Poster Abstract Presentations
47
VOLUME-BY-VOLUME BIOPRINTING OF CHONDROCYTES-
ALGINATE BIOINKS IN HIGH TEMPERATURE
THERMOPLASTIC SCAFFOLDS FOR CARTILAGE
REGENERATION
J. Baena
Biopathology and Regenerative Medicine Institute (IBIMER), Centre for
Biomedical Research, University of Granada, Granada, Armilla, Spain
Background & Aim: Biofabrication technologies with layer-by-layer simulta-
neous deposition of a polymeric matrix and cell-laden bioinks (also known as
bioprinting) offer an alternative to conventional treatments to regenerate carti-
lage tissue. Thermoplastic polymers, like poly-lactic acid, are easy to print using
fused deposition modeling, and the shape, mesh structure, biodegradation time,
and stiffness can be easily controlled. Besides some of them being clinically
approved, the high manufacturing temperatures used in bioprinting applications
with these clinically available thermoplastics decrease cell viability. Geometric
restriction prevents cell contact with the heated printed fibers, increasing cell
viability but comprising the mechanical performance and biodegradation time
of the printed parts. The objective of this study was to develop a novel volume-
by-volume 3D-biofabrication process that divides the printed part into different
volumes and injects the cells after each volume has been printed, once the tem-
perature of the printed thermoplastic fibers has decreased.
Methods, Results & Conclusion: A bioprinter with three syringes and one
FDM extruder (REGEMAT 3D, Granada, Spain), was configured for PLA
and used two syringes with needles to inject embedded chondrocytes in algi-
nate bioink and a calcium solution . The Designer software can be set up to
print anatomical structures, selecting how and when the bioinks are deposited.
In order to avoid cell contact with the high temperature thermoplastic printed
parts, VbVallows the deposition of x layers of the thermoplastic and afterwards
the filling of the resulting volume with chondrocytes. The syringe containing
the cell-loaded hydrogel solution will penetrate into the scaffold in different
points (N points), filling it up uniformly.
Human articular chondrocytes were isolated. Chondrocytes were grown and
incubated at 37࿽C humidified atmosphere and expanded in a monolayer for
7 10 days before the experiment.
This new procedure avoids the contact between the thermoplastic and the
cells at a higher temperature than is normally physiologically viable, it can be
used with already clinically approve biomaterials and does not have restrictions
in geometries that could limit the clinical application of 3D bioprinting in car-
tilage TE. The use of a VbV 3D-biofabrication process might accelerate the
clinical application of the technology and overcome the current limitation
when using thermoplastics as scaffolds.
55
CONSTRUCTION AND FUNCTIONAL EVALUATION OF A
POLYCISTRONIC MODIFIED MRNA FOR HUMAN IPS CELL
GENERATION
B. Mahdavi12, N. Rezaei1, M. Nasr-Esfahani1 & K. Dormiani1
1
Department of Molecular Biotechnology, Cell Science Research Center,
Royan Institute for Animal Biotechnology, ACECR, Isfahan, the Islamic
Republic of Iran, 2Department of Biology, Faculty of Science and Technology,
ACECR Institute of Higher Education, Isfahan, the Islamic Republic of Iran
Background & Aim: During the last decade, induced pluripotent stem cells
(iPSCs) had an enormous impact on the progress of cell biology and regenera-
tive medicine. Several approaches are accomplished for iPSCs induction.
Modified mRNAs (mmRNA) has shown a potential to derive safe and high
efficient integration-free iPSCs. However, low stability of mRNAs resulted in
daily transfection that is costly and time-consuming. To overcome this limita-
tion, we have constructed a polycistronic mmRNA containing WPRE element
as a potential tool for safe iPSCs induction.
Methods, Results & Conclusion Methods: We developed a 2A-mediated
polycistronic plasmid containing a single expression cassette with open reading
frames of four human pluripotency transcription factors (LIN28, NANOG,
SOX2, and OCT4) along with the EGFP coding sequence and WPRE ele-
ment. We cloned the polycistronic DNA fragment in an appropriate vector
containing T7 promoter, untranslated regions and poly (A) tail for in vitro
transcription. Transcripts are produced using T7 RNA polymerase, modified
nucleotides and cap analog. Phosphatase treatment was employed to reduce
the immune responses. Evaluating integrity and quantity, the transcript was
run on a denaturing agarose gel. Finally, mmRNAs were transfected into
S23
HEK293T cells and EGFP expression was assessed by fluorescent microscopy
and flow cytometry.
Results: Primary polycistronic plasmid was successfully constructed. Correct
orientation of each cloned fragments in the vector was confirmed using PCR
and sequence analysis. Subsequently, the total ORF subcloned into another
plasmid designed for IVT reaction, and the accuracy of new construct was con-
firmed using restriction digestion. By in vitro transcription, the mmRNA tran-
script was produced efficiently with expected size. Transfection of synthesised
mmRNA into HEK293T cells was carried out and the fluorescent signal was
effectively detected using fluorescent microscope and flow cytometry. The
results demonstrated that mmRNA was efficiently expressed in HEK293T
cells. Moreover, the presence of WPRE resulted in significantly enhanced
mmRNA stability and higher level of protein expression.
Conclusions: We developed a polycistronic mmRNA for producing safe iPSCs.
The cap, modified nucleotides and poly (A) tail in the mRNA enhanced its stability
and increased translation efficiency. Moreover, WPRE element in the expression
cassette increased the mmRNA stability and the duration of protein expression.
57
ENABLING STEM CELL BASED THERAPIES: END-TO-END
SOLUTION FOR HUMAN PLURIPOTENT STEM CELLS
MANUFACTURING
I. Friedrich Ben Nun
Cell Therapy R&D, Lonza, Rockville, Maryland, United States
Background & Aim: Enabling stem cell-base therapies requires innovative
solutions to close the gaps existing between research and commercialization.
Allogeneic cell therapy indications that target large patient populations will
necessitate the use of flexible cell production platforms to meet required cell
quantities. Developing a truly scalable, controlled bioreactor platforms for cell
expansion enables meeting cell quantity demand for clinical applications while
allowing comparability between the various scales. Likewise, it enhances pro-
cess automation and allows integration of online monitoring systems. The
capability of effectively culturing adherent stem cells, namely pluripotent stem
cells, will be presented.
Methods, Results & Conclusion: Our platform take advantage of 3D, stirred
tank bioreactors, expanding hPSCs in suspension. Our data shows that hPSCs
expand rapidly and efficiently in a 3L bioreactor, while retaining normal kar-
yotyping and hPSC-associated characteristics. hPSCs are fed with an animal-
free hPSC media, perfused into a closed, monitored and controlled, bioreac-
tor, with no need for cell passaging. The platform supports expansion of
hPSCs seeded in a bioreactor either when harvested from 2D cell culture or
when thawed directly into the suspension vessel. Thawing cryopreserved cells
directly into a suspension vessel eliminates the need for a 2D seed train and,
therefore, greatly reduces labor, time and contributes to process aseptic, moni-
toring and automation. Furthermore, we show that 3D seed train from one
suspension vessel to another is a feasible and viable solution for large-scale
expansion of hPSCs. hPSCs expanded in a bioreactor are shown to have the
capability to be directly differentiated in suspension. This expansion platform
and associated attributes provides end-to-end, cryo-to-cryo solution for hPSC
manufacturing. Expanding hPSCs in suspension, in a controlled bioreactor,
results in high fold expansion without compromising cell quality, and the
capacity of the cells to be further differentiated. This platform avoids 2D cell
culture steps, reduces footprint, labor and cost, while enhancing process con-
trol and cell product quality.
58
NEW FN1 BASED CULTUREWARE FOR ANIMAL-FREE
EXPANSION OF HUMAN MESENCHYMAL STEM CELLS
N. Mellies1, A. Tacheny2, W. Ben El Mostapha2 & F. De Longueville2
1
Eppendorf AG, Hamburg, Germany, 2Eppendorf Application Technologies
SA, Namur, Belgium
Background & Aim: A newly developed human- and animal-component-free,
ready-to-use cultureware was specifically designed to allow reliable and robust
expansion of human mesenchymal stem cells (hMSCs). Based on synthetic
peptides, the FN1 motifs surface supports stem cell attachment by mimicking
the native ECM protein fibronectin. hMSCs represent a promising stem cell
population in various cell applications. Present at relatively low abundance in
their natural tissue of origin, hMSCs require a robust in vitro expansion system
to obtain sufficient high-quality cell numbers for research and clinical applica-
tions. Well-defined like serum-free (SF), xenogenic-free (XF) or even animal-