Bottom-up strategies in tissue engineering

Tissue fabrication techniques

• Top down tissue engineering

• Bottom up (or modular) tissue engineering

• Scaffold-Free (spheroids aggregation and printing ; cell
sheet)
• Scaffold-Based (cell-laden based approaches; cell-
seeded micro carriers based approaches)
Bottom-up strategies in tissue engineering

Tissu
e fab actors
ricat re
BOTTOM UP ion t / Bio
echn
ique process
TOP DOWN e
s Tissu
The initial cellular construct need to be
To organize cells in a 3D context guided toward the final tissue

Bioreactors
Material
Cellular Process
Cell material
microenvironment variables
interaction
Fluid
transport
Strategy
Top down Nutrient
/ transport
3D cellular
constructs Bottom up
Cell font Control
Tissue
growth and
The design of these remodeling
variable applies @ Time …..Toward the final tissue
0!!!!
Bottom-up strategies in tissue engineering

Top-Down: cells are inserted in a 3D preformed structure (scaffold): dimension centimeters

Bottom up: living biological entities with at least one dimension minaor than 100 microns: are
named building blocks, lving lego, mictotissuesm micromodules. Such enttites are then
assembles in a larger structures.
Scaffold Cell-Laden Cell-Seeded
Free Hydrogels Micro-carriers

Spheroids Cell-sheet Living Bio-ink Living Legos Cell-beads Cell-Fiber

Bottom-up strategies in tissue engineering

Worldwide strategies
Scaffold Free Cell-Laden Hydrogels

Tissue
Units
Spheroids Cell-sheet Living Bio-ink Living Legos Cell-beads Cell-Fiber

Culture on non- Confluent 2D cells Cell entrapped Cell encapsulation

Micro-molding Co-axial flow in
Fabrication adhesive on responsive in pre-gel at T-Junction in
Flow-lithography microfluidic
Method surface surface solution microfluidic

3D Deposition Cross-link
Stacking cell Molding Fiber weaving
of sintered during the Molding
sheets 3D-Printing
Macro- spheroids printing
Tissue
Formation

Worldwide
Lab Leaders
Forgacs G., Univ. Okano T., Women’s Khademhosseini A., Takeuchi S.,
of Missouri Med. Univ., Tokyo MIT - Boston Univ. Of Tokyo
Bottom-up strategies in tissue engineering

BOTTOM-UP/SCAFFOLD-FREE/Spheroids
Preparation techniques
Bottom-up strategies in tissue engineering

BOTTOM-UP/SCAFFOLD-FREE/Spheroids
Cells are able to self assemble by means of cell – cell junctions
Bottom-up strategies in tissue engineering

BOTTOM-UP/SCAFFOLD-FREE/Spheroids
Cells are able to self-organize depending on the adhesion properties of the surface
Spontaneous formation of endothelial lumen

Shadows: Self-Orientation of les

Endothelial cells membrane with low adhesive membranes in
density of adhesion the inner region
proteins
Bottom-up strategies in tissue engineering

BOTTOM-UP/SCAFFOLD-FREE/Spheroids
self-sorting: depends on adhesion properties of different cell populations

Cell “mixture”
- Green: normal cells
- Red: modified cells with, less adhesive

Spontaneous evolution: less adhesive cells migrate to the outer region

Bottom-up strategies in tissue engineering

BOTTOM-UP/SCAFFOLD-FREE/Spheroids
Self-Assembly and Sorting: formation of complex structures

200 micron
sintering sorting

17h 42h 73h

Black: epithelial cells

Gray: neural cells
Bottom-up strategies in tissue engineering
BOTTOM-UP/SCAFFOLD-FREE/Spheroids
Self-Assembly and Sorting: tissue fluidity theory

Adhesion force can be

considered as “surface
tension” of the cells.

Cell segregation depends

on surface tension values
Bottom-up strategies in tissue engineering
BOTTOM-UP/SCAFFOLD-FREE/Spheroids

Pro’s
• Easy to prepare
• High yielding
• Replication of necrotic core (tumoral tissues)
• Can be printed forming complex architectures
• Useful to reproduce tissue with high cell density

Con’s
• Low ECM density
• Low interaction with the ECM
• Not adequate to describe transpor or mechanical properties
Bottom-up strategies in tissue engineering

BOTTOM-UP/SCAFFOLD-FREE/Spheroids
Printing techniques by using single cells: high planar resoltion
Bottom-up strategies in tissue engineering

BOTTOM-UP/SCAFFOLD-FREE/Spheroids
Controlled deposition of building-blocks od fused spheroids

Extruder bio-printing
Bottom-up strategies in tissue engineering
BOTTOM-UP/SCAFFOLD-FREE/Spheroids

Use of systems formed by single cells or cell aggregates to form complex architectures

Extruder bioprinting

Micro-cylinders

Tissue fibers
Bottom-up strategies in tissue engineering

BOTTOM-UP/SCAFFOLD-FREE/Spheroids
Examples layer-by-layer desposition of spheroids
Problems: instability due to structure collapse

Stacking of tissue fibers or spheroids on non-adhesive hydrogels template.

More stable structures
Bottom-up strategies in tissue engineering

BOTTOM-UP/SCAFFOLD-FREE/Spheroids
Examples

Hetero-typic
cardiomyocites Ramified structures Capillary like
structures

0h

Red:
60-70 h endothelium
Green:
SMC
Bottom-up strategies in tissue engineering

BOTTOM-UP/SCAFFOLD-FREE/Spheroids
Examples Non adhesive gel

SMC

ENDOTHELIUM
Bottom-up strategies in tissue engineering
In vitro meat
BOTTOM-UP/SCAFFOLD-FREE/Spheroids
Examples of companies that use this
approach

In vitro Hamburger

https://www.youtube.com/watch?v=u31RXdazMRo

Leather

https://www.youtube.com/watch?v=7gXq1ml6B1E