Bioprinting: Chapter 3

The start of yet another grind

What is involved in bioprinting

1. Biomaterials

2. Cells

3. Growth factors

Bioprinting

The use of material transfer processes for patterning and assembling biologically relevant materials (molecules, cells, tissues, and biodegradable biomaterials) with a prescribed organisation to accomplish one or more biological functions

Organ printing definition

Computer aided 3D tissue engineering of living organs based on the simultaneous deposition of cells and hydrogels with the principles of self-assembly

Advantages of Bioprinting

• Ability to create 3D structures with living biological elements

• ability to manipulate material and cells

• find solutions to solve limited cell in-growth and nutrient exchange

• can use for disease testings

• can fabricate organs

Bioprinting Process Flow

• Preprocessing: Design

• Processing: Biofabrication

• Post-processing: Maturation

Bioprinting Process Flow Steps

1. Imaging

2. Design approach

3. Material selection

4. Cell selection

5. Bioprinting

6. Application

Basic Techniques of Bioprinting

• extrusion

• inkjet

• laser-based

Types of materials extruded for bioprinting

• biomaterials/hydrogels (envisiontec)

• cell spheroid strands (organovo)

EnvisionTEC’s 3D-Bioplotter System

• most work in sterile environments (laminar flow box)

• 0.001mm positioning accuracy

• 5 different material cartridges can be used!

process:

• deposits material from a syringe using air or mechanical pressure

• material is in strand form

Bioplotter System Principles

• extrude soft bioink

• print material into supporting liquid, which holds soft material in place using buoyancy

• solidify the bioink using ionic transfer or crosslinking

• once solidified, the structure will hold its shape

Organovo’s NovoGen MMX BioprinterTM

• small, compact, and sterile,

• extrudes spherical aggregates (diameter of 500 or 260μm) and preloaded in micropipette-cartridges (75mm long)

• aggregates preloaded in micropipette-cartridges

• hydrogels can be printed with the purpose of being removable support structures

• Fabricated into a 24 well-plate

NovoGen MMX BioprinterTM Spheroid Printer

1. make bio ink: grow spheroids from the cells of specific tissues

2. the printer has multiple dispensing heads (bio ink: cells and bio-inert hydrogel: supports/agarose rods) and prints the material layer by layer

Traits of tissue spheroids

• are visco-elastic-plastic soft matter

• are a complex fluid

• THEY CAN FUSE (in fact you want them to!)

NovoGen MMX BioprinterTM Advantages & Disadvantages

Advantages:

• high efficiency (better than inkjet)

• high cell density (allows for organised cellular feature: intercellular tight junctions, microvascular networks)

• can make thick tissue constructs

• can print multiple geometries

• scaffold-free

Disadvantages

• need a controlled environment

• lack of stability in vertical printing

• can control position of each cell

• cant print fine features

Disadvantages with Spheroid Specific Approaches

• need a spheroid of a certain size

• shrinkage

• spheroid fusion required before

Cyfuse Biomedical K.K. Regenova

• assemble spheroids by placing the in a prepared needle array

• let the cells mature/culture

• needle array: 100-200μm

Inkjet Printing

• non-contact printing

• drop-on-demand manner (dropped where and when it needs to)

• not a new technology (used in electronics + micro-engineering)

Types of inket printing

• thermal

• piezoelectric

Piezoelectric inkjet printing

• ink drops ejected through the piezoelectric actuator (converts energy into motion)

• ejected from contraction of reservoir, and then regains original shape

• no heat!

Thermal inkjet printing

• elements: heating unit, ink chamber, small nozzles (diameter 30-200μm)

• heater raises temp (300°C) within 10 microseconds.

  • fluid temperature increases by 5-10°C)

• small air bubble is created, bubble expands then collapses

• chamber refills and repeats

Inkjet Printing Process & Principle (4 Steps)

1. generating pressure to eject fluids

2. bubble forms and collapses through the orifice

3. droplet is deposited onto a substrate

4. repeat

Fujifilm’s Dimatix Materials Printer (DMP)

• has a disposable piezoelectric inkjet cartridge

  • users can fill their own fluids!

  • catridge capacity: 1.5ml

  • cartridge nozzles: 16 spaced at 254 micros

    • drop sizes are 1 and 10 picoliters

    • as small as 20μm

• area: 200 × 300 mm

• substrates: up to 25mm thick

• temperature: up to 60°C

• variety of patterns possible

Inkjet Printing Advantages & Disadvantages

Advantages:

• Low cost, high reproducibility, no-contact

• High automation

• Wide variety of materials

Disadvantages:

• Nozzle clogging

  • lead to cell damage

• Cell degradation

  • through force of droplet hitting substrate

  • difficulties in cell aggregation and sedimentation

• High shear strain

• low resolution of droplet

• low efficiency

• can’t print high-cell density constructs

nScrypt’s Tabletop and 300 Series Printers

• opens and closes with the Smart PumpTM

  • uses positive pressure + computer controlled needle valve

• extrudes with pressure (12 nL/s to 1 mL/s)

• uses optical and piezoelectric sensors

• volume in pump: 0.024 to 0.1 cubic cm

• viscocity range: 1 to 1 million centipoise

• resolution: less than 5μm

Laser-assisted Bioprinting Advantages & Disadvantages

Advantages

• single cell resolution

• fast and precise

• no-contact

Disadvantages

• small working volume

• highly skilled operation

• costly

• need safety equipment

Laser Guidance Direct Write (LGDW)

• uses radiation pressure to guide particle deposition

  • foce: more than 10pN (much much larger then cell)

• receiving substrate and particle suspension

• 2 components:

  • radial pulls particles towards center of laser (towards center)

  • axial guides particles along laser beam (straight)

Laser Induced Forward Transfer (LIFT)

• moves material from an optically transparent quartz disk to a receiving substrate

• pulsed laser → mirror → lens → ribbon → substrate

• interface:

  • quartz disk (absorbs laser, 1-100 nm thickness) functions as laser absorption

  • biomaterial layer (underneath disk, 10-100 μm thic)

• 5-7 cells per droplet

• high resolution

Light Processing - Photopolymerization

• UV light to crosslink hydrogels

Requirements of Photo Initiators in Bioprinting

• soluble in water

• low cytotoxicity

• high extinction (absorption) coefficient at visible-light wavelength

VAT Photopolymerisation Bioprinting

• plate is lowered from the top of the resin vat

• uv light cures resin layer by layer

• layers are built on top of previous

• 2.5 dimensional structure can be built and 50 μm resolution

VAT Photopolymerisation Advantages & Disadvantages

advantages:

• high resolution

• fast

• can make complex structures

• can make scalable products

disadvantages

• limited bio-ink/photoinitator

• can’t print multiple materials

• difficult to achieve high cell density

RegenHU’s BioFactory Combination System

• both contact and non-contact

• uses UV and laser beams

• uses in vivo morphology

• does all! (inkjet, extrusion, laser)

Material viscosity classifications

• inkjet

  • low viscosity

• extrusion

  • medium/high viscocity

• melt extruder

  • solid