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3D CELL CULTURE
ENGINEERING:Microenvironment directed manipulation
and mathematical models
By: Naghmeh Poorinmohammad
We are going to talk about...
• Why three-dimensional cell cultures?
• What is the technology used for?
• Factors affecting SC differentiation in 3D cultures
• How can it help us—microbial biotechnologists?
• Summary
We are going to talk about...
• Why three-dimensional cell cultures?
• What is the technology used for?
• Factors affecting SC differentiation in 3D cultures
• How can it help us—microbial biotechnologists?
• Summary
Why three-dimensional cell cultures?
• Growing cells on flat surfaces is artificial and unnatural.
• Naturally ECM plays an important role in regulating cellular behaviors by influencing cells with: biochemical signals and topographical cues.
• In 3-D cultures, we can control scaffold morphology, architecture and components(haman).
• Therefore cells behave and respond more like they would in vivo to stimuli.
Blagovic, Katarina, et al. "Engineering cell–cell signaling." Current opinion in biotechnology 24.5 (2013): 940-947.
Why three-dimensional cell cultures?
Lee, Jungwoo, Meghan J. Cuddihy, and Nicholas A. Kotov. "Three-dimensional cell culture matrices: state of the art." Tissue
Engineering Part B: Reviews 14.1 (2008): 61-86.
Why three-dimensional cell cultures?
• 2D culture substrates not only fall short of reproducing the complex and dynamic environments of the body, but also are likely to misrepresent findings to some degree by forcing cells to adjust to an artificial flat, rigid surface 1.
• These matrices, or scaffolds, are porous substrates that can support cell growth, organization, and differentiation on or within their structure. Architectural and material diversity is much greater on 3D matrices than on 2D substrates 1.
• Other than physical properties, chemical/ biochemical modification with specific biological motives to facilitate cell adhesion, cell-mediated proteolytic degradation and growth factor binding and release 2 .
[1] Lee, Jungwoo, Meghan J. Cuddihy, and Nicholas A. Kotov. "Three-dimensional cell culture matrices: state of the art."
Tissue Engineering Part B: Reviews 14.1 (2008): 61-86.
[2] Lienemann, Philipp S., et al. "A Versatile Approach to Engineering Biomolecule‐Presenting Cellular Microenvironments."
Advanced healthcare materials 2.2 (2013): 292-296.
We are going to talk about...
• Why three-dimensional cell cultures?
• What is the technology used for?
• Factors affecting SC differentiation in 3D cultures
• How can it help us—microbial biotechnologists?
• Summary
What is the technology used for?
Clinical
applicationin vitro studies
Regenerative
medicine
Drug
Discovery
process
Analysis of cell
biology at the
molecular level
Lee, Jungwoo, Meghan J. Cuddihy, and Nicholas A. Kotov. "Three-dimensional cell culture matrices: state of the art."
Tissue Engineering Part B: Reviews 14.1 (2008): 61-86.
3D culture engineering to regenerate tissues
Adopted from: Tandon, Nina, et al. "Bioreactor engineering of stem cell environments."Biotechnology advances 31.7 (2013):
1020-1031.
3D culture engineering to regenerate tissues
• In order to induce cell growth in the third dimension and to support tissue development, it is critical to provide mass transport to and from all cells using dynamic culture systems such as bioreactors.
• In bioreactors, stirring, perfusion, and dynamic loading have been applied to provide convective transport and allow tissue development on a millimeter to centimeter scale
Tandon, Nina, et al. "Bioreactor engineering of stem cell environments."Biotechnology advances 31.7 (2013): 1020-1031.
Figure adopted from: http://www.tissuegrowth.com/prod_systems.cfm
We are going to talk about...
• Why three-dimensional cell cultures?
• What is the technology used for?
• Factors affecting SC differentiation in 3D cultures
• How can it help us—microbial biotechnologists?
• Summary
1. Matrix elasticity/stiffness
CASE STUDY A:
• Cells experience a wide range of matrix mechanics, from soft (e.g. brain 0.1 kPa) to stiffer (e.g. precalcified bone 80 kPa) tissues, which direct many aspects of cellular function. az guvendiren
• Hydrogel mechanics can be easily controlled by increasing crosslinking density.
Engler, Adam J., et al. "Matrix elasticity directs stem cell lineage specification." Cell 126.4 (2006): 677-689.
Cigognini, Daniela, et al. "Engineering in vitro microenvironments for cell based therapies and drug discovery." Drug
discovery today 18.21 (2013): 1099-1108.
1. Matrix elasticity/stiffness
2. Mechanical properties
Cigognini, Daniela, et al. "Engineering in vitro microenvironments for cell based therapies and drug discovery." Drug
discovery today 18.21 (2013): 1099-1108.
3. Matrix topology
Lee, Jungwoo, Meghan J. Cuddihy, and Nicholas A. Kotov. "Three-dimensional cell culture matrices: state of the art."
Tissue Engineering Part B: Reviews 14.1 (2008): 61-86.
CASE STUDY B:
• Neural stem cells (NSCs) are capable of self-renewal and differentiation into three principle central nervous system cell types under specific local microenvironments.
3. Matrix topology
Chi-F
Chi-MC
Chi-PS
Wang, Gan, et al. "The effect of topology of chitosan biomaterials on the differentiation and proliferation of neural
stem cells." Acta biomaterialia 6.9 (2010): 3630-3639.
Wang, Gan, et al. "The effect of topology of chitosan biomaterials on the differentiation and proliferation of neural
stem cells." Acta biomaterialia 6.9 (2010): 3630-3639.
3. Matrix topology
Cigognini, Daniela, et al. "Engineering in vitro microenvironments for cell based therapies and drug discovery." Drug
discovery today 18.21 (2013): 1099-1108.
4. soluble signaling molecules
• Natural ECM hosts soluble signaling molecules, including growth factors (GFs).
• They play significant roles in tissue development by triggering a wide range of cellular responses.
• Therefore, it is important to introduce well-controlled GF presentation into hydrogels to instruct encapsulated stem cell behavior.
• Direct encapsulation is the traditional method of GF presentation in hydrogels.
• Limitations in this approach (e.g. lack of control over delivery profiles) led to the development of micro/ nano delivery vehicles within hydrogels for GF delivery.
Guvendiren, Murat, and Jason A. Burdick. "Engineering synthetic hydrogel microenvironments to instruct stem
cells." Current opinion in biotechnology24.5 (2013): 841-846.
CASE STUDY C:
• Amsden and colleagues reported rapid induction and enhancement of chondrogenesis of encapsulated adipose-derived stem cells (ASCs) in chitosan-based hydrogels with coencapsulation of microspheres containing either BMP-6 or TGF-b3 .
4. soluble signaling molecules
Guvendiren, Murat, and Jason A. Burdick. "Engineering synthetic hydrogel microenvironments to instruct stem
cells." Current opinion in biotechnology24.5 (2013): 841-846.
4. soluble signaling molecules
Cigognini, Daniela, et al. "Engineering in vitro microenvironments for cell based therapies and drug discovery." Drug
discovery today 18.21 (2013): 1099-1108.
Analytical/Mathematical modeling of 3D cultures
bioreactor
Supporting 3D scaffold
cells
The choice of cells
concerns mainly their
capability to proliferate
and the preservation of
biological activity
The scaffold is not only a
physical support for the
cells, but it also affects cell
metabolism, differentiation,
and morphogenesis
its function is to provide
suitable nutrients and
oxygen flow to the cells in
the scaffold to ensure their
growth and to remove
catabolic products
Analytical/Mathematical modeling of 3D cultures
• Mathematical models are very useful in order to better understand the complex chemical, mechanical, and biological factors involved in engineered tissue cultures.
• Since many studies have highlighted, especially for osteoblasts, the sensitivity of cell growth to mechanical stress, several studies have been made of fluid dynamics inside the bioreactor.
• Botchwey calculated the shear stress inside the construct, describing the velocity with Darcy’s law for porous media.
• Critical for fluid dynamic studies is the identification of scaffold geometry. For this purpose, optical methods have been used. Raimondi captured a light microscopy image of a cross section of the construct’s histological sample to generate a computation fluid dynamics (CFD) model and, then, computed the shear stress inside a perfused scaffold.
Coletti, Francesco, Sandro Macchietto, and Nicola Elvassore. "Mathematical modeling of three-dimensional cell cultures
in perfusion bioreactors." Industrial & engineering chemistry research 45.24 (2006): 8158-8169.
Analytical/Mathematical modeling of 3D cultures
• Cell growth and mass transport are the two major phenomena that affect bioreactor performance.
• For this reason, efforts have been made to describe cell growth and the supply of metabolites to the growing cells.
• Nehring proposed a reaction/diffusion model in a spherical chondrocytes pellet.
• Malda developed a mathematical model for oxygen gradient calculations in a three dimensional polymeric scaffold and compared simulated with experimental data given by a glass microelectrode.
• Using the method of volume averaging, Pathi developed a dynamical mathematical model for the growth of haematopoietic cells in a perfusion bioreactor in which the scaffold is placed between two perfusion chambers where the medium flows.
•
Coletti, Francesco, Sandro Macchietto, and Nicola Elvassore. "Mathematical modeling of three-dimensional cell cultures
in perfusion bioreactors." Industrial & engineering chemistry research 45.24 (2006): 8158-8169.
Analytical/Mathematical modeling of 3D cultures
• Various types of bioreactors have been used to culture cells for tissue
regeneration or repair.
• Recent studies show the importance of perfusion (the forcing of medium
flow through the scaffold) in growing a uniform and high-density tissue.
•
Intlet
Outlet
Scaffold
Coletti, Francesco, Sandro Macchietto, and Nicola Elvassore. "Mathematical modeling of three-dimensional cell cultures
in perfusion bioreactors." Industrial & engineering chemistry research 45.24 (2006): 8158-8169.
Analytical/Mathematical modeling of 3D cultures
•
Coletti, Francesco, Sandro Macchietto, and Nicola Elvassore. "Mathematical modeling of three-dimensional cell cultures
in perfusion bioreactors." Industrial & engineering chemistry research 45.24 (2006): 8158-8169.
Analytical/Mathematical modeling of 3D cultures
Coletti, Francesco, Sandro Macchietto, and Nicola Elvassore. "Mathematical modeling of three-dimensional cell cultures
in perfusion bioreactors." Industrial & engineering chemistry research 45.24 (2006): 8158-8169.
We are going to talk about...
• Why three-dimensional cell cultures?
• What is the technology used for?
• Factors affecting SC differentiation in 3D cultures
• How can we—microbial biotechnologists— involve?
• Summary
How can we involve?
• Problem: scaffolds can be colonized by bacteria, and the ensuing
infection can have catastrophic consequences.
• Solution case study: “Development of bioactive glass based
scaffolds for controlled antibiotic release in bone tissue engineering
via biodegradable polymer layered coating”.
• Our involvement: Antibiotics can affect ell adhesions and
differentiation....*.
Antimicrobial scaffolds for tissue engineering
[*] Xing, Zhi-Cai, et al. "In vitro assessment of antibacterial activity and cytocompatibility of quercetin-containing PLGA
nanofibrous scaffolds for tissue engineering." Journal of Nanomaterials 2012 (2012): 1.
How can we involve?
• Appropriate topology for neuroblastoma differentiation generated by
using a nanocellulose extracellularly excreted by Gluconacetobacter
xylinus.
• Case study: “3D Culturing and differentiation of SH-SY5Y
neuroblastoma cells on bacterial nanocellulose scaffolds”.
• Our involvement: Other potential bacterial products to be used in
3D matrice with optimized geometry for a specific application....
Bacterial nanofibers
How can we involve?
• Problem: Immobilization of microbial cells in membranes and
bioreactors provides enhanced catalytic activity and stability,
protecting microorganisms from mechanical degradation and
deactivation and allowing for an overall intensification of biochemical
reactions **.
• Pattern and rate of proliferation and function can be depended on
the matrix properties which should be studied.
Scaffolds with Immobilized Bacteria for 3D
Cultures!
[**] Gutiérrez, María C., et al. "Hydrogel scaffolds with immobilized bacteria for 3D cultures." Chemistry of
materials 19.8 (2007): 1968-1973.
We are going to talk about...
• Why three-dimensional cell cultures?
• What is the technology used for?
• Factors affecting SC differentiation in 3D cultures
• How can we—microbial biotechnologists— involve?
• Summary
Summary
• 2D cell cultures differ greatly from natural state of cells
in vitro studies are not much reliable.
• 3D culture preparation for any application must be
comprehensively optimized it is complex
• No general mathematical analysis exists.
• Researches are still being conducted in tuning the
cultures.
Thank you