Transcript LBS Walkersville Overview - The Stem Cell Training Course

4D-Nucleofector® –
Stretching Transfection Dimensions
Dr. Nazim El-Andaloussi
Lonza Cologne, Germany
Agenda

Introduction



The Principle of the Amaxa® Nucleofector® Technology
Available platforms
Details about the 4D-Nucleofector®





The Core Unit
The X-Unit
The Operation Software
Consumables
Protocol Transfer

Nucleofection and Stem Cells

Summary: System key benefits
slide 2
Nucleofection® – The Principle
Notebook
 High transfection efficiency combined with low mortality
Control Unit
 DNA is directed into the nucleus giving faster gene expression
Nucleofection®
Other transfection methods
slide 3
DNA is Delivered Into Cytoplasm and Nucleus
GFP
(R)R-DNA
DAPI
Merge
2 hrs
Primary NHDF-neo cells were transfected with TMR-labeled
plasmid DNA encoding GFP, fixed after 2h in 3.5% PFA and
analyzed by confocal microscopy.
slide 4
Simple Handling – Optimized Protocols
step 1
step 2
Harvest cells of interest
Mix
Nucleofector®
Solution with
Supplement
& combine with:
cells
step 3
transfer to an
amaxa certified
cuvette
DNA or siRNA
step 4
Select Nucleofector® program
Insert cuvette
Press start button “X”
Rinse cuvette
with culture
medium and
transfer to
culture dish
expression
detected as early
as 3-8 hours
slide 5
Our Product Family – A Technical Comparison
Nucleofector® 2b*
96-well
Shuttle®
4DNucleofector®
HT
Nucleofector®
Very low (1)
Medium (96)
Low (1-16)
High (384)
100µl
20µl
100µl and 20µl
20µl
Aluminum
Conductive
polymer
Conductive
polymer
Conductive
Polymer
Cell numbers
(orders of magnitude)
105 to 107 (2)
104 to 106 (2)
104 to 107 (3)
104 to 106 (2)
Adherent
Nucleofection
No
Yes
Yes
Yes
Shuttle compatibility
No
-
Yes
No
Device
Throughput
Reaction volume
Electrode material
* Nucleofector without Shuttle connectivity
slide 6
The Amaxa® Nucleofector® Technology

Efficient transfection of primary cells & cell lines


Versatility in substrate delivery

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Applications in Neurology, Metabolic diseases,
Cancer, Immunology, Cardiology and other
Therapeutic areas
Notebook
Control Unit
DNA, siRNA, miRNA, mRNA, morpholinos
Peptides, proteins, antibodies
Small molecules
Applicable for

Assay development
RNAi and cDNA screening

Target validation

slide 7
Agenda

Introduction



The Principle of the Amaxa® Nucleofector® Technology
Available platforms
Details about the 4D-Nucleofector®





The Core Unit
The X-Unit
The Operation Software
Consumables
Protocol Transfer

Nucleofection and Stem Cells

Summary: System key benefits
slide 8
Overview 4D-Nucleofector® System
A new, modular system offering advanced performance and
convenience. Comprising one Core Unit and several Functional
Modules the system is designed for maximum flexibility.
Features

1D Easy
 Transfection of various cell numbers with same conditions

2D Fast
 Different throughput, from one to 16 wells in 10 seconds

3D Flexible
 Nucleofection of cells in adherence for assays at various stages

4D Future-proof
 Modular system for upcoming transfection challenges
slide 9
4D-Nucleofector® System
Core Unit
X-Unit
Y-Unit
Straight, very elegant, valuable and distinguishable

Fold-away Graphical User Interface
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Electrically driven drawer for cuvette/sample retainer
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Could be assembled side by side or on top of each other
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Noble housing, shell made of metal
slide 10
Amaxa® Nucleofector® 96-well Shuttle® System
Notebook
Control Unit
USB
Amaxa® Nucleofector®
II.S
4D-Nucleofector®
Core and X-Unit
Amaxa® Nucleofector®
96-well Shuttle®

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+
Amaxa® Nucleocuvette®
Plate
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
Intuitive Software
96 different programs /
plate
3-4 min / plate
Connection via USB
slide 11
4D-Nucleofector® System –
The Core Unit
A
B
C
The Control Unit of the 4D-Nucleofector® System


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5,7’’ foldable touch screen to operate the system (A)
Operated by intuitive operation software
Controls several functional units
Comprises USB and serial connectivity for the 96-well Shuttle® (B)
USB port for software update and data transfer (C)
slide 12
4D-Nucleofector® System –
The X-Unit
A
B
C
The Unit supporting Nucleofection® of various cell numbers
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Features positions for Nucleocuvette® Strips (A) and 100µl CP-cuvettes (B)
Comprises HV connectivity for the Shuttle (C)
Seamless transfer of conditions between Nucleofection® Vessels
Suited for transfection of cells in adherence (ACT-Strips)
slide 13
4D-Nucleofector® System –
Operation Software
The intuitive tool operating the 4D-Nucleofector® System

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Easy-to-use through up-to-date touch screen interface
Comes with predefined Nucleofection® Parameters and Experiments
Supports data transfer / software update via USB storage device
PC-Editor software included to predefine experiments off-line
slide 14
4D-Nucleofector® System —
Consumables
A
B
Consumables tailored to customer needs

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Kits supporting Nucleofection in 16-well Nucleocuvette Strips (20µl) (A)
Kits supporting Nucleofection in single CP-cuvettes (100µl) (B)
Three kits for cell lines; five kits for primary cells
Cell line and primary cell optimization kits
Kits for adherent cell Nucleofection
slide 15
Reduction of Specific Kits
4D-Nucleofector® Kit Concept:
 5 Primary Cell Kits (P1-5)
 1 Primary Cell Optimization Kit
 3 Cell Line Kits (SE, SF, SG)
 1 Cell Line Optimization Kit
4D-Nucleofector OPs:
 Modified 96-well Shuttle OPs with ordering information for 20µl
and 100µl Kits.
slide 16
Cell Optimization Strategy
Cell Line Optimization
SF
SE
SG
FINE TUNE
Primary Cell Optimization
P1
P2
P3
P4
P5
FINE TUNE
Easy Optimization

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Test three solutions for cell lines or five solutions for primary cells each with
15 different programs
Based on best solution and program combination, fine tune transfection
efficiency or viability
slide 17
Cell Line Optimization Strategy
Sol.
SE
SF
SG
Row
1
2
1
2
1
2
A
CA-137
DS-150
CA-137
DS-150
CA-137
DS-150
B
CM-138
DS-120
CM-138
DS-120
CM-138
DS-120
C
CM-137
EH-100
CM-137
EH-100
CM-137
EH-100
D
CM-150
EO-100
CM-150
EO-100
CM-150
EO-100
E
DN-100
EN-138
DN-100
EN-138
DN-100
EN-138
F
DS-138
EN-150
DS-138
EN-150
DS-138
EN-150
G
DS-137
EW-113
DS-137
EW-113
DS-137
EW-113
H
DS-130
Control
DS-130
Control
DS-130
Control
Strip 1
Strip 2
Strip 3
slide 18
4D-Nucleofector® System –
Optimized Protocols
% Transfection efficiency
100
90
80
70
60
50
40
30
20
10
0
Jurkat
4D-Nucleofector®
HUVEC
T Cells
NIH-3T3
K562
96-well Shuttle®
Excellent performance on various cell numbers
 4D-Nucleofector® performs equally compared to the 96-well
Shuttle®
slide 19
4D-Nucleofector® System –
Protocol Transfer
100
TE
80
VIA
%
60
40
20
0
CHO-K1
Human T cells
Rat neurons
TE 20µl
TE 100µl
Jurkat
VIAB 20 µl
HeLa-S3
NIH-3T3
NHDF-neo
VIAB 100 µl
Same conditions for different volumes / cell numbers
 20µl and 100µl Nucleocuvettes® working with the same
parameters – no additional optimization required
slide 20
Agenda

Introduction



The Principle of the Amaxa® Nucleofector® Technology
Available platforms
Details about the 4D-Nucleofector®





The Core Unit
The X-Unit
The Operation Software
Consumables
Protocol Transfer

Nucleofection and Stem Cells

Summary: System key benefits
slide 21
Nucleofection of Stem Cells
Transfection
Efficiency *
Adipose Stem Cells
CD34+
MSC
(human)
(human)
(human)
Viability
76 %
71% – 83%
62%
47% – 78%
65 – 78%
ES Cells
(human)
45% – 78%
50 – 98%
ES Cells
(mouse)
77% - 90%
68 – 95%
75%
NSC
(cow)
65%
NSC
(human)
90%
NSC
(mouse)
60%
80%
NSC
(rat)
42% – 90%
75%
* pmaxGFP
slide 22
Nucleofection™
of human Embryonic Stem Cells
100
90
80
70
60
50
40
30
20
10
Data for Nucleofection™
of human stem cells
are compiled from
experiments performed
by leading stem cell
research customers
0
Efficiency
Viability
slide 23
GFP
SSEA4
OCT4
Nuclei
% Positive cells (flow cytometry)
Human ES cells maintain pluripotency
100
80
60
40
20
0
SSEA4
GFP
SSEA4/GFP
H9 cells express pluripotency markers SSEA4 and OCT4 24 hours post
Nucleofection™ of pmaxGFP™ reporter vector
Data kindly provided by Jennifer Moore, Rutgers University, Piscataway, USA
slide 24
Nucleofection™ for generation of iPS cells
Transfection Efficiency and viability (%)
High transfection efficiencies for human chondrocytes, dermal
fibroblasts (NHDF) or mouse embryonic fibroblasts – potential for
generation of iPS via Nucleofection™
100
80
60
40
20
0
NHDF-adult*
NHDF-neo*
Transfection Efficiency
Human chondrocytes*
Mouse Embryonic Fibroblast
Viability
Photograph courtesy of Dr. H. Hermanns and Prof. P.H. Heinrich, University of Aachen, Germany
slide 25
Nucleofection™ for generation of iPS cells
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Virus-free induction of pluripotency and subsequent excision
of reprogramming factors; Nature, 9 Apr 2009; 458 (7239)

Generation of mouse-induced pluripotent stem cells by
transient expression of a single nonviral polycistronic vector
PNAS June 2, 2009 vol. 106 no. 22 8918-8922

Efficient human iPS cell derivation by a non-integrating
plasmid from blood cells with unique epigenetic and gene
expression signatures Cell Research (2011) 21:518-529.

Efficient Feeder-Free Episomal Reprogramming with Small
Molecules (2011) PLoS ONE 6(3): e17557.
slide 26
Agenda

Introduction



The Principle of the Amaxa® Nucleofector® Technology
Available platforms
Details about the 4D-Nucleofector®





The Core Unit
The X-Unit
The Operation Software
Consumables
Protocol Transfer

Nucleofection and Stem Cells

Summary: System key benefits
slide 27
System Key Benefits
Major Benefits of the 4D-Nucleofector®

Easy-to-use through intuitive software and touch screen interface

Upgradeable by adding new Functional Modules

Customizable through its modular architecture

Uses existing 96-well Shuttle® Protocols

The use of CP electrodes allows fewer solutions for primary cells

Supports Nucleofection® of various cell numbers with same
conditions

Allows Nucleofection® of cells in adherence

Excellent technical and application support by Nucleofection®
Experts
slide 28
Thank you for your kind attention!