optimisation of current good manufacturing practice ... · the generation of clinical grade human...
TRANSCRIPT
Introduction
The generation of clinical grade human embryonic stem cell lines
(hESCs) to a level of current Good Manufacturing Process (cGMP)
requires a validated, reproducible and safe method of freezing cellsfor the creation of a Master Cell Bank. In combination with a suitable
chemically-defined cryopreservation reagent, Controlled RateFreezing offers the highest level of compliance with cGMP standards
for human cells. The Asymptote EF600 is a compact, cleanroom-
compliant controlled rate freezer that is not dependent on liquidnitrogen for operation. Our aim in this study was to determine the
efficiency in recovery of hESCs using the EF600 in combination with
a suitable defined cryopreservant. In addition, to optimise hESCviabilities by assessing the effect of nucleation and different freeze
profiles on the recovery post-thaw.
Cryostor CS10 yields the highest viability of hESCs post-thaw
Three commercially available xeno-free and defined products - Synth-a-Freeze (Invitrogen), Profreeze
(Lonza) and Cryostor CS10 (Biolife Solutions) were evaluated in addition to our own x2 Freeze reagent
using passive freezing in a Mr Frosty. Immediate recovery was not significantly different with eachreagent, however Cryostor CS10 provided the highest viability when assessing post-thaw recovery and
growth (figure 1).
Efficient nucleation is essential for high levels of hESC viability
The EF600 was used to freeze hESC in a linear freeze profile, using Cryostor CS10, with an ice
nucleation step at -9.0°C where indicated. Efficient nucleation of the vials resulted in an improvementof immediate viability of 26% compared to that of non-nucleated cells (from 39.9 ± 2.0% to 65.9 ± 4.5%
- See Table 1) and a consequent increase in hESC colony survival following the 48 hr culture period(figure 2).
Improvements to viabilities using non-linear freeze profiles
To investigate further optimisation of the recovery rate, two additional freeze profiles were evaluated
in conjunction with nucleation. Using Cryostor CS10 as the cryopreservant, hESCs were frozen
down in a non-linear !A" profile (with an initial shallow temperature drop) or in a !B" type profile (with asteeper initial temperature drop) with and without nucleation (figure 3).
Materials and Methods
Culture and freezing - RCM-1 hESC line was cultured in a feeder-
free system comprising StemPro (Invitrogen) supplemented with 8ng/ml bFGF, using CELLstart (Invitrogen) as the matrix. Cells were
cultured in a 12-well plate (approximately 1x106 cells/well), refreshed
with fresh supplemented media 1 hour prior and harvestedmechanically using an EZ passage tool (Invitrogen). The hESCs
from each of the wells were processed discretely in triplicate orquadruplicate. The cells were centrifuged and either resuspended
into ice-cold media and an equivalent volume of x2 freeze reagent
(RC x2 Freeze) was added or cells were resuspended according tomanufacturers instructions (Synth-a-Freeze, Invitrogen; Profreeze,
Lonza and Cryostor CS10, Biolife Solutions). The cells were frozen
down either using the programmed profiles indicated on the EF600controlled rate freezer before being stored in a -150°C freezer, or
alternatively placed in a !Mr Frosty" isopropanol tub (Nunc) and thenplaced in a -80°C freezer overnight, before being transferred to a -
150°C freezer for at least 24 hours.
Resuscitation and assessment of viability – Cells were resuscitated
and 10% of the cells were reserved, trypsinised and subjected to a
Trypan Blue (Invitrogen) assessment of immediate viability. Theremaining cells were re-seeded into a 12-well plate and cultured for
48 hours in StemPro media supplemented with 8 ng/ml bFGF onCELLstart. Following 48 hours incubation, the cells were subjected
to an Alkaline Phosphatase stain (Sigma) to visualise successfully
plated and growing ES cell colonies, to determine post-thaw (i.e.longer term) viability. These wells were imaged and a viability
assessment was made qualitatively. Each experiment wasperformed at least in triplicate and a student t-test performed to
indicate statistically significant differences (*p<0.05, **p<0.001,
***p<0.005).
Temperature measurements were made using a Squirrel data logger
(Grant Instruments) with the thermocouples situated within thesample vials for each run, and subjected to nucleation, where
indicated as above.
Conclusions
Cryostor CS10 provides the highest hESC viability post-thaw in a comparison of commercial defined,xeno-free cryopreservants.
Efficient nucleation is essential for high recovery of cells when applying controlled rate freezing.
Different freeze profiles can provide varying viabilities in combination with nucleation.
The Asymptote EF600 represents an efficient solution for the cryopreservation of hESCs within a
cleanroom environment.
Figure 1: The immediate viabilities of hESCs does not vary significantly when frozen in various cryopreservants and assessed with Trypan Blue (A),images taken following an incubation period post-thaw indicate that Cryostor CS10 provides the highest level of recovery (B).
RC x2 Freeze Profreeze
Synth-a-Freeze Cryostor CS10
Cryopreservant comparison
RC x2 Freeze Profreeze Synth-a-Freeze Cryostor CS100
10
20
30
40
50
60
70
80
90
100V
iab
ilit
y o
f cells (
%)
A
B
Optimisation of current Good Manufacturing Practice compliant controlled rate freezing
for human embryonic stem cellsDaniel Collins
a, Adriana Bos-Mikich
a, John Morris
b and Paul de Sousa
a
aRoslin Cells, Roslin Institute, Roslin, Midlothian, EH25 9PS and bAsymptote Ltd, St John"s Innovation Centre, Cowley Road, Cambridge, CB4 0WS
Temperature profiles within cryovials - non nucleated samples
-70.0
-50.0
-30.0
-10.0
10.0
30.0
00:00:00 00:15:00 00:30:00 00:45:00 01:00:00 01:15:00 01:30:00
Time
Tem
pera
ture
(D
eg
C)
linear
non-linear 'B'
non-linear 'A'
Mr Frosty
Temperature profiles within cryovials - nucleated samples
-70.0
-50.0
-30.0
-10.0
10.0
30.0
00:00:00 00:15:00 00:30:00 00:45:00 01:00:00 01:15:00 01:30:00
Time
Tem
pera
ture
(D
eg
C)
linear
non-linear 'B'
non-linear 'A'
A B
Figure 3: Temperature profiles of samples frozen in Cryostor CS10 using the linear,and non-linear type !A" and type !B" freeze profiles without (A) and with (B) a nucleation step. The temperature profile of a sample frozen using a !Mr Frosty" is included for comparison (A).
Interestingly the profiles yielded different viabilities depending on whether a nucleation step wasincluded (figure 4). The profile !A" was highly dependent on efficient nucleation, with nucleation
providing a significant increase in viability from 38.6 ± 3.2% to 75.2 ± 5.7%. This contrasted with
profile !B" where recovery was not significantly improved by a nucleation step (56.4 ± 8.5% to 68.8 ±1.7%).
Figure 2: Efficient nucleation of vials results in a significant increase in hESC viability when compared to non-nucleated control samples (A). Images
of cells following an extended culture period (B).
Non nucleated Nucleated
Mr Frosty
A
B
Nucleation Efficiency
Non nucleated Nucleated Mr Frosty0
10
20
30
40
50
60
70
80
90
100
*** **
Via
bilit
y o
f cells (
%)
Profile !A" Non nucleated Profile !A" Nucleated
Profile !B" Non nucleated Profile !B" Nucleated
Profile comparison
Profile A Profile B
0
10
20
30
40
50
60
70
80
90
100
Non-nuc Nuc Non-nuc Nuc
***
Via
bilit
y o
f C
ells (
%)
A
B
Figure 4: Immediate viabilities (A) and images from an extended culture period following recovery of cells (B) indicating the differences
between profile !A" and profile !B". Nucleation significantly improves recovery of cells frozen using profile !A" but not that of !B".
References
G.J Morris et al., (2006) Reprod. Biomed. Online 13, 421-426
J.M. Crook et al., (2007) Cell Stem Cell 1, 490-494
Table 1: A summary of the immediate viabilities of
hESCs frozen using the various EF600 profilesPost-thaw viability
Freeze profile Non nucleated (%) Nucleated (%)
Linear 39.9 ± 2.0 65.9 ± 4.5
Type A 38.6 ± 3.2 75.2 ± 5.7
Type B 56.4 ± 8.5 68.8 ± 1.7
Mr Frosty 63.4 ± 6.7