Biomanufacture

Question 1

Bioprinting techniques

Answer 1

Ink-jet
Extrusion
Laser-assisted/LIFT (laser induced forward transfer)
Stereolithography

Question 2

Possible filament types during extrusion

Answer 2

Well-defined filament
- Swelling filament
- Equivalent diameter filament
- Stretched filament

Irregular filament
- Rough surface filament
- Over-deposited filament
- Compressed filament
- Discontinuous filament

Question 3

Printing considerations

Answer 3

Printing:
Extrudable
Laminar flow
Self-supporting
Can span gaps

Structure:
Few-to-many layers
Biocompatible
Supports cell function
Can be handled

Cell Encapsulation:
Cell-compatible
Sterile
Temperature-controlled

Question 4

Printability requirements

Answer 4

Different requirements on:
1. Complexity (complex versus simple)
2. Resolution (um versus mm)
3. Model scale (cm versus sub-mm)

Question 5

General requirements for extrusion

Answer 5

1. Material flows out of a nozzle;
2. Extruded material “settles” after extrusion, no “spreading out”;
3. Printed model should recapitulate designed model.

Question 6

Requirements extrusion before during after

Answer 6

1. Before extrusion:
   Material stays in a cartridge WITHOUT flowing out.
   —-Material’s gravity needs to be balanced by the friction between piston and cartridge wall + adhesive force between material and cartridge wall
2. During extrusion:
   Material flows out of nozzle, and be “shaped” by the nozzle geometry.
   —-Material must flow -> it undergoes plastic deformation —-Extrudate follows nozzle’s geometry
   To start the flow:
   The stress exerted on the material must exceed its yield strength
3. After extrusion:
   Material settles on a platform, without “spreading out”

Question 7

Assumptions for material to be extruded

Answer 7

• Homogeneous, isotropic, and incompressible. (hydrogels and pastes good)
• Steady state laminar flow. (true bioprinting bc low reynolds number)
• The lubrication boundary condition is satisfied. This means there is no slippery on the wall of the nozzle.
• Isothermal during extrusion

Question 8

Does higher viscosity stop post extrusion flow

Answer 8

Higher viscosity makes it =ow more “slowly”, but it does NOT stop the =ow

Question 9

Criteria for printing

Answer 9

Material’s intrinsic properties
viscosity;
flow index;
flow consistency;
cell type;
cell concentration; MW;
shear thinning; thixotropy;
stiffness;
gelationtime;
material concentration; integrity;
yield strength; toughness;
G’,G”;
material density;

Printing setup:
nozzle type; nozzle gauge; nozzle geometry; cartridge type; piston type;
pressure (pneumac driven) OR piston speed (piston driven); temperature (cartridge and plaZorm); translaonal speed;

Output: flow rate; Filament width; Filament height;

Question 10

Cell densities in biofabrication process

Answer 10

A key component of any biofabrication process is the living cells, often required in the 10 million–20 million cells per ml quantities

Need to reach cell densities greater than 200 million cells for any potent post-biofabricated tissue or organoid structure

Question 11

The major challenges in the design of bioinks for bioprinting

Answer 11

(i) designing materials that can be processed with current or developing biofabrication techniques at desired resolutions, (ii) maintaining the viability of cells during and after processing, and (iii) providing the appropriate cellular environment to guide desired cell behaviour.

Balancing printability with cell viability and function has been challenging, as important cellular processes, such as proliferation, di*erentiation, and ECM deposition, can be impeded when cells are embedded in dense polymer networks, while dense networks often support the best shape-,delity and long-term stability after printing

Challenge: Many in vivo tissue functions are not yet replicated in biofabricated tissues.
Importance: These functions are essential for evaluating drug responses and tissue mechanics.

Question 12

Integration of long-term cell culture with microfluidic devices.

Answer 12

Purpose: Provides a long-term and simulated physiological environment for culturing printed models.

Question 13

How to mimic biological relationships in vitro.

Answer 13

Use microfluidic systems and perfusion chambers to simulate in vivo environments.
Outcome: Facilitates the study of cell and tissue interactions, incorporating cell types, fluid flows, and biomolecules.

Question 14

Biohybrid tissues

Answer 14

Combination of 3D-printed non-living parts (e.g., polymers/electrodes) with biofabricated cellular components.
Function: Leverage mechanical/electrical properties for specific tissue functions.

Question 15

Collagen for organ engineering

Answer 15

Collagen is an ideal material for biofabrication because of its critical role in the ECM, where it provides mechanical strength, enables structural organization of cell and tissue compartments, and serves as a depot for cell adhesion and signaling molecules.
*
However, it is difficult to 3D-bioprint complex scaffolds using collagen in its native unmodified form
because gelation is typically achieved using thermally driven self-assembly, which is difficult to control.

Question 16

FRESH bioprinting

Answer 16

FRESH works by extruding bio-inks within a thermoreversible support bath composed of a gelatin microparticle slurry that provides support during printing and is subsequently melted away at 37°C

Question 17

What is final extrusion width related to

Answer 17

(1) material’s intrinsic properes (n, K, τy)
(2) nozzle geometry (R, L)
(3) printer input (ΔP, Vtoolhead, h)

Question 18

Electrospinning setup

Answer 18

high-voltage power supply (AC or DC), a syringe pump, a spinneret (usually, a hypodermic needle with blunt tip), and a conductive collector.

Question 19

Electrospinning

Answer 19

Electrospinning is a voltage-driven, fabrication process governed by a specific electrohydrodynamic phenomenon where small fibers are yielded from a polymer solution.

Question 20

How does the process of electrospinning work

Answer 20

An electric field is established between the needle tip and collector by applying a specified voltage.

Pump causes the solution to flow at a constant rate, charges accumulate at the surface of the liquid.

When the electrostatic repulsion is larger than the surface tension, the liquid meniscus deforms into a conically shaped structured known as a Taylor cone from which a charged jet is ejected.

• The jet initially extends in a straight line and then undergoes vigorous whipping motions because of bending instabilities.

Question 21

4 steps of electrospinning

Answer 21

(i) charging of the liquid droplet and formation of Taylor cone or cone- shaped jet;
(ii) extension of the charged jet along a straight line;
(iii) thinning of the jet in the presence of an electric field and growth of electrical bending instability (also known as whipping instability);
(iv) solidification and collection of the jet as solid fiber(s) on a grounded collector

Question 22

Effect of solvent on electrospinning

Answer 22

Manipulates fiber morphology
Solvents that have more interaction with the polymer structure also tend to result in a solution with higher viscosity.

• A solvent able to dissolve the polymer(s) and additive(s) while maintaining a homogeneous solution is always desired.
• Ideally, the solvent in the polymer solution should completely evaporate during the electrospinning process.
• If it does not evaporate the generated fibers may undergo fused fiber- fiber bonding.

Question 23

Effects of Viscosity on electrospinning

Answer 23

• Viscosity influenced by the molecular weight of the polymer (along with solution concentration and solvent selection of course).

higher molecular weight or higher concentration translates into higher viscosity.

• Processing with a high viscous solution encourages polymer chain entanglements to occur during the jet formation -> source of the fiber structures characteristic of electrospinning.

Question 24

Key parameters to maintain batch-to-batch consistency for electrospun nano- and microfiber development

Answer 24

viscosity, conductivity, and surface tension — all of which are affected by polymer, solvent, and additive selection

Solid content and solvent selection are measured to maintain solution reproducibility and manipulate fiber morphology, respectively.

solution should be well mixed and homogeneous

Question 25

What happens when polymer viscosity is too low during electrospinning

Answer 25

• Processing a solution with a low viscosity translates to insufficient polymer entanglements during the process, resulting in the polymer jet breaking and forming particles instead of fibers.
• This is a different process known as electrospraying.

Question 26

Effects of conductivity on electrospinning

Answer 26

• Solvent and polymer content can affect the overall solution conductivity.
• Solutions with higher conductivity tend to generate bead-free fibers.
• Some solvents contain additives or stabilizers that then affect solution conductivity and its spinnability.
• Inorganic and organic salts, along with surfactants, can be used to selectively manipulate the solution’s conductivity.
• When increasing the overall charge density of the solution, jets formed during the electrospinning process can be optimized to promote bead-free fibers.

Question 27

Effects of Surface Tension on electrospinning

Answer 27

• Electrospinning is dependent upon the electrostatic force overcoming the surface tension of the solution.
• As a consequence, solutions with low surface tensions are usually preferred.
• Surface tension is affected by polymer concentration, solvent selection (single or co-solvent systems), and additives (ex. surfactants).