4D-Nucleofector ® – Stretching Transfection Dimensions Dr. Nazim El-Andaloussi Lonza Cologne,...

4D-Nucleofector® – Stretching Transfection Dimensions

Dr. Nazim El-Andaloussi

Lonza Cologne, Germany

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

Agenda

NotebookControl Unit

Nucleofection® – The Principle

Other transfection methods

High transfection efficiency combined with low mortality DNA is directed into the nucleus giving faster gene expression

Nucleofection®

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.

(R)R-DNA GFP DAPI Merge

DNA is Delivered Into Cytoplasm and Nucleus

2 hrs

Simple Handling – Optimized Protocolsstep 1

Harvest cells of interest

step 3

Nucleofector® Solution with Supplement

Mix

cells DNA or siRNA

transfer to an amaxa certified cuvette

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

& combine with:

step 2

step 4

Device Nucleofector® 2b*96-well Shuttle®

4D-Nucleofector®

HT Nucleofector®

Throughput Very low (1) Medium (96) Low (1-16) High (384)

Reaction volume 100µl 20µl 100µl and 20µl 20µl

Electrode material AluminumConductive

polymerConductive

polymerConductive

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

Our Product Family – A Technical Comparison

* Nucleofector without Shuttle connectivity


The Amaxa® Nucleofector® Technology

Efficient transfection of primary cells & cell lines Applications in Neurology, Metabolic diseases,

Cancer, Immunology, Cardiology and other Therapeutic areas

Versatility in substrate delivery DNA, siRNA, miRNA, mRNA, morpholinos Peptides, proteins, antibodies Small molecules

Applicable for Assay development RNAi and cDNA screening

Target validation





Agenda

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

Straight, very elegant, valuable and distinguishable Fold-away Graphical User Interface

Electrically driven drawer for cuvette/sample retainer

Could be assembled side by side or on top of each other

Noble housing, shell made of metal

Core Unit

X-Unit

4D-Nucleofector® System

Y-Unit


Amaxa® Nucleofector®

II.S

Amaxa® Nucleofector® 96-well Shuttle®

Amaxa® Nucleocuvette® Plate

Intuitive Software 96 different programs /

plate 3-4 min / plate Connection via USB

Amaxa® Nucleofector® 96-well Shuttle® System

USB

4D-Nucleofector® Core and X-Unit

+

4D-Nucleofector® System – The Core Unit

The Control Unit of the 4D-Nucleofector® System 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)

A

B

C

4D-Nucleofector® System – The X-Unit

The Unit supporting Nucleofection® of various cell numbers

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)

A

B

C

4D-Nucleofector® System – Operation Software

The intuitive tool operating the 4D-Nucleofector® System 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

4D-Nucleofector® System — Consumables

Consumables tailored to customer needs 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

BA

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.

Cell Optimization Strategy

FINE TUNE

Cell Line Optimization

Easy Optimization 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

FINE TUNE

Primary Cell Optimization

P1 P2 P3 P4 P5

SE SF SG

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

4D-Nucleofector® System – Optimized Protocols

Excellent performance on various cell numbers 4D-Nucleofector® performs equally compared to the 96-well

Shuttle®

% T

rans

fect

ion

effic

ienc

y

4D-Nucleofector® 96-well Shuttle®

0

10

20

30

40

50

60

70

80

90

100

Jurkat HUVEC T Cells NIH-3T3 K562

Same conditions for different volumes / cell numbers 20µl and 100µl Nucleocuvettes® working with the same

parameters – no additional optimization required

4D-Nucleofector® System – Protocol Transfer

0

20

40

60

80

100

CHO-K1 Human T cells Rat neurons Jurkat HeLa-S3 NIH-3T3 NHDF-neo

TE 20µl TE 100µl VIAB 20 µl VIAB 100 µl

TEVIA

%





Agenda

Transfection Efficiency *

Viability

Adipose Stem Cells (human) 76 %

CD34+ (human) 71% – 83% 62%

MSC (human) 47% – 78% 65 – 78%

ES Cells (human) 45% – 78% 50 – 98%

ES Cells (mouse) 77% - 90% 68 – 95%

NSC (cow) 65% 75%

NSC (human) 90%

NSC (mouse) 60% 80%

NSC (rat) 42% – 90% 75%

Nucleofection of Stem Cells

* pmaxGFP

Nucleofection™ of human Embryonic Stem Cells

0

10

20

30

40

50

60

70

80

90

100

H1H9

H9.2

Hues-

9

BG01V

Custo

mer

Efficiency Viability

Data for Nucleofection™ of human stem cells are compiled from experiments performed by leading stem cell research customers

Data kindly provided by Jennifer Moore, Rutgers University, Piscataway, USA

GFPSSEA4OCT4Nuclei

H9 cells express pluripotency markers SSEA4 and OCT4 24 hours post Nucleofection™ of pmaxGFP™ reporter vector

Human ES cells maintain pluripotency

0

20

40

60

80

100

SSEA4 GFP SSEA4/GFP% P

osi

tive

ce

lls (

flow

cyt

om

etr

y)

Nucleofection™ for generation of iPS cells

High transfection efficiencies for human chondrocytes, dermal fibroblasts (NHDF) or mouse embryonic fibroblasts – potential for generation of iPS via Nucleofection™

Mouse Embryonic Fibroblast

Photograph courtesy of Dr. H. Hermanns and Prof. P.H. Heinrich, University of Aachen, Germany

Tra

nsf

ect

ion

Eff

icie

ncy

an

d v

iab

ility

(%

)

0

20

40

60

80

100

NHDF-adult* NHDF-neo* Human chondrocytes*

Transfection Efficiency Viability

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.

Nucleofection™ for generation of iPS cells





Agenda

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

System Key Benefits

Thank you for your kind attention!

4D-Nucleofector ® – Stretching Transfection Dimensions Dr. Nazim El-Andaloussi Lonza Cologne,...

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Transcript of 4D-Nucleofector ® – Stretching Transfection Dimensions Dr. Nazim El-Andaloussi Lonza Cologne,...