers as scaffold materials

Answer 16

1. Gives you a high degree of control over degradation rates, mechanism, chemical functionality and biological signals
2. Reproducible - less batch to batch variation
3. Bulk processing is easier - scalable option
4. Can incorporate interesting properties through the making process

Question 17

What is the most studied synthetic co-polymer for scaffolds?

Answer 17

PLGA
poly lactic-glycoic acid