A new class of compounds for the protection of cells
during storage at 2 – 8 °C
Introduction
P.C. Vogelaar1
A.C. van der Graaf1
R.H. Henning2
S. Stienstra3
Cell preservation/shipping techniques include live-culture
or cryopreservation. Inspired by hibernation1, nature’s
solution to low temperature and lack of nutrition, we have
designed and synthesized a new class of small molecules
(henceforth, SUL-compounds)2 complying with Rule-of-5.
Efficacy in storage of mammalian cells at ultraprofound
hypothermia (2 – 8°C) was tested in a high-throughput
screening and compared with compounds with known
protective capacities (Trolox, dopamine).
1Sulfateq
B.V., Groningen, The
Netherlands;
2Dept. of Clinical
Pharmacy & Pharmacology,
UMCG, Groningen, The
Netherlands;
3BBBeratung, Beek Ubbergen,
The Netherlands
Contact:
[email protected]
Table 1. Chemicophysical properties of SUL-compounds.
% V ia b ilit y ( r e la t iv e O D 5 5 0 )
2. Patent no. WO2014098586 A1
3. Dugbarty, G.J. et al. (2014)
Dopamine and SUL-121 protect
kidney against in vivo
hypothermia/rewarming-induced
injury via CBS/H2S pathway.
FIGON Dutch Medicine Days (page
25).
4. Tolouee, M. et al. (2014)
Dopamine and SUL-121 protect
against DNA damage during
cellular hypothermia and
rewarming in cultured smooth
muscle cells. FIGON Dutch
Medicine Days (page 62, poster P017).
T r o lo x
100
Compound
MW (kDa)
ClogP
TEAC
EC50 (M)
SUL-121
SUL-128
SUL-138
SUL-132
SUL-134
SUL-122
SUL-125
SUL-135
SUL-137
SUL-95
SUL-142
SUL-131
SUL-141
SUL-139
Trolox
Dopamine
< 400
< 400
< 400
< 400
< 400
< 450
< 400
< 400
< 450
< 400
< 400
< 400
< 400
< 450
250,29
189,4
2,60
4,06
1,84
1,84
3,05
1,56
2,91
3,05
3,57
2,84
0,04
3,88
0,04
1,01
n.d.
n.d.
0,84
1,05
0,82
0,90
0,90
0,52
1,01
0,54
0,22
0,41
0,97
n.d.
0,98
0,91
1,00
n.d.
5,66E-09
1,08E-08
2,47E-08
3,00E-08
5,05E-08
5,48E-08
1,23E-07
2,08E-07
3,30E-07
5,45E-07
6,06E-07
6,23E-07
6,57E-07
3,37E-06
1,17E-05
1,54E-05
D o p a m in e
80
S U L -1 2 1
S U L -1 2 8
60
S U L -1 3 2
40
20
0
-12
-10
-8
-6
-4
lo g ( m o la r c o n c e n t r a t io n )
Figure 1. SUL-compounds protect cells from cooling/rewarming at high potency.
HEK293 were pre-incubated for 2 hours in standard cell culture before storage at 2–
8°C for 24 hours, followed by 4 h rewarming at 37°C. Cell viability was assessed by
Neutral Red uptake. High doses do not show toxicity. Data are mean ±SEM (n=6).
Results
Cell line
SUL-compounds protect cells stored at 2–8°C at 1.000 and
10.000 fold potency compared to dopamine and Trolox (fig 1). HUVEC
13 days
Model for endothelial damage
HEK293
7 days
Host for production of recombinant proteins
SH-Sy5y
7 days
7 days
In vitro cytotoxicity cell assays
Model for neuronal function and differentiation
MS5
5 days
Feeder layer for stem cells and iPS cells
CaCo-2
7 days
Model for human intestinal drug absorption
Cell growth rate and morphology is maintained after storage
3T3-L1
at 2–8°C in cells protected by SUL-compounds (fig 2).
SUL-compounds were successfully used to store 7 human
and rodent cells at 2 – 8° for up to 21 days.
EC50 of SUL compounds did not correlate with Trolox
Equivalent Antioxidant Capacity (table 1).
NRK-52E 21 days
Model for kidney epithelial damage
V ia b ilit y o f t ry p s in iz e d 3 T 3 -L 1 c e lls
V ia b ilit y o f tr y p s in iz e d H E K 2 9 3 c e lls
N e u t r a l R e d u p t a k e o f 3 T 3 -L 1 c e lls fo llo w in g
C e ll c o u n t o f H E K 2 9 3 c e lls fo llo w in g
a ft e r 4 d a y s a t 2 - 8 °C w it h S U L -1 3 8
a ft e r 3 d a y s a t 2 - 8 °C w it h S U L -1 3 8
4 d a y s a t 2 - 8 °C
3 d a y s a t 2 - 8 °C
1 .5  1 0 6
D e a d c e lls
2 .0  1 0 6
2 .0  1 0 5
1 .2
L iv e c e lls
1 .0  1 0 6
65%
65%
81%
61%
5
5 .0  1 0 5
l
ro
nt
Co
µ
µ
0
nt
Co
M
M
l
0
ro
M
µ
10
10
0
µ
M
0
10
17 %
+ SUL
1 .0
3 7 °C C o n tro l
C e ll c o u n t ( m L
nm)
86%
10
5 .0  1 0
6
V ia b ilit y ( O D 5 5 0
)
1 .0  1 0
6
S U L -1 3 8 1 0 0 µ M
S U L -1 3 8 1 0 0 µ M
1 .5  1 0
-1
C e ll c o u n t ( m L
Stored days Application
viability >70%
)
1.Talaei, F.et al. (2011) Serotonin
and dopamine protect from
hypothermia/rewarming damage
through the CBS/H2S pathway.
PLoS One 6, e22568
6. www.Rokepie.com
MW, molecular weight; TEAC, Trolox Equivalent Antioxidant Capacity.
V ia b ilit y o f H E K 2 9 3 c e lls a f t e r 2 4 h o u r s s t o r a g e a t 2 - 8 ° C
-1
References
5. Han, B. et al. (2014)
Exploration of anti-inflammatory
and broncho-relaxing properties of
SUL-121. FIGON Dutch Medicine
Days (page 93, poster P-080)
The chemical structure of 60
SUL-compounds2. R1 and R2
consist of CH3 or C3H7. All
compounds were synthesized
by Syncom B.V., Groningen.
0 .8
0 .6
0 .4
1 .5  1 0
3 7 ° C C o n t r o ls
5
1 .0  1 0 5
5 .0  1 0 4
0 .2
0
0 .0
0
1
2
3
4
C u lt u r e d a y s
5
6
7
0
1
2
3
4
5
6
7
8
C u lt u r e d a y s
Figure 2. (Top Left) Cells were immediately stored at 2 – 8°C after trypsinization. At this
temperature, attachment of cells to the culture vessel is absent. SUL-compounds protect freshly
trypsinized cells during storage at 2 – 8 °C. (Top Right) Cell growth of cells treated with 100 µM SUL138 showed unaltered growth characteristics as compared to 37°C cells. (Lower left) Morphology of
NRK-52E and HUVEC cells after storage at 2 – 8°C.
Conclusions
NRK-52E cells after 21 days at 2 – 8°C
No SUL compound
NRK-52E cells after 21 days at 2 – 8°C
100 µM SUL-138
+ SUL
HUVEC cells after 13 days of at 2 – 8°C
No SUL compound
HUVEC cells after 13 days of at 2 – 8°C
10 µM SUL-138
SUL-compounds represent a new class that protect cells
during storage at 2–8°C, independent of antioxidant
characteristics in the micro- to nanomolar range.
SUL-compounds are now tested to protect kidney against
hypothermia/rewarming injury3 and to hypothermic DNA
damage4.
SUL-compound 121 protects from ROS associated damage in
a model for chronic obstructive pulmonary disease5.
SUL-109 is launched as a Research Only product to store
mammalian cells and tissues at 2 – 8°C (ROKEPIE®-S01)6.