Abstract
compounds microfluidic QPlate unique integrated Figure Transiently Transfect up to 1E10 Cells in <30 Minutes MaxCyte
ion STX reproducibility three patch cryopreservation formation transfected is space internal technology, HYPER culturing HYPER demonstrate Transfected protein electroporation The bulk scale electroporation viability reproducibility range of types, a Figure QPatch QPatch Instrument Fully Automated Patch Clamp System Sophion QPatch
Mark Rothenberg. Corning Inc James Brady, Peer Heine,Rama convenient cellculture usingCorning Rapid generation ofcells forion channelefficient, assays:large proprietary, approximately
interest QPlates channel
system
MaxCyte
-
transfections
clamp
of saving, QPatch exchange
including
2 independent Flask Flask 1 expression culture
greater . . cell
large MaxCyte solutions
. and
Sophion
®
or
system assays with
which MaxCyte
cells
system offers ® types, STX™ Scalable Transfection System
Cell the
.
increasing
of 10 ®
automated scalable Cell
technology or strong
numbers
environment
primary .
a the facilities Transfection - than
can
STX™ layer
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utilizes
.
plasmid a for ®
. Culture
simplifying
plasmids,
for
enables
transfection
QPatch STX™ time use
High
be channel times
has large
90 basic of
potassium film
ion
electroporation
cells,
concentrations assayed
%
in enabling
of Vessel the Scalable patch
a and with suction
Bottom channel inlet developed viability
that
.
design
and extracellular flow channels flow and extracellular intra with sites clamp 48 patch 16 or
encoding Vessel
full ., Kennebunk, USA.ME, channel
gas
that Scalable Ion adherent
and
Shivakumar
fast scale
Transfected activity MaxCyte with system efficiencies as - labor
assay scale, can
connection
clamp
Channel
/ permeable pressure
for process
inlet Channel Bottom
features
of immediately
the well
currents several
and liquid results
DNA, to
transfect
future saving a Transfection
drug
development
electroporations,
was a screening
provide . electroporation external
Transfection
system CHO normal
as consistent
, Angelia on
K technology Sophion STX
application ( Top
inlet Channel Top . Compound
RNA, research
exchange
v
Corning’s
These in 1 hours use assayed
other • • • • • discovery are
the channel inlet cells
. alternative QPlate liquid handling QPlate liquid following
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GFP or an )
has same
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The and
Capillary to -
to of
- channel Bottom cryopreserved molecules, Hervør can stop Capillary results 175
intracellular -
levels efficient
provides
Rear
cm System and
while before
provide an unattended 1
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on fusion flow channel
HYPER
to
Madhusudan System E
profiling MaxCyte
and cell
( Front ( stop and
intracellular
2 be 10 large MEAS
protocols
flask
integrated in
to
the flow channel proteins assay cell Lykke (co)transfect
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demonstrate
extracellular maximizing illustrating extracellular
cells
greater
coordination
stable
used
allows
safety
and and
expression protein . Sophion
gas growth
) advanced
uses scale . (High environment . Olsen, We
in
The
development STX
with
operation
economical
)
after
exchange for immediately less or
Peshwa REF
cell
optimized
cell also
Breather testing
than testing
MaxCyte
Yield Nikolaj
exhibited other electroporation a
surface Waste future
can target,
QPatch
line performance
than a preparation transfection
robustness that
® proprietary
were
show , Karen, Donato and KristaSteger. MaxCyte Inc., Gaithersburg,MD,
with products
perform 85 variety
PERformance .
.
biomolecules generation
. Nielsen, KristinaNielsen, Christensen,Jeffrey Webber and HYPERFlask™ vessels
Breather
use
The Breather of
% the
between
reporter The
30
for area,
Sophion’s
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solution
obtained
STX
automated Corning’s good following
.
that multiple and
MaxCyte perform QPlate
minutes
Here a
of unique,
small
. which uses
wide
and and
and
cells cell cell seal flow
The and and and
the we for for
in - ®
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High Viability, Reproducible Efficiency and Functional Ion Channel Activity Expression of a K Expansion in HYPERFlask Vessels and Transfection via Flow Electroporation Rapid and Scalable Approach to Generating Cells for Ion Channel Assays cryopreservation data transfection GFP with transfected Figure cultured processing expression for traditional exchange Hyper harvested pre Figure MaxCyteSTX CL • • • vessel HYPER cells in ExpandHEK 293H electroporation small by achieved those to equivalent are efficiency andviability oftransfection Levels minutes 30 in< 1E10 cells Variable to 1E8 of transfection enables capacity contamination prevents design use single closed Sterile,
-
three A. FACS AnalysisPrior to Cryopreservation
a and generated
expression Flasks K
Corning Corning
Flask 4 v 3
1 for .
.
post .
large 3 Analysis 175 with
Large directly
- plasmid
assemblies 24 efficiency GFP with
contain
-
hrs
transfection
cm , androbusttargetactivity assayedbytheSophion QPatch. trypsin on scale
does
Morten - K expression - scale
2 2 Processing Assembly electroporation therusing MaxCyteSTX. processingassemblyand transfect by Kv1.3 buffer 150with µg/mL of plasmidencoding Suspendcells10 3E8 in mL MaxCyte of and
- and
v into
the
flask scale transfectionusing theMaxCyte 1 were of
v .
3 ten 1.3
not transfections -
and
GFP fusion protein,loadinto CL . - and Propidium Iodide cryopreserved
Sophion Propidium Iodide
GFP Propidium Iodide GFP Sunesen viability the electroporation Following .
performed In interconnected - impact
GFP Fusion Protein Following Flow Electroporation
high resuspended
. medium,
this DNA HEK plasmid USA. expression, . Sophion Bioscience A/S , GFP GFP GFP
viability
QPatch experiment, prior the
.
- 293
scale, static static scale, a HEK
(
20 with 3 performance
using
in and H E to
8
min 90 293 cells
in
in 61%GFP+ 91%viable 64%GFP+ 95%viable 68%GFP+ 95%viable Transfection#3 Transfection#2 Transfection#1
in the polystyrene of
cells
% cryopreservation
offer viability
three MaxCyte single .
H MaxCyte’s
flow flow
- HEK recovery, FBS/ were 2 five MaxCyte
cells
per
1720
sets
HYPER of
10 hole
cells expanded
were
• • • sample)
and culture 24h andcryopreserve Hyper Seed transfectedinto cells transfected % flow
between the Flaskette & the air gap through a film through gap air & the Flaskette the between occurs exchange Gas Flaskette. each beneath atmosphere to the open space the is gap air The HYPER the form to together grouped are 10 Flaskettes A compartment culture is a cell “Flaskette”
Ballerup film
of cm
Corning HYPER Principle of Operation STX cells electroporation DMSO
mode
Flasks cultured harvested
ion transfected Flask
electroporation 2 B. QPatch with AssayFrozen Cells
growth
growth Flask . system
were
, showed
in Transfections (1.7E8per cells flask), channel Denmark. . with
vessels
Corning
cells
Vessel. 2400 pA
returned surfaces
in thawed 24
area
Max (Peak) [A] in
Current (pA) Ave. (tail) [A] on cells Current voltage,vs. activation phase HYPER
Flask hrs reproducible sterile,
buffer
Current voltage,vs. tail current activity
yielded
HYPER .
the
in
A . post
. cells to B
that with
the Vessel Qpatch Raw data trace
FACS
.
Flask time (ms) time at
V (mV) single a
Representative
electroporation
sufficient
Flask in 3 HYPER footprint allow illustrate a Qpatch assayon Thawandcells E
HEK 7 analysis ®
K
- platform
levels use v
cells/mL
1
vessels, vessels ® for
. 900 ms 3
Flask Sophion Sophion
- cells
CL
STX™ system, GFP cells of that
gas
of of - . a 2
, .
MaxCyte & MaxCyte STX are registered trademarks and/or trademarks of MaxCyte, Inc. Corning & Corning & Inc. MaxCyte,of trademarks and/or trademarks registered STX are & MaxCyteMaxCyte Inc. ©2012 MaxCyte, All Reserved. Rights Correspondence: Jim Brady at [email protected] Summary • • • were 61 immediately hole harvested were expression 293 Figure impacted by cryopreservation. by impacted QPatch. Sophion the on currents not robust was exhibited performance Assay and seals tight formed channels sodium or potassium either with transfected Cells integrity. membrane good results. Transfected viability, high exhibit cells and expression transgene robust reproducible and consistent produces process MaxCyte’s flowelectroporation minimal enable Corning liquid transiently illustrates protocol channel channel time hrs of cells expression QPatch half for Copenhagen channel plasmid Cells Figure A. Na High expression and strong currents detected by Sophion QPatch Robust Sodium Channel Activity in Transiently Transfected Cells Cryopreservation does not inhibit ion channel activity Analysis of Na A. Na ** Percentage ** Percentage block compared saline to of period a mMsingle 20 addition TTX * Measured mV @ 0 in depolarizing simple step protocol [
% [DNA] Temp.[ m condition
250 200 150 100 250 200 150 100 DNA g/ml)]
24
prior
-
Test mode . and cells were 37 28 also current transferred
-
v v
were junction Optimal
1.5 CryopreservationActivityBefore
1.5 hrs Activity48 post electroporation hrs
° 6
the 5 C]
.
and
consumption stepped assays
activity HYPER blocker .
60
to
Na
consistent Na
Temp. [ condition encoding
48
at
Analysis kept were
University)
immediately plasmid
Transfection
typical transfected % Test transfected rapid analysis
37 37 37 37 28 28 28 28 and efficiency
v after plot v
37 shifting 1 1 , hrs
°
[%] corrected . 80 93 results . C]
respectively
5 5
°
at
C
to
and
from transfected in
channel (TTX) Flask robust
from post
. thawing generation
(kindly
37 . . 28
Half v
Transfection
single sodium . Transfected
efficiency 1.5 Activity in Freshly Transfected and Frozen Cells between Transfected
of
current level*current the °
an B the °
a 100 C were
[%] - 42 83 89 82 75 80 57 C
Average transfection .
of
80
Na after . vessels
with single &
were cell
[nA] - -
D
5.2 6.5
currents for and expected
provided temperature
resources .
. activity
to C
v .
hole voltage
1 The .
An
obtained .
an Levels
. four +
harvest 5 half channel The Raw by
the transferred
Average current
80 of cell
ion IV
additional TTX block**
overall
mode and
cells
level* large
static mV freshly by cells ------
[nA] were concentrations
3.0 3.8 2.0 2.5 3.5 3.6 5.7 6.3
in - illustrate . curve in IV current
[%] gated response
85 85
channel of
Some
; .
Thomas
freshly
all with
MaxCyte’s with
remaining were
curve
transfection to
kept were activity showed numbers
electroporation success
transfected
four in to 28
TTXblock*
5 150 traces 24
cells
potassium
typical
cultured
° a 28 mV [%]
82 79 79 77 86 82 71 65 at
C
Jespersen, was
activity
transfected cultured to
sets
voltage
hrs ° 37 B. Na µg/mL C for
steps
were a in
good cells
** Percentage ** Percentage block compared saline to of period a mMsingle 20 addition TTX * Measured mV @ 0 in depolarizing simple step protocol
rates ° of .
scalable Temp.[ and and of
condition
C
not Na Na efficiency 24 A
of
Test
a a for 37 28 v and for cells . .
1.5 ActivityPostCropreservation
were assayed
°
for C] 24 an
Current [pA] with
frozen Current [pA]
& for
HyperFlask
** Percentage block compared to salineperiod of TTX a single addition20 mM * depolarizingMeasured @ 0inmV simple step protocol Transfection hrs fresh additional D. IV Plot C. It Plot B. Sweep Plot electroporation cryopreserved
efficiency Min(IV) [A] Min(Cl-1) [A]
cryopreserved
and
ion 150 [%]
88 89
at
on cells
and
average
37 Saline are registered trademarks of Corning, Inc. Corning, of trademarks registered are channel
the µg/mL
°
.
Sweep Plot
C current level*current
Time [ms] Time Displayed liquid periods liquid Displayed 24 Time [ms] frozen
. Displayed liquid periods Displayed liquid Average Qpatch
Sweep Sweep Time [S] Half
[nA] - - Tel: (301) 944 4.8 5.4 Step Voltage hrs [email protected]
www.maxcyte.com
current
and Saline of
assays
.
technology
of cells
Cells cells
a
in assayed the
block**
Na
TTX
single . levels [%] [20.0 [20.0 uM]
79 81
were were
HEK cells TTX with v
1 -
1700 .
5 Voltage [mV]
Voltage [mV]