3D cell culture engineering

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

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.

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.

• 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.

• Summary

What is the technology used for?

Clinical

applicationin vitro studies

Regenerative

medicine

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."

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

• 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

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."

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-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

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 .

Guvendiren, Murat, and Jason A. Burdick. "Engineering synthetic hydrogel microenvironments to instruct stem

cells." Current opinion in biotechnology24.5 (2013): 841-846.

discovery today 18.21 (2013): 1099-1108.

Analytical/Mathematical modeling of 3D cultures

bioreactor

Supporting 3D scaffold

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

• 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.

• 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.

• 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

• 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.

• 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

3D cell culture engineering

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