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Bio-printing with Stem Cells

Michael Peters & Olivia Marais

Olivia Marais

on 8 April 2014

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Transcript of Bio-printing with Stem Cells

The Dream
3D Bioprinting with Stem Cells
Use of
printing technology
to deposit any of the following on a receiving solid or gel substrate or liquid resevoir:
Living cells
Extracellular matrix (ECM) components
Biochemical factors

What is Bioprinting?
Photolithography (Ravi et al., 1999)
highly developed
expensive equipment
requires clean room
not suitable for delicate ligands
Old Technologies
High throughput
Geometrically well-defined
Multiple layers
Allows for spatial gradients
Advantages of 3D Bioprinting
ommercial colour inkjet printers

Precise control of drop volume & position
Multiple ink cartridges
Non-contact -> print on any medium

Inkjet Printing
Seiko Epson inket printer
Bovine vascular endothelial cells
Dot-pattern printed onto cell culture disk
Proof of concept
Determine if printing damages cells
Determine if cells stay alive after printing
Nakamura et al., 2005
Solve problems with inkjet nozzles
nozzle clogging with cells
"flight bending"
Yamaguchi et al., 2012
(Tasoglu & Demirci, 2013)
Source: http://www.explainingthefuture.com/visions/vision_bioprinter.html
Cell viability
Maintenance of long-term functionaily after printing
Apoptotic effects during/after printing
Inkjet-based printing
Laser printing
Submerged printing
Valve-based printing
New Technologies
Printed organs/tissue for transplant
Disease modeling
Drug testing

Bioprinting Applications
Feasbility confirmed
Printing does
change cell morphology
Some printed cells adhered to cell culture disk -> viable
Important Considerations
Heat damage
to cells from inkjet
Use injket head made of glass/silicone
(Nakamura et al., 2005)
(Nakamura et al., 2005)

Constructed piezoelectric inkjet head with glass capillary for viewing cells
Developed system to detect cell position in nozzle
Investigated push-pull vs. pull-push method for ejection
"Flight Bending"
384-well plate, 1000 droplets per well
Cells stained with calcein-AM/propidium iodide
Cells counted at 20 & 60 min
living cells = green (calcein)
dead cells = red (PI)
Printing has
no effect
on cell viability after 20 & 60 min
Unprinted & printed cells = 90% viable

Cell Viability
Printing Technologies
Submerged Printing
Laser Printing
Aggregation of
Embryonic Stem Cells
Why create aggregates?
A link to UBC research
Balance between shear forces and nozzle size
Cellular Interaction
Cell solution injection
Agarose-> Flurocarbon
ECM formation
Structural integrity issues
- Solution?
Mouse endothelial cells, Human stem cells
"Revealed cell-cell interactions and morphological dependency..."
Application to tumor metastasis
Protein output
Inkjet printing
Submerged printing
Laser printing
Tissue Engineering
An engineering problem
Permeability vs. Support
and Stereolithography
All particles exhibit dielectrophoretic activity when in electric fields
Ability to create large patterns of cells
Bajaj, P
et al.
Cellular interaction
Stem cell aggregation
Tissue engineering
Application limited by imagination
Drug testing
Stem cells
Tissue Engineering
Cell interaction
Technology limited by innovation
Inket printing
Submerged printing
Laser printing
Image from: http://3dprintinginsider.com/files/2013/08/bioprinting.jpg
Image from: http://www.3dprinter.net/wp-content/uploads/2013/03/iowa-biomanufacturing-laboratory.jpg
Bajaj P, Marchwiany D, Duarte C, Bashir R. (2012). Patterned three-dimensional encapsulation of embryonic stem cells using dielectrophoresis and stereolithography. Adv Healthc Mater.;2(3):450-8. doi: 10.1002/adhm.201200318. Epub. PubMed PMID: 23463644.

Binder, K. et al. (2011). Droplet-on-demand Inkjet Bioprinting: A Primer. Gene Therapy and Regulation. Gene Ther. Reg. 06:01, 33-49.

Duarte Campos DF, Blaeser A, Weber M, Jäkel J, Neuss S, Jahnen-Dechent W, Fischer H. Three-dimensional printing of stem cell-laden hydrogels submerged in a hydrophobic high-density fluid. Biofabrication. 2013 Mar;5(1):015003. doi:10.1088/1758-5082/5/1/015003. Epub 2012 Nov 21. PubMed PMID: 23172592.

Fabien Guillemot, Bertrand Guillotin, Aurélien Fontaine, Muhammad Ali, Sylvain Catros, Virginie Kériquel, Jean-Christophe Fricain, Murielle Rémy, Reine Bareille and Joëlle Amédée-Vilamitjana. (2011) Laser-assisted bioprinting to deal with tissue complexity in regenerative medicine. MRS Bulletin, 36, pp 1015-1019.

Faulkner-Jones A, Greenhough S, King JA, Gardner J, Courtney A, Shu W. (2013). Development of a valve-based cell printer for the formation of human embryonic stem cell spheroid aggregates. Biofabrication;5(1):015013.

Hong S, Song SJ, Lee JY, Jang H, Choi J, Sun K, Park Y. (2013). Cellular behavior in micropatterned hydrogels by bioprinting system depended on the cell types and cellular interaction. J Biosci Bioeng;116(2):224-30. doi: 10.1016/j.jbiosc.2013.02.011. Epub. PubMed PMID: 23562089.

Karoly Jakab, Cyrille Norotte, Francoise Marga, Keith Murphy, Gordana Vunjak-Novakovic and Gabor Forgacs. (2010). Tissue engineering by self-assembly and bio-printing of living cells. Biofabrication 2 022001.

Koch L, Gruene M, Unger C, Chichkov B. (2013). Laser assisted cell printing. Curr Pharm Biotechnol. 14(1):91-7.
Kucukgul C., Ozler B, Karakas H.E., Gozuacik D, Koc B. (2013). 3D Hybrid Bioprinting of Macrovascular Structures. Procedia Engineering, Volume 59, Pages 183-192, ISSN 1877-7058.

Nakamura, M. et al. (2006). Biocompatible Inkjet Printing Technique for Designed Seeding of Individual Living Cells. Tissue Engineering. Volume 11, Number 11/12.

Ozbolat, I.T., & Yu, Y. (2013). Bioprinting toward organ fabrication: challenges and future trends. IEEE Trans Biomed Eng.

Why would you want
to inhibit Cadherins?
Why are the cells in
the middle dead?
Laser causes rapid evaporation
Two-plate apparatus
Multi-layer printing
Stem Cell Ethics
Please Welcome:
Liavio Saraim— Bioethicist, Lawyer
Dr. Peter Kozlo— Physician
Stem cell ethics experts

Tasoglu, S. & Demirci, U. (2013). Bioprinting for stem cell research. Trends Biotechnol. http://www.ncbi.nlm.nih.gov/pubmed/23260439

Xu F., Sridharan B., Wang S, Gurkan U.A., Syverud B, Demirci U. (2011). Embryonic stem cell bioprinting for uniform and controlled size embryoid body formation. Biomicrofluidics. 022207.

Yamada, M. et al. (2013). Microarrays of phospholipid bilayers generated by inkjet printing. Langmuir. 29 (21), pp 6404–6408.

Yamaguchi, S. et al. (2012). Cell patterning through inkjet printing of one cell per droplet. Biofabrication 4. 045005

Stem Cell Ethics
Embryonic stem cells (ESCs)
Extraction destroys embryo
Moral status of embryo?
Limited sources for research
Leftover IVF embryos
Creation of embryos NOT allowed
Image from: http://www.textually.org/3DPrinting/2013/05/03/4d68a12d2acb7cd0119bc54571bdc39f_vice_630x420.jpg
New Hope (2008)
Induced Pluripotent Stem Cells (iPSCs)
-> derived from skin cells
using growth factors

Image from: Duarte, C.
et al.
Image from: Duarte, C.
et al.
Image from: Duarte, C.
et al.
Image from: Duarte, C.
et al.
More Hope (2014)
Stimulus-triggered acquisition of pluripotency (STAP) cells
Lower the pH
Validity unconfirmed

Image from: Koch L.,
et al
Image from: Koch L.,
et al
Image from: Koch L.,
et al
Image from: Koch L.,
et al
iPSCs & STAP-SCs are not always
100% equivalent to embryonic SCs

Embryonic SCs = gold standard

Image from: Hong S.,
et al
Image from: Hong S.,
et al.
Image from: Faulkner-Jones A.,
et al
Image from: Faulkner-Jones A.,
et al
Image from: Kucukgul C.
et al.
Image from: Kucukgul C.
et al.
Why use 3D cell cultures instead of 2D cultures?
Name some challenges for bioprinting!
Hint: Don't just say low pH
What could bioprinting be used for?
Source: http://s1.ibtimes.com/sites/www.ibtimes.com/files/styles/v2_article_large/public/2013/05/01/023d-bio-printer1.jpg
Source: http://www.kurzweilai.net/images/diybioprinter.jpg
Full transcript