Poster: Novel Engineered Basic Fibroblast Growth Factor...
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Thermo Fisher Scientific • 7305 Executive Way • Frederick, MD 21704 • thermofisher.com Brittany Balhouse, Diana Navarro, Richard Josephson, and Matthew Dallas, Thermo Fisher Scientific, Frederick, MD, USA, 21704 Novel Engineered Basic Fibroblast Growth Factor Improves Stability and Enables Improved Cell Culture Outcomes Introduction HS bFGF: Engineered for greater stability • Basic fibroblast growth factor (bFGF) is used in NSC media to maintain multipotency and is known to be present in the tumor microenvironment • Native bFGF rapidly loses biological activity when exposed to culture conditions (37°C); we found only ~20% bioactivity after 72 hours • HS bFGF maintains > 90% homology to the native protein and ≥ 80% biological activity, even after 72 hours of exposure to 37°C Figure 1. HS bFGF maintained 95% activity after 72 hours at 37°C. Dose-response of Balb/3T3 mouse embryonic fibroblast cells to native (top) and HS (bottom) bFGF stored at 4°C or 37°C for 72 hours. Analysis by PrestoBlue® assay after 18 h stimulation. Mean ± SEM. HS bFGF Native bFGF Figure 4. HS bFGF allows for normal proliferation of multipotent NSCs with less bFGF. At 5 ng/mL, HS bFGF (A) reduced the doubling time by ~30% (back to 20 ng/mL standard, ~45 h) and (B) had more cells at each passage versus native bFGF at the same concentration, (C) while maintaining a multipotent morphology and SOX1 expression. Mean ± SEM. ** = p < 0.01. Arrows denote neurite outgrowth. (A) SOX1 DAPI 100 μm (B) (C) Conclusions • In primary rat NSCs, using HS bFGF allows for a more user-friendly workflow while maintaining multipotency • In human ESC-derived NSCs, HS bFGF can maintain multipotency and standard doubling times with reduced bFGF concentrations • After expansion, HS bFGF can be removed just as easily as native bFGF to allow for downstream differentiation into neurons and glial cells • HS bFGF can be used for spheroid culture, or other systems where media changes are undesirable Primary Rat Neural Stem Cell (NSC) Culture © 2018 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. For Research Use Only. Not for use in diagnostic procedures. P1870 New Technologies in Cell Biology 2 Figure 2. HS bFGF maintains multipotent NSCs with fewer feeds. Using 10 ng/mL bFGF, HS bFGF decreased the doubling time and maintained NSC multipotency with feeds every 48 h. Native bFGF, 48 h feed Native bFGF, 24 h feed HS bFGF, 48 h feed MAP2 DAPI MAP2 DAPI MAP2 DAPI Figure 3. HS bFGF does not impact NSC differentiation. Three days after the removal of HS bFGF, the NSCs were stained for differentiation markers and showed trilineage potential. Neuronal lineage Astrocyte - lineage Oligodendrocyte - lineage MAP2 DAPI GFAP DAPI GALC DAPI Human Embryonic Stem Cell-Derived NSC Culture Human Breast Cancer Spheroid Culture Medium only Native bFGF HS bFGF Figure 5. HS bFGF maintained stimulation of MCF7 cells over 8 days. Without media changes, HS bFGF enhanced spheroid growth, as shown by (A) live-dead assay, (B) image analysis of spheroid size, and (C) PrestoBlue® assay. Scale bars = 250 μm. (C) (B) (A)
Transcript of Poster: Novel Engineered Basic Fibroblast Growth Factor...
Thermo Fisher Scientific • 7305 Executive Way • Frederick, MD 21704 • thermofisher.com
Brittany Balhouse, Diana Navarro, Richard Josephson, and Matthew Dallas, Thermo Fisher Scientific, Frederick, MD, USA, 21704
Novel Engineered Basic Fibroblast Growth Factor Improves Stability and Enables Improved
Cell Culture Outcomes
IntroductionHS bFGF: Engineered for greater stability
• Basic fibroblast growth factor (bFGF) is used in NSC media to maintain
multipotency and is known to be present in the tumor microenvironment
• Native bFGF rapidly loses biological activity when exposed to culture
conditions (37°C); we found only ~20% bioactivity after 72 hours
• HS bFGF maintains > 90% homology to the native protein and ≥ 80%
biological activity, even after 72 hours of exposure to 37°C
Figure 1. HS bFGF maintained 95% activity after 72 hours at 37°C. Dose-response
of Balb/3T3 mouse embryonic fibroblast cells to native (top) and HS (bottom) bFGF
stored at 4°C or 37°C for 72 hours. Analysis by PrestoBlue® assay after 18 h
stimulation. Mean± SEM.
HS
bF
GF
Native b
FG
F
Figure 4. HS bFGF allows for normal proliferation of multipotent NSCs with less bFGF. At 5 ng/mL,
HS bFGF (A) reduced the doubling time by ~30% (back to 20 ng/mL standard, ~45 h) and (B) had more
cells at each passage versus native bFGF at the same concentration, (C) while maintaining a multipotent
morphology and SOX1 expression. Mean± SEM. ** = p < 0.01. Arrows denote neurite outgrowth.
(A)SOX1
DAPI
100 µm
(B) (C)
Conclusions
• In primary rat NSCs, using HS
bFGF allows for a more
user-friendly workflow while
maintaining multipotency
• In human ESC-derived NSCs,
HS bFGF can maintain
multipotency and standard
doubling times with reduced
bFGF concentrations
• After expansion, HS bFGF
can be removed just as
easily as native bFGF to
allow for downstream
differentiation into neurons and
glial cells
• HS bFGF can be used for
spheroid culture, or other
systems where media changes
are undesirable
Primary Rat Neural Stem Cell (NSC) Culture
© 2018 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the
property of Thermo Fisher Scientific and its subsidiaries unless otherwise
specified. For Research Use Only. Not for use in diagnostic procedures.
P1870
New Technologies in Cell Biology 2
Figure 2. HS bFGF
maintains multipotent
NSCs with fewer
feeds. Using 10 ng/mL
bFGF, HS bFGF
decreased the doubling
time and maintained
NSC multipotency with
feeds every 48 h.Native bFGF, 48 h feed Native bFGF, 24 h feed HS bFGF, 48 h feed
MAP2
DAPI
MAP2
DAPIMAP2
DAPI
Figure 3. HS bFGF
does not impact NSC
differentiation. Three
days after the removal
of HS bFGF, the NSCs
were stained for
differentiation markers
and showed trilineage
potential.Neuronal lineage Astrocyte-lineage Oligodendrocyte-lineage
MAP2
DAPIGFAP
DAPI
GALC
DAPI
Human Embryonic Stem Cell-Derived NSC Culture
Human Breast Cancer Spheroid Culture
Me
diu
m o
nly
Na
tive
bF
GF
HS
bF
GF
Figure 5. HS bFGF
maintained
stimulation of MCF7
cells over 8 days.
Without media
changes, HS bFGF
enhanced spheroid
growth, as shown by
(A) live-dead assay,
(B) image analysis of
spheroid size, and (C)
PrestoBlue® assay.
Scale bars = 250 µm.
(C)(B)
(A)
