The New Eppendorf Cell Culture Consumables

No. 41 – 2014

(BN 41) July 2014

Your local distributor: www.eppendorf.com/contact eppendorf ag · 22331 Hamburg · germany · e-mail: [email protected] · www.eppendorf.com

Cultivation of Human CAP® Cells: Evaluation of Scale-Down Capabilities Using BioBLU® Single-Use Bioreactors

Abstract

Increasing process complexity coupled with rising cost pressures and rapidly evolving regulatory requirements makes today’s process development efforts a special challenge. The pressure of achieving faster time-to-market for new and innovative biotechnological products has led to the need to optimize every element of the total development workflow.

The following application note illustrates how the DaSbox® Mini Bioreactor Sys-tem combined with the BioBlu 0.3c single-use vessels supports bioprocess development in human cell culture. Scale-down capabilities were investi-gated by comparison of 500 ml cultures in a DaSgIP® Parallel Bioreactor System (PBS) with 170 ml cultures in the DaSbox using the BioBlu 0.3c single-use vessel.

Introduction

CeVeC® Pharmaceuticals gmbH (Cologne, germany) has established a master cell bank of CaP cells growing in suspension, tested and certified ac-cording to ICH guidelines and european Pharmacopeia. The platform expression technologies CaP and CaP-T are based on specific, amniocyte-derived human cell lines. CaP and CaP-T were designed for stable and transient protein produc-tion and achieve highest protein yields with authentic human glycosylation patterns.

Simple and reliable protocols allow for the fast generation of customized producer cell lines for pharmaceutically relevant proteins based on the parental permanent CaP cells under controlled and optimized conditions. For the required human cell line screening as well as for media opti-mization, the small working volumes of 100 – 250 ml make the extendable 4-fold DaSbox and the BioBlu 0.3c single-use vessel a perfect fit. Bioprocesses are controlled as precisely and effectively as they are in larger scale bioreactors while cell material, media and supple-ments as well as lab space are saved.

Several experiments were carried out aiming at verifying the scale-down capabilities from the DaSgIP Parallel Bioreactor System, which CeVeC gen-erally uses in process development, to the Mini Bioreactor System DaSbox. To overcome the risk of cross-contami-nation and to reduce time for cleaning, sterilization and assembly they evalu-ated BioBlu 0.3c single-use vessels facilitating full industry standard instru-mentation.

a specifically designed port including a gas permeable membrane allows for DO measurement using a reusable probe which can easily be plugged in directly on the bench. Recuperation of liquid from exhaust gas is carried out via a liquid-free Peltier condenser.

Materials and methods

To evaluate the scale-down capability of the DaSbox Mini Bioreactor System and the usability of the BioBlu 0.3c single-use vessel experimental series with two different systems were carried out and compared. a 4-fold Parallel Bioreactor System for cell culture was used in 500 ml scale experiments (PBS).

The corresponding small-scale approach-es were carried out in a DaSbox system

using single-use vessels with 170 ml (DaSbox Su). The recombinant human CaP cells producing a pharmaceutically relevant protein were batch cultivated for 7 d (170 h) in CeVeC's serum-free, chemically defined CaP medium supple-mented with 40 mM glucose and 6 mM glutamine at 37 °C. Initial viable cell den-sity was 3 x105 cells/ml.

The DO set-point of 40 % was main-tained by a constant stirrer speed and the oxygen concentration in the inlet gas. Stirrer speed was adjusted to 160 rpm (PBS) and 150 rpm (DaSbox Su).

The pH value was regulated to 7.1 by addition of 1 M Na2CO3 (feeding, speed rate regulated) and CO2 (submerged gassing). Inlet gas (air, O2, CO2 and N2) was mixed continuously mass flow-con-trolled. The bioreactors were equipped with pitched blade impellers and liquid-free operated exhaust gas condensers. The pre-cultures were cultivated in 125 ml erlenmeyer flasks (Corning®) with 25 ml working volume using a shaker incubator (37 °C, 5 % CO2) agi-tating at 185 rpm (Multitron® 2, Infors®

ag). The cells were expanded up to a viable cell density of 3 x 106 cells/ml in the same medium used for bioreactor runs.

ClauDIa M. HüTHeR-FRaNkeN*, ag BIOPROCeSS CeNTeR, JuelICH, geRMaNy

HelMuT keweS aND MICHael SCHOMBeRg, CeVeC PHaRMaCeuTICalS gMBH, COlOgNe, geRMaNy

*CORReSPONDINg auTHOR: [email protected]

Fig. 1: Viable cell numbers of all experiments with DaSgIP Parallel Bioreactor Systems (PBS) and BioBlu 0.3c vessels with average growth rate of 0.02 h-1

Via

ble

cel

l nu

mb

er (

mL-1

)

Process time (h)

Viable cell number

8.0 e+06

7.0 e+06

6.0 e+06

5.0 e+06

4.0 e+06

3.0 e+06

2.0 e+06

1.0 e+06

0.0 e+00

PBS-1

PBS-2

PBS-3

PBS-4

DASBox SU-1

DASBox SU-2

0 20 40 60 80 100 120 140 160 180

The New Eppendorf Cell Culture Consumables> Parallel Dimension in Cell Culture> The New Classics – Eppendorf Tubes® 5.0 mL> Perfect Asymmetry: Mastercycler® nexus X2

Application NotesCultivation of Prostate Cancer Cells under Hypoxic Conditions in the Galaxy® 170 R CO2

Incubator · DNA Purification from Sample Material of Different Marine Animals · etc.

Imprint

Editorial team

Berrit Hoff (Editor-in-Chief), Axel Jahns,

Jochen Müller-Ibeler, Natascha Weiß

Publisher

Eppendorf AG, Barkhausenweg 1,

22339 Hamburg, Germany

Telephone: (+49) 40-53801-636

Fax: (+49) 40-53801-840

E-mail: [email protected]

Internet: www.eppendorf.com

We welcome all readers' articles for this

publication. However, no responsibility is

accepted for unsolicited manuscripts.

Important note

The new products described may be

launched at different times in various

countries. Please contact your local

Eppendorf organization or distributor

for details.

Technical specifications subject to change.

Errors and omissions excepted.

All rights reserved,

including graphics and images.

© Copyright Eppendorf AG, July 2014.

Carbon neutrally printed in Germany.

2

Dear Readers,

For nearly 70 years, Eppendorf has been using innovative technologies and premium products to make an important contribution to improving work processes in liquid, cell and sample handling in laboratories and research facilities all over the world. In this edition of BioNews we are introducing some of these products to you.

> Experience a new dimension in cell culture with Eppendorf Cell Culture Consumables. The portfolio answers your needs for excellent cell performance and reliability of your experiments as well as for advanced protection against contamination (pages 4 – 5).

> Do you already know our new classic, the Eppendorf Tube 5.0 mL? It closes the gap between the currently common tube types, and it enables safe, practical and efficient handling of sample volumes up to 5.0 mL. Moreover, it may be easily integrated as a complete system into the lab routine (pages 6 – 7).

> The DASGIP® product line for cell culture applications combines the advantages of small working volumes with the full functionality of industrial bioreactors (page 8).

> As the latest addition to the popular Mastercycler® range, the Mastercycler nexus X2 continues the success story of Eppendorf thermal cyclers (page 12).

Additionally you will find detailed Application Notes and our popular competition with great prizes.

Enjoy reading!

Your Eppendorf BioNews Editorial Team

EdItorIAl · DEAR READERS,

4 126

(BN 41) July 2014 Page 1

Your local distributor: www.eppendorf.com/contact eppendorf ag · 22331 Hamburg · germany · e-mail: [email protected] · www.eppendorf.com


Abstract

Increasing process complexity coupled with rising cost pressures and rapidly evolving regulatory requirements makes today’s process development efforts a special challenge. The pressure of achieving faster time-to-market for new and innovative biotechnological products has led to the need to optimize every element of the total development workflow.

The following application note illustrates how the DaSbox® Mini Bioreactor Sys-tem combined with the BioBlu 0.3c single-use vessels supports bioprocess development in human cell culture. Scale-down capabilities were investi-gated by comparison of 500 ml cultures in a DaSgIP® Parallel Bioreactor System (PBS) with 170 ml cultures in the DaSbox using the BioBlu 0.3c single-use vessel.

Introduction

CeVeC® Pharmaceuticals gmbH (Cologne, germany) has established a master cell bank of CaP cells growing in suspension, tested and certified ac-cording to ICH guidelines and european Pharmacopeia. The platform expression technologies CaP and CaP-T are based on specific, amniocyte-derived human cell lines. CaP and CaP-T were designed for stable and transient protein produc-tion and achieve highest protein yields with authentic human glycosylation patterns.

Simple and reliable protocols allow for the fast generation of customized producer cell lines for pharmaceutically relevant proteins based on the parental permanent CaP cells under controlled and optimized conditions. For the required human cell line screening as well as for media opti-mization, the small working volumes of 100 – 250 ml make the extendable 4-fold DaSbox and the BioBlu 0.3c single-use vessel a perfect fit. Bioprocesses are controlled as precisely and effectively as they are in larger scale bioreactors while cell material, media and supple-ments as well as lab space are saved.

Several experiments were carried out aiming at verifying the scale-down capabilities from the DaSgIP Parallel Bioreactor System, which CeVeC gen-erally uses in process development, to the Mini Bioreactor System DaSbox. To overcome the risk of cross-contami-nation and to reduce time for cleaning, sterilization and assembly they evalu-ated BioBlu 0.3c single-use vessels facilitating full industry standard instru-mentation.

a specifically designed port including a gas permeable membrane allows for DO measurement using a reusable probe which can easily be plugged in directly on the bench. Recuperation of liquid from exhaust gas is carried out via a liquid-free Peltier condenser.

Materials and methods

To evaluate the scale-down capability of the DaSbox Mini Bioreactor System and the usability of the BioBlu 0.3c single-use vessel experimental series with two different systems were carried out and compared. a 4-fold Parallel Bioreactor System for cell culture was used in 500 ml scale experiments (PBS).

The corresponding small-scale approach-es were carried out in a DaSbox system

using single-use vessels with 170 ml (DaSbox Su). The recombinant human CaP cells producing a pharmaceutically relevant protein were batch cultivated for 7 d (170 h) in CeVeC's serum-free, chemically defined CaP medium supple-mented with 40 mM glucose and 6 mM glutamine at 37 °C. Initial viable cell den-sity was 3 x105 cells/ml.

The DO set-point of 40 % was main-tained by a constant stirrer speed and the oxygen concentration in the inlet gas. Stirrer speed was adjusted to 160 rpm (PBS) and 150 rpm (DaSbox Su).

The pH value was regulated to 7.1 by addition of 1 M Na2CO3 (feeding, speed rate regulated) and CO2 (submerged gassing). Inlet gas (air, O2, CO2 and N2) was mixed continuously mass flow-con-trolled. The bioreactors were equipped with pitched blade impellers and liquid-free operated exhaust gas condensers. The pre-cultures were cultivated in 125 ml erlenmeyer flasks (Corning®) with 25 ml working volume using a shaker incubator (37 °C, 5 % CO2) agi-tating at 185 rpm (Multitron® 2, Infors®

ag). The cells were expanded up to a viable cell density of 3 x 106 cells/ml in the same medium used for bioreactor runs.

ClauDIa M. HüTHeR-FRaNkeN*, ag BIOPROCeSS CeNTeR, JuelICH, geRMaNy

HelMuT keweS aND MICHael SCHOMBeRg, CeVeC PHaRMaCeuTICalS gMBH, COlOgNe, geRMaNy

*CORReSPONDINg auTHOR: [email protected]

Fig. 1: Viable cell numbers of all experiments with DaSgIP Parallel Bioreactor Systems (PBS) and BioBlu 0.3c vessels with average growth rate of 0.02 h-1

Via

ble

cel

l nu

mb

er (

mL-1

)

Process time (h)

Viable cell number

8.0 e+06

7.0 e+06

6.0 e+06

5.0 e+06

4.0 e+06

3.0 e+06

2.0 e+06

1.0 e+06

0.0 e+00

PBS-1

PBS-2

PBS-3

PBS-4

DASBox SU-1

DASBox SU-2

0 20 40 60 80 100 120 140 160 180

3CoNtENts

CLAuDIA M. HüTHER-FRANkEN, HELMuT kEWES, MICHAEL SCHOMBERG

Cultivation of Human CAP® Cells: Evaluation of Scale-Down Capabilities using BioBLu® Single-use Bioreactors

1 – 2

uLRIkE GAST, MuRIEL ART

Decontamination of Pipettes: Influence of Material and Design on Decontamination Efficiency

3 – 4

CECILE REBOuT, CHRISTOPHE CARPENTIERI

DNA Purification from Sample Material Obtained from a Selection of Different Marine Animals using the epMotion® 5075 VAC

5 – 6

STACEY S. WILLARD, LINETTE PHILIP, MA SHA

Cultivation of Prostate Cancer Cells under Hypoxic Conditions in the New Brunswick™ Galaxy® 170 R CO2 Incubator from Eppendorf

7 – 8

IN thE sPotlIght The New Eppendorf Cell Culture Consumables 4 – 5

INNovAtIoN Parallel Dimension in Cell Culture 8

New Systems for Micromanipulation of Cells 11

Perfect Asymmetry: Mastercycler® nexus X2 12

strAIght from thE lAb The New Classics – Eppendorf Tubes® 5.0 mL 6 – 7

epMotion® – Automated Liquid Handling at its Best 10

NEws / tIPs Two Catalogs – One Source: Eppendorf 5

A Great Team for Cell Culture Applications 9

CO2 Incubator Performance Plans 9

Go Farther with us! 12

Eppendorf Research Prizes: Winners 2013 13

sErvICE Prize Competition 14

Reply Fax / Readers' Service 15

4

The New Eppendorf Cell Culture Consumables

How can we make our customers' work processes simpler and more secure, quicker and more reproducible? How can we make work more enjoyable for users? When we are working on new product ideas, we ask users and ourselves these and another thousand questions. And if necessary, we are prepared to go back right to the beginning. This is exactly what we did when we were developing the new Eppendorf Cell Culture Consumables. In spite of our 50 years of experience in the field of plastics technology we want-

Experience a new dimension in cell culture work. In the development of the new Eppendorf Cell Culture Consumables

we put a special focus on your needs for excellent cell growth as well as for advanced protection against contami-

nation. our Cell Culture Consumables provide you with proven Eppendorf Quality™ at a new premium level. Premium

in all aspects: product, performance and packaging.

DANIEL WEHRHAHN, EPPENDORF AG

IN thE sPotlIght · THE NEW EPPENDORF CELL CuLTuRE CONSuMABLES

ed to question everything once again. Now we have reached our goal. And we are convinced that the all new product line will also impress your cells!

Eppendorf Cell Culture dishes: safety in all situations

Whenever you need direct access to your cells, dishes are the format of choice. We focused on an improved handling and stacking performance to ensure a new level of safe and carefree usage of those formats in cell culture. It all starts with

the packaging: opening is easy and tool- free. An innovative resealable top and side make tape-free closing possible.

Working with dishes is not always easy due to the small sizes and hard to differ-entiate lids and dish bottoms. With the new Eppendorf dishes, a corrugated handling ring provides an unsurpassed safety in handling during transportation and expansion of cells. A SplashProtect™ ring inside the dish lid prevents spillage that may cross-contaminate other samples.

An outstanding stacking performance completes these products in their ease, safety and convenience in handling.

Eppendorf Cell Culture Plates: efficient, reproducible assays

We optimized the optical performance by enhancement of the planarity, material clarity and reduction of the meniscus of liquids in the wells to facilitate every step where manual or automated read-out is necessary.

The proven contrast-rich alphanumeric OptiTrack® labeling known from other Eppendorf Plates™ allows easy and fast identification of individual wells. Further-more, the outer moat of the 96-well plate can be filled with liquid due to the inno-vative new chimney-well design. Thus in-homogeneous assay results due to the »edge effect« can be minimized. By filling the complete inter well space the temper-ature is more stable throughout the plate when the cells are outside the incubator.

Eppendorf Cell Culture Consumables offer proven Eppendorf Quality at a new premium level. Premium in all aspects: product, performance and packaging.

tip

Two Catalogs – One Source: Eppendorf

For the first time, Eppendorf has published

two catalogs in 2014. In our »Catalog 2014:

Liquid Handling, Sample Handling, Cell

Handling« we are presenting you a variety

of new and innovative products that will

make your laboratory work easier and more

efficient.

The catalog itself offers some new features:

in addition to a different appearance, the

product pages have been restructured.

The new, clear presentation of the products

helps you locate important information in

a fast and easy manner.

From now on, you will find all the Eppendorf

bioprocessing products in our »Catalog

2014: Bioprocess products«. It contains

comprehensive information on our fermen-

tor and bioreactor system families, which

can be used for volumes of 0.1 to 3,000

liters. Here you will also find our new

range of single-use bioreactor vessels and

comprehensive bioprocess software

solutions.

See for yourself at

www.eppendorf.com/catalog!

Catalog 2014Bioprocess products

We know Bioprocessing

5THE NEW EPPENDORF CELL CuLTuRE CONSuMABLES · IN thE sPotlIght

This will help you increase the efficiency and reproducibility in your assays.

Carrying the new Eppendorf Plates is ex-tremely comfortable and safe due to the enlarged corrugated gripping area of the plate. Lid and plate bottom can easily be distinguished providing extra safety in transportation and stacking.

Eppendorf Cell Culture flasks: easy access and improved protection

unsurpassed protection of your cells from contaminants was one of the key require-ments in developing the new Eppendorf Cell Culture Flasks. A new, high efficiency air filter technology combines excellent protection with reliable gas exchange. A 100% in-line pressure test for each flask guarantees leakage-free flasks for maximum workflow safety. The plug-seal lids are equipped with an arrested and marked venting position that prevents unintended closing.

Workflow safety is combined with solu-tions for facilitated and ergonomic access to your cells in the new Eppendorf Cell Culture Flasks.

The new Eppendorf Cell Culture Flasks combine workflow safety with facilitated and ergonomic access to your cells.

Handling Eppendorf Cell Culture Dishes is easy, safe and convenient

OptiTrack labeling known from other Eppendorf plates allows easy and fast identification of individual wells. Furthermore, the outer moat of the 96-well plate can be filled with liquid to minimize inhomogeneous assay results due to the »edge effect«.

The special ConvexAccess™ shape signifi-cantly facilitates the access to the growth area and makes cell seeding and media exchange much more convenient, safe and reliable. Also it is more ergonomic and helps to prevent unintended disrup-tion of the cell layer during any workflow step performed in the flasks.

Packaging: easy and reliable

The packaging of the new Eppendorf Cell Culture Consumables supplements the outstanding product quality in many aspects. It is compact, resealable and shrinkable for space saving and safe stor-age of remaining consumables.

Color coded icons allow for easy identifi-cation of formats and surfaces. Improved protection against breakage during transport and an innovative tray for easy removal of products from the box and carrying in the lab will make your work in a cell culture lab easier.

Last but not least all product boxes are clearly labeled for easy identification of products when stacked on the shelf or in the storage facility.

Discover the new dimension in cell cul-ture work! For more information see the new brochure »Impress Yourself« or visit www.eppendorf.com/ccc to order your free product sample!

Eppendorf Cell Culture Consumables • Ref. no. 270

6 strAIght from thE lAb · THE NEW CLASSICS – EPPENDORF TuBES® 5.0 mL

The New Classics – Eppendorf Tubes® 5.0 mL

NILS GERkE, EPPENDORF AG

with the development of the first Eppendorf tube more than 50 years ago, Eppendorf sparked the evolution of

microtubes! since then, Eppendorf tubes have been used throughout the world every day. the new Eppendorf

tubes 5.0 ml system closes the long-standing gap in the processing of medium sample volumes between 2.0 ml

and 5.0 ml. for this volume range, too, users are now able to count on the same exceptional qualities which have

made the classic Eppendorf tubes a worldwide premium standard in laboratories.

Tubes in the volume range between 0.5 mL and 2.0 mL are established standard for-mats for processing and storage of small volumes in molecular and microbiological as well as cell biological applications. For medium-sized sample volumes, users were compelled to resort to disproportionately large vessels with screw cap.

Processing of medium-sized volumes

Of course sample volumes between 2.0 mL and 5.0 mL may be divided among several smaller tubes. However, the workload will increase considerably when handling twice or three times the number of tubes. Furthermore, the risk of contamination rises, and demand for space and racks also increases.

Another alternative is represented by the commonly used large volume conical screw cap tubes (15 mL and 50 mL). When using these relatively narrow and deep vessels, the pipette cone occasionally reaches

Clear advantages through low total height. The pipette tip reaches to the bottom of the Eppendorf Tube 5.0 mL. The pipette cone does not come in contact with the tube’s inner wall. Sample or supernatant can be completely removed, without the risk of contamination.

deeply into the tube during sample and reagent transfer, making it nearly impos-sible to avoid contact with the tube's inner wall. As a consequence the risk of contamination is also increased. In cell culture, where 15 mL tubes are often used for passaging of cells, the user is thus forced to use longer serological pipettes or Pasteur pipettes.

The comparatively low total height of the new Eppendorf Tube 5.0 mL, thus,

Same shape, same exceptional performance! The new Eppendorf Tube 5.0 mL and »the classic« for over 50 years, the Eppendorf Tube 1.5 mL

brings along with it obvious advantages: a 300 µL epT.I.P.S.® pipette tip reaches to the bottom of the tube [1] without the pipette cone entering the tube. Sample or supernatant may be completely re-moved without contamination.

An additional perk of the Eppendorf Tube 5.0 mL: the snap cap. This is easier to close with just one hand than a screw cap lid which – as a separate component – may act as a further source of contamination.

++

7THE NEW CLASSICS – EPPENDORF TuBES® 5.0 mL · strAIght from thE lAb

Safe centrifugation, e.g. in the Centrifuge 5430 with rotor FA-45-16-17, for 16 × Eppendorf Tubes 5.0 mL, up to 21,191 × g (rotor may be sealed aerosol-tight).

The ideal Starter Package: Eppendorf Tubes 5.0 mL Starter Pack, PCR clean. Includes 400 Eppendorf Tubes 5.0 mL (PCR clean) + 8 universal adapters for rotors with bores for conical 15 mL vessels + 2 practical Tube Racks 5.0 mL (with 16 spaces each), white.

is free of plasticizers, biocides and mold release agents. For good reason – after all, standard laboratory methods such as photometric detection of nucleic acids and proteins may be distorted by leach-able substances.

By using a simple test system which in-cluded heating water in reaction vessels (with standard laboratory methods) and a subsequent absorbance scan in the uV-range we were able to show that, in some cases, significant amounts of leachables washed out of conical 15 mL tubes sourced from different manufacturers whereas Eppendorf Tubes 5.0 mL resulted in no noteworthy interference of photometric analyses. An important condition for reli-able experimental results [2]!

versatile and robust

The Eppendorf Tubes 5.0 mL fulfill the requirements of various methods in the areas of cell culture and bacterial culture, as well as those employed with nucleic acids and proteins. For example, by in-creasing the volume of the bacterial starter culture to 5.0 mL, considerably higher plasmid yields can be obtained [3].

Besides variants made from Eppendorf LoBind® material and the purity grades established for the Eppendorf Safe-Lock tubes (Eppendorf Quality™, PCR clean, Biopur®) the Eppendorf Tubes 5.0 mL are available in the purity grade »sterile« for which each individual lot is additionally tested and certified pyrogen-free. The selection is completed by the availability of amber tubes. These are ideally suited for processing, incubating and archiving light sensitive samples.

The Eppendorf Tubes 5.0 mL display high safety and stability during centrifugation up to 25,000 x g and thus offer the option of performing centrifugation steps in a secure and time-saving fashion [1].

And when are you going to switch?

Conclusion: Our Eppendorf Tube 5.0 mL is the »missing link« in sample process-ing. It closes the gap between the cur-rently common tube types, and it en-ables safe, practical and efficient handling of sample volumes up to 5.0 mL. The Eppendorf Tube 5.0 mL offers great advantages for many applications, and thanks to comprehensive accessory

components, it may be easily integrated as a complete system into the lab routine.

Ask for free samples today or order the attractively priced Starter Pack!

well thought-out system

Ideally the bottom shape of the Eppendorf Tube 5.0 mL is equivalent to that of a typical conical 15 mL tube. This way, many instruments and accessories al-ready present in the laboratory may be used directly, or via an adapter, in a cost-effective manner. In order to pursue the Eppendorf system concept further, matching accessories have been devel-oped for new instruments as well for in-struments already available on the mar-ket. Thus, the new 5.0 mL system is easily integrated into daily laboratory routine.

Select from:

> Matching high-speed rotors for the Eppendorf Centrifuges 5427 R, 5430/R, 5804/R and 5810/R

> 5.0 mL adapters and inserts for all existing Eppendorf rotors with bore holes for 15 mL or 50 mL tubes

> Elegant and functional racks for the laboratory bench

> Storage boxes for freezing

> 5.0 mL thermoblocks for Eppendorf ThermoMixer® C and Thermomixer® comfort as well as for the Eppendorf ThermoStat™ C and ThermoStat plus

> Reservoir rack module 5.0 mL for auto-mated pipetting with the epMotion®

high quality material for reproducible, reliable results

The Eppendorf Tubes 5.0 mL are manu-factured from exceptionally high-quality, transparent polypropylene. The material

[1] Application Note 265. Applications of the Eppendorf 5.0 mL system

[2] Application Note Nr. 264. Comparison of Eppendorf Tubes® 5.0 mL to conical 15 mL tubes with a focus on releasable uV-absorbing substances (leachables)

[3] Application Note 262. Increased yield with isolation of a low-copy plasmid using Eppendorf Tubes® 5.0 mL compared to the 1.5 mL and 2 mL formats

These and further Application Notes are available for downloading at www.eppendorf.com/applications.

Eppendorf Tubes® 5.0 mL • Ref. no. 264

8 INNovAtIoN · PARALLEL DIMENSION IN CELL CuLTuRE

Parallel Dimension in Cell Culture

CLAuDIA M. HüTHER-FRANkEN, EPPENDORF AG, BIOPROCESS CENTER EuROPE

The DASGIP product line for cell culture applications combines the advantages of small working volumes with the full functionality of industrial bioreactors. With our DASbox we also offer the most compact mini bioreactor system on the market.

faster results, shorter time-to-market

Parallel operation reduces development times while the modular design and inter-connectivity to external devices allows individual solutions.

Our superior DASGIP Control Software and the DASware® Software Suite support sophisticated process control, comprehen-sive data and information management as well as Design of Experiments (DoE).

Adaptable to any application

With working volumes of 35 mL to 3.8 L and specific solutions for the cultivation of stem cells the Eppendorf parallel operated bioreactor systems fit many bioprocess applications in cell culture. Our reusable and single-use bioreactors feature variable stirring speeds starting at 20 rpm, active heating and cooling options, accurate control of pH, dissolved oxygen and filling level as well as parallel processing in batch, fed-batch and continuous mode including perfusion.

Next generation bioprocess software

DASGIP Control offers reliable control of parallel bioprocesses including parallel recipe management and simultaneous

calibration of probes and pumps. Our DASware suite offers smart and flexible solutions to accelerate bioprocess devel-opment.

It enables interconnectivity of bioreactors with external lab devices, comprehensive data- and information management, DoE and remote control of bioprocesses. DASware can be used with any Eppendorf benchtop bioreactor solution.

Additional information can be found in the »Bioprocess« section of your local Eppendorf website or in our comprehen-sive brochure.

the simultaneous operation of multiple bioreactors is one of the key features of the Eppendorf dAsbox® and dAsgIP®

Parallel bioreactor systems. designed for benchtop applications in research and process development, our systems

are used in labs around the world. the modular design offers flexible solutions for the cultivation of mammalian,

insect and human cells as well as stem cells.

Eppendorf DASGIP Parallel Bioreactors Systems: flexible benchtop solutions for cell culture

Eppendorf DASbox: our most compact mini bioreactor system, perfectly fits DoE approaches

A dream come true: remote control of bioprocesses with Eppendorf DASware

Brochure »We know Bioprocessing« • Ref. no. 274

(BN 41) JuLY 2014 PAGE 1

Your local distributor: www.eppendorf.com/contact Eppendorf AG · 22331 Hamburg · Germany · E-mail: [email protected] · www.eppendorf.com


Abstract

Increasing process complexity coupled with rising cost pressures and rapidly evolving regulatory requirements makes today's process development efforts a special challenge. The pressure of achieving faster time-to-market for new and innovative biotechnological products has led to the need to optimize every element of the total development workflow.

The following application note illustrates how the DASbox® Mini Bioreactor Sys-tem combined with the BioBLu 0.3c Single-use Vessels supports bioprocess development in human cell culture. Scale-down capabilities were investi-gated by comparison of 500 mL cultures in a DASGIP® Parallel Bioreactor System (PBS) with 170 mL cultures in the DASbox using the BioBLu 0.3c Single-use Vessel.

Introduction

CEVEC® Pharmaceuticals GmbH (Cologne, Germany) has established a master cell bank of CAP cells growing in suspension, tested and certified ac-cording to ICH guidelines and European Pharmacopoeia. The platform expression technologies CAP and CAP-T™ are based on specific, amniocyte-derived human cell lines. CAP and CAP-T were designed for stable and transient protein produc-tion and achieve highest protein yields with authentic human glycosylation patterns.

Simple and reliable protocols allow for the fast generation of customized producer cell lines for pharmaceutically relevant proteins based on the parental permanent CAP cells under controlled and optimized conditions. For the required human cell line screening as well as for media opti-mization, the small working volumes of 100 – 250 mL make the extendable 4-fold DASbox and the BioBLu 0.3c Single-use Vessel a perfect fit. Bioprocesses are controlled as precisely and effectively as they are in larger scale bioreactors while cell material, media and supple-ments as well as lab space are saved.

Several experiments were carried out aiming at verifying the scale-down capabilities from the DASGIP Parallel Bioreactor System, which CEVEC gen-erally uses in process development, to the Mini Bioreactor System DASbox. To overcome the risk of cross-contami-nation and to reduce time for cleaning, sterilization and assembly they evalu-ated BioBLu 0.3c Single-use Vessels facilitating full industry standard instru-mentation.

A specifically designed port including a gas permeable membrane allows for DO measurement using a reusable probe which can easily be plugged in directly on the bench. Recuperation of liquid from exhaust gas is carried out via a liquid-free Peltier condenser.

materials and methods

To evaluate the scale-down capability of the DASbox Mini Bioreactor System and the usability of the BioBLu 0.3c Single-use Vessel experimental series with two different systems were carried out and compared. A 4-fold Parallel Bioreactor System for cell culture was used in 500 mL scale experiments (PBS).

The corresponding small-scale approach-es were carried out in a DASbox system

using Single-use Vessels with 170 mL (DASbox Su). The recombinant human CAP cells producing a pharmaceutically relevant protein were batch cultivated for 7 d (170 h) in CEVEC's serum-free, chemically defined CAP medium supple-mented with 40 mM glucose and 6 mM glutamine at 37 °C. Initial viable cell den-sity was 3 x105 cells/mL.

The DO set-point of 40 % was main-tained by a constant stirrer speed and the oxygen concentration in the inlet gas. Stirrer speed was adjusted to 160 rpm (PBS) and 150 rpm (DASbox Su).

The pH value was regulated to 7.1 by addition of 1 M Na2CO3 (feeding, speed rate regulated) and CO2 (submerged gassing). Inlet gas (air, O2, CO2 and N2) was mixed continuously mass flow-con-trolled. The bioreactors were equipped with pitched blade impellers and liquid-free operated exhaust gas condensers. The pre-cultures were cultivated in 125 mL Erlenmeyer flasks (Corning®) with 25 mL working volume using a shaker incubator (37 °C, 5 % CO2) agi-tating at 185 rpm (Multitron® 2, Infors®

AG). The cells were expanded up to a viable cell density of 3 x 106 cells/mL in the same medium used for bioreactor runs.

CLAuDIA M. HüTHER-FRANkEN*, EPPENDORF AG BIOPROCESS CENTER, JuELICH, GERMANY

HELMuT kEWES AND MICHAEL SCHOMBERG, CEVEC PHARMACEuTICALS GMBH, COLOGNE, GERMANY

*CORRESPONDING AuTHOR: [email protected]

fig. 1: Viable cell numbers of all experiments with DASGIP Parallel Bioreactor Systems (PBS) and BioBLu 0.3c vessels with average growth rate of 0.02 h-1

via

ble

cel

l nu

mb

er (

ml-1

)

Process time (h)

viable cell number

8.0 E+06

7.0 E+06

6.0 E+06

5.0 E+06

4.0 E+06

3.0 E+06

2.0 E+06

1.0 E+06

0.0 E+00

Pbs-1

Pbs-2

Pbs-3

Pbs-4

dAsbox sU-1

dAsbox sU-2

0 20 40 60 80 100 120 140 160 180

(BN 41) JuLY 2014



The critical process parameters were monitored, controlled and visualized on-line while additionally offline parame-ters were added manually for collective analysis and storage in a joint database. Daily samples were taken in place.

Viable cell numbers, the concentrations of glucose as well as the target protein were determined via semi-automated trypan blue cell counting (Cedex® XS, Roche® Innovatis®), an automated glu-cose biosensor (YSI® 7100 MBS, YSI Life Sciences) and ELISA, respectively.

results and discussion

The highly comparable results shown in figures 1 and 2 prove the reliability of the process control in both indepen-dent experimental series. The viable cell density increases exponentially within all cultivation studies in a reproducible manner with an average growth rate of 0.02 h-1. The corresponding anti-cyclic glucose consumption thereby illustrates the similar metabolism of the different cultures. Cell viabilities ranged in be-tween 90 – 95 % for each sample.

As shown in figure 3 the final product yield reached 80 – 121 % in respect to the average protein concentration gained with the Parallel Bioreactor System (PBS) commonly used at CEVEC. No differ-ences in cell growth, metabolic activity and protein expression could be observed using the BioBLu 0.3c Single-use Vessels.The results show the successful scale-down from a 500 mL (PBS) to 170 mL (DASbox Su) bioreactor working volume.

Conclusion

Summarized, the presented results give direct evidence to the scale-down capability of the DASbox Mini Bioreactor System used with Single-use Vessels. This proves the DASbox to be a superi-or tool for process development with human cell cultures. The small working volumes save material and consumable costs while utilizing Single-use Vessels drastically reduces turnover-times and thereby labor costs and development times.

For detailed information please visit the »Bioprocess« section of your local Eppendorf website.

glu

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45

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fig. 2: Comparison of metabolic activity by glucose consumption

fig. 3: Product yield normalized to the average protein concentration gained using the DASGIP Parallel Bioreactor System (PBS)

readers' serviceBrochure »We know Bioprocessing« • Ref. no. 274




Abstract

In this study we investigated the effi-ciency of chemical decontamination methods against bacterial contamination of pipettes. It was shown that the effi-ciency of bacterial decontamination is influenced not only by the disinfectant but also by the instrument's material and design. The Eppendorf Reference® 2 pipette with its smooth PTFE-containing surface and a handle made in one pour, showed the most reliable efficiency in bacterial decontamination.

Introduction

Pipettes are basic tools in all kinds of labs. In contrast to consumables like tips and tubes, pipettes are not dis-carded after use but may need to be decontaminated frequently.

The presence of pathogens can have serious consequences on diagnostic results as well as on the user's safety. Autoclaving is a time-consuming process and not all pipette models can be auto-claved completely, only their lower parts. Thus most commonly the decontamina-tion is performed by intensive disinfec-tion of materials and surfaces. Here it is important that the disinfectant can reach all possibly contaminated areas. Some instrument suppliers offer equipment

with antimicrobial surfaces or a special design. An antimicrobial surface inhibits bacterial growth but does not avoid con-tamination with bacteria. A special pi-pette design, like that of the Reference 2, avoids insufficient decontamination by eliminating critical locations where dis-infection becomes difficult.

Adhesion and growth of bacteria are very complex processes influenced by many factors. One of these factors is the material the bacteria come into contact with. Here the physicochemical proper-ties as well as the surface topography and surface roughness play an impor-tant role [1].

Since adhesion and growth of different bacteria types vary on different materi-als, three different bacteria species were chosen to analyze the decontamination efficiency of different pipette models: Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa.

methods

The handles of four different pipettes were contaminated with bacteria solu-tions and dried for 1 h. Both sides of the handles were sampled by contact slides. A commercial bacterial disinfectant, Meliseptol® rapid (B. Braun® Melsungen AG) was used for decontamination.

The disinfectant was sprayed on a cloth and the pipette wiped clean. After disin-fection both sides of the handles were sampled by contact slides again. The number of colonies before and after decontamination were counted and compared.

In order to ensure confident results, each test was performed twice. In a sec-ond analysis the pipettes were contami-nated with E. coli and decontaminated with Meliseptol rapid. After disinfection three locations per pipette suspected to be favorable for bacteria growth were sampled using swabs. A contact slide was applied on the handles as described above.

For more detailed information please see Application Note No. 328 (in prepa-ration); available soon as download pdf at www.eppendorf.com/applications.


It was shown that the influence of the pipette model, driven by material and surface, plays a role in efficient cleaning (Table 1). Rougher surfaces, like those of competitors B and C, are more difficult to decontaminate. This influence rises with decreasing efficiency of disinfection (e. g. due to incorrect use of disinfectant; data not shown).

uLRIkE GAST, EPPENDORF AG, HAMBuRG, GERMANY

MuRIEL ART, EPPENDORF APPLICATION TECHNOLOGIES, NAMuR, BELGIuM

Degree of contamination by E. coli Degree of contamination by P. aeruginosa Degree of contamination by S. aureus

Test 1 Test 2 Test 1 Test 2 Test 1 Test 2

Pipette model Area sampled Before After Before After Before After Before After Before After Before After

Eppendorf Reference 2

Pipette 1 - Area 1 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0

Pipette 1 - Area 2 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0

Pipette 2 - Area 1 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0

Pipette 2 - Area 2 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0

Competitor A

Pipette 1 - Area 1 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0

Pipette 1 - Area 2 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0

Pipette 2 - Area 1 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0

Pipette 2 - Area 2 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0

Competitor B

Pipette 1 - Area 1 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0

Pipette 1 - Area 2 > 42 0 > 42 0 > 42 0 > 42 0 > 42 > 42 > 42 0

Pipette 2 - Area 1 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0

Pipette 2 - Area 2 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0

Competitor C

Pipette 1 - Area 1 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0

Pipette 1 - Area 2 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0

Pipette 2 - Area 1 > 42 1 > 42 0 > 42 0 > 42 0 > 42 0 > 42 0

Pipette 2 - Area 2 > 42 0 > 42 1 > 42 0 > 42 0 > 42 0 > 42 0

table 1: Number of colonies detected before and after Meliseptol rapid treatment. Note: P. aeruginosa were growing within a biofilm, a precise counting was impossible.

After decontaminationPipette model Location sampled

Eppendorf Reference 2

Handle 0

user adjustment display 0

Volume locking button 0

Ejection sleeve upper part 0

Competitor A

Handle 0

Volume adjustment 0

Counter display 0

Ejection button edges > 42

Competitor B

Handle 0

Volume adjustment 0

Counter display > 42

Edges connection of upper and lower part 3

Competitor C

Handle > 42

Volume locking 0

Counter display 0

Tip ejector / housing junction > 42

PAGE 4 (BN 41) JuLY 2014



When focusing on the design of pipettes it was found that E. coli bacteria hidden in cavities (e. g. counter display), grooves (e. g. housing junctions) or sliding parts (e. g. ejection button edges) survived the decontamination (Table 2).

These bacteria represent a contamina-tion risk on a disinfected pipette. The Reference 2 pipette is the only pipette for which no microorganisms were detected no matter which instrument part was sampled. This is the result of a smooth PTFE-containing housing made in one pour: The handle of the

literature

[1] katsikogianni M., Missirlis Y.F.: Concise review of mechanisms of bacterial adhesion to biomaterials and of techniques used in estimat-ing bacteria-material interactions. European Cells and Materials, 2004, Vol. 8, pp. 37– 57.

Reference 2 has no cavities or grooves, and its display for user adjustment is perfectly integrated in the housing (Fig. 1).

On all other tested pipette models bac-terial contamination after disinfection was observed. The handles of competi-tors A, B and C cannot be autoclaved. The remaining methods commonly used for decontamination are uV-irradiation or the use of disinfectants. However, a decontamination of certain locations by using disinfectants is obviously in-sufficient (Table 2).

Conclusion

From the present data it can be conclud-ed that the pipette model influences the efficiency of bacterial decontamination. This is driven on the one hand by the instrument's material and its surface roughness/topography. On the other hand the design of a pipette itself plays an important role.

In general, the less effective the disinfec-tion method, the bigger the influences of the pipette's material, surface and design become. When pipettes are used in areas with bacterial contamination, especially in Class A/B rooms, instru-ments with non-porous surfaces and a design without grooves and angles are recommended.

4

2

1

3

table 2: Number of E. coli colonies detected on critical locations of four pipette models after Meliseptol rapid treatment and according to pipette location

readers' serviceEppendorf Reference® 2 • Ref. no. 269

fig. 1: Locations on the Reference 2 pipette where samples were taken:

1 – Handle 2 – user adjustment display 3 – Volume locking button 4 – Ejection sleeve upper part



DNA Purification from Sample Material Obtained from a Selection of Different Marine Animals Using the epMotion® 5075 VAC

Introduction

DNA purification from marine animals such as corals, crustaceans, echinoderms and sponges is exquisitely difficult. Final-ly, this purification was successfully performed using the MACHEREY-NAGEL NucleoSpin® 8/96 Tissue kit on the epMotion® 5075 VAC.

The NucleoSpin kit is based on vacu-um filtration with binding, wash and elution steps. This application aids in making a statement about changes in ecosystems and about marine animal populations.

The yield and purity of earlier attempts tended to be of very low quality. Prob-lems arise from the pigments, keratins and sponge-needles of the invertebrate life forms.

Studies of the genetic populations of invertebrate animals can only be per-formed with very specific genetic mark-ers, which can only be amplified from high quality purified DNA.

Purification using the NucleoSpin 8/96 Tissue kit on the epMotion 5075 VAC with integrated vacuum station yields very good quality DNA. Automation of the kit enables processing of a large number of samples within a short period of time while achieving reproducible results.


For our experiments, an epMotion 5075 VAC with the respective accessories was employed, in combination with either the MACHEREY-NAGEL NucleoSpin 8 Tissue kit or the NucleoSpin 96 Tissue kit, depending on the number of samples to be purified.

The following samples, conserved in ab-solute alcohol at –20 °C, were used:

> 20 mg tissue from the red coral (Corallium rubrum)

> 20 mg tissue from the violescent sea whip (Paramuricea clavata)

> 20 mg tissue from the yellow sea whip (E. cavolinii)

> 30 mg tissue from the white sea fan (E. singularis)

> 45 mg tissue from the sponge (Spongia sp.)

> 3 mg tissue from the lobster (Hemimysis margalefi)

> 45 mg tissue from the smooth brittle star (Ophioderma longicauda)

The samples were first filled into a MA-CHEREY-NAGEL Tube-Strips plate. PBS buffer was then pipetted into the indi-vidual samples by the epMotion.

The different samples were subsequent-ly broken up using the Tissue Lyser II (QIAGEN®). Following brief centrifuga-tion, the plate was placed back into the

epMotion. Now the lysis buffer was added in an automated fashion. The plate was subsequently removed from the epMotion, sealed, centrifuged and mixed over night at 56 °C and 600 rpm on the Eppendorf ThermoMixer® comfort.

Note: If an epMotion with integrated thermomixer and vacuum station is used, the entire process may be carried out on the epMotion.*

The following morning, the plate was centrifuged for 10 minutes before it was placed back into the epMotion. In the following step, supernatant from the lysis plate was transferred into a MACHEREY-NAGEL square-well block using the epMotion. Subsequently, the binding buffer and ethanol were added. This mixture was pipetted into a Nucleo-Spin Tissue Binding Plate (vacuum sta-tion), followed by several wash and vac-uum steps at 400 millibar for 2 minutes. Several vacuum and pipetting steps were performed during the purification process. Following the final wash step another vacuum step of 10 minute dura-tion was carried out in order to remove residual ethanol from the filter.

In the meantime the elution buffer was pre-warmed to 70 °C (thermo module). In order to optimize yield the elution step designed to release the DNA from the filter was carried out in two steps.

CECILE REBOuT, uMR IMBE, SCBM, MARSEILLE, FRANCE

CHRISTOPHE CARPENTIERI, MACHEREY-NAGEL, MARSEILLE, FRANCE

fig. 1: Overview of the DNA purities (A260/A280) obtained for the individual samples. The A260/280 ratio achieved a mean of 1.83.

fig. 2: Overview of the mean total DNA yields for the individual samples

dNA purity

2.5

2

1.5

1

0.5

0

C. ru

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A26

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Sample Initial weight (mg)

n DNA purity A260/A280

Mean DNA concentration

(ng/µl)

Mean total yield (µg)

C. rubrum 20 8 1.95 34.51 6.90

P. clavata 20 40 1.90 36.42 7.28

E. cavolinii 20 88 1.98 45.43 9.09

E. singularis 30 15 2.04 110.03 22.01

Spongia sp. 45 14 1.95 114.16 22.83

H. margalefi 3 8 1.53 8.29 1.66

O. longicauda 45 8 1.91 74.6 14.92

PAGE 6 (BN 41) JuLY 2014


Yield and purity

Purity and concentration of the samples were determined using the Eppendorf BioPhotometer® plus in combination with a Hellma® TrayCell® cuvette. 4 µL of each sample were used for this measure-ment. Purity was determined by the ratio A260/A280, and concentration was measured by absorbance at 260 nm.

PCR analysis

Amplification of the samples derived from each species was carried out using the universal Primer COI Iocus (LCO1490/HCO2198). A mastermix (Green GoTaq® Flexi Buffer and GoTaq®

DNA polymerase by Promega®) and 2 µL genomic DNA were added.

In the second step, the respective am-plifications were carried out on the Eppendorf Mastercycler® gradient pro S with species-specific markers.

DNA Purification from Sample Material Obtained from a Selection of Different Marine Animals Using the epMotion® 5075 VAC

Agarose gel electrophoresis

Analysis of the PCR results occurred on a 1 % TBE agarose gel.

Cross contamination

Possible cross contaminations were detected by the inclusion of negative control samples in each extraction procedure.


DNA concentration und purity

The samples of the different invertebrate marine animals could be purified in a very efficient manner on the epMotion in combination with the NucleoSpin kits. The method delivered consistently highly concentrated DNA results of very high purity and without major protein contam-ination. The A260/A280 ratio achieved a mean value of 1.83 for all tissue samples tested (Table 1; Fig. 1 and 2).

Cross contamination

No DNA could be detected in either of the negative control samples; neither in the PCR reaction, nor during photo-metric measurement.

PCR results

The results of the universal COI PCR kit showed no degraded DNA samples (Fig. 3).

Fragments of ~700 bp were amplified from samples derived from each marine animal. The use of these non-specific primers explains the presence of non-specific bands.

Conclusion

The combination of the epMotion VAC and the MACHEREY-NAGEL Nucleo-Spin 8/96 Tissue kit has delivered very good results which are of high quality with respect to yield and purity. The NucleoSpin 8 Tissue kit is recommend-ed for a smaller number of samples, whereas the NucleoSpin 96 Tissue kit is ideal for higher sample throughput.

The DNA of the invertebrate marine animals is of exceptional quality and suitable for a number of downstream applications such as PCR or DNA se-quencing.

C.r P.c E.c E.s S.s H.m O.l Extraction control

PCR control

* Since 2014 the epMotion VAC has been sold under the product name epMotion 5075v. The Version 5075vt con-tains an additional integrated thermomixer (see fig. 4).

readers' serviceepMotion® • Ref. no. 254

fig. 4: epMotion 5075vt with integrated vacuum station and thermomixer

table 1: DNA concentration and purity

fig. 3: Agarose gel electrophoresis of the amplified DNA (one sample per species; extraction and PCR controls).

The Smartladder by Eurogentec® served as the size standard.



Cultivation of Prostate Cancer Cells under Hypoxic Conditions in the New Brunswick™ Galaxy® 170 R CO2 Incubator from Eppendorf

STACEY S. WILLARD, LINETTE PHILIP AND MA SHA

EPPENDORF, INC., ENFIELD, CT, uSA

Introduction

Oxygen is a critical regulator of cellular homeostasis and as such, oxygen depri-vation is lethal to normal cells. Decades of cancer research have established that tumor cell growth, survival, motil-ity, the recruitment of blood vessels (angiogenesis), energy metabolism and cellular differentiation are affected by the surrounding oxygen level [1].

LNCaP, a prostate cancer cell line, has been widely used in drug discovery and is uniquely sensitive to androgen levels. It has been shown that androgen stimu-lated LNCaP cells grow faster under hypoxic conditions (2 % O2) than cells maintained under normal oxygen condi-tions (20.9 % O2) [2].

We show here that low O2 cell culture conditions are easy to establish using the New Brunswick Galaxy 170 R CO2 incubator from Eppendorf with the 1– 19 % O2 control option. Although in our experiment LNCaP cells grown at 2 % O2 showed the same growth dynam-ics as cells cultured under normal O2 conditions, they displayed a significant morphology difference.


Two Eppendorf New Brunswick Galaxy 170 Liter incubators were used: The Galaxy 170 R variant with high temper-ature disinfection, 4-split inner door and the oxygen control option from

1– 19 % O2 was used for hypoxic condi-tions (Fig. 1).

The Galaxy 170 S variant with high tem-perature disinfection and 4-split inner door was used for normoxic conditions. Setpoints were 37 °C and 5 % CO2, re-spectively. For hypoxic conditions, the Galaxy 170 R was used at 2 % O2 and allowed to stabilize at setpoint for 72 h before cells were introduced into the in-cubator. using Eppendorf BioCommand®

SFI, O2, temperature and CO2 process values were tracked for the course of the experiment. The O2 concentration inside the Galaxy 170 R never exceeded 2.1 %.

LNCaP clone FGC cells were grown in appropriate medium in T75 flasks until 80 % confluency. At target density, the cells were seeded in quadruplicate 6-well dishes at a density of 300,000 cells/well and two plates were placed in the same position in both the normoxia and hypoxia incubators. Cells were counted and photographed every 3 days for 9 days. After 6 days, one well from each treatment was stained using the phallotoxin, phalloidin (which selectively binds f-actin) and the double-stranded DNA intercalator, 4',6-diamidino-2- phenylindole (DAPI).

For detailed information on procedures, reagents and instruments used, please see Application Note 331 document available at www.eppendorf.com/ applications).


LNCaP cells grown at 2 % O2 showed growth dynamics indistinguishable from those grown in normal atmospheric O2 (20.9 %) (Fig. 2).

It is possible that we did not see the pre-viously published growth bias in hypoxia because we did not stimulate the cells with androgens prior to exposure to O2 deprivation. Importantly, no differences were seen in the attachment of the cells in hypoxia, as evidenced by the phase contrast micrographs in Fig. 3.

Interestingly, we noted a significant morphology difference between the cells grown in hypoxic and normoxic conditions. Grown in atmospheric O2 conditions, LNCaP cells are known to form large clusters or colonies of cells wherein they display no contact inhi-bition.

These colonies are themselves loosely attached to the substrate and can be-come quite large. In contrast, LNCaP cells grown in 2 % O2 do not form such 3-dimensional structures and tend to cover the substrate and grow in more of a packed monolayer.

To document these morphological changes, we stained cells grown in both O2 concentrations with rhodamine-con-jugated phalloidin and DAPI to visualize the actin cytoskeleton (red, Fig. 4) and the cell nucleus (blue, Fig. 4).

growth of lNCaP cells in hypoxic and normoxic conditions

time (days)

Normoxia Hypoxia Normoxia Hypoxia

via

ble

cel

ls /w

ell

% v

iab

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2.5E+06

2.0E+06

1.5E+06

1.0E+06

5.0E+05

0.0E+00

100

90

80

70

60

50

40

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00 3 6 9

fig. 1: The New Brunswick Galaxy 170 R CO2 incubator

fig. 2: Growth and viability of LNCaP cells grown in hypoxia and normoxia. This graph shows the viable cell density and % viable cells in each well. Each data point represents the mean of 3 wells. Error bars indicate standard error of the mean.

ANOVA analysis revealed that no significant dif-ference was observed between the two growth conditions.

(BN 41) JuLY 2014


Cultivation of Prostate Cancer Cells under Hypoxic Conditions in the New Brunswick™

Galaxy® 170 R CO2 Incubator from Eppendorf

As documented in Fig. 4, large clusters of cells are observed in normoxic con-ditions while a flatter monolayer is seen in hypoxic conditions. Furthermore, the cytoplasm-to-nucleus ratio in the cells grown in 2 % O2 appears to be larger than in those grown in 20.9 % O2, al-though this observation was not quan-titated. We conclude that morphological changes have occurred in LNCaP cells grown with O2 deprivation that result in diminished colony forming behavior and a flatter appearance.

Conclusion

We have shown that LNCaP cells grown in hypoxic conditions display normal growth as compared to those grown in atmospheric O2 concentrations.

Growth in 2 % O2 also resulted in mor-phological changes and changes in growth characteristics such as colony formation.

The ease of setup and the tight O2 con-centration control displayed by the Galaxy 170 R incubator provided ideal conditions for this experiment.

literature

[1] kim Y, Lin Q, Glazer PM, Yun Z. Hypoxic tumor micro environment and cancer cell differ-entiation. Curr Mol Med 2009; 9(4):425-434.

[2] Danza G, Di Serio C. Rosati F, Lonetto G, Sturli N, kacer D, et al. Notch signaling modu-lates hypoxia-induced neuroendocrine differ-entiation of human prostate cancer cells. Mol Cancer Res 2012; 10(2):230-238.

day 3 day 6 day 9

Nor

mox

iah

ypox

ia

readers' serviceNew Brunswick™ Galaxy® 170 R CO2 Incubator • Ref. no. 273

fig. 3: LNCaP cells grown in hypoxic conditions display normal attachment and homogenous growth. In the top row, LNCaP cells grown in 2 % O2 show similar density as compared to those grown in atmospheric O2 (bottom row). Photos were taken at 100 x magnification and the scale bar in each panel represents 100 μm.

fig. 4: Growth characteristics of cells in hypoxia and normoxia. Panels A (fluorescence) and B (phase contrast) show examples of cells grown in 20.9 % O2 for 3 days. The yellow arrowheads denote areas where 3-dimensional colonies are observed, as evidenced by the overlapping cell nuclei (blue) and high concentration of actin (red). Panels C and D show monolayers grown in 2 % O2 where 3-dimensional growth is not seen. In addition, cells grown in hypoxia appear flatter and with larger cytoplasmic volume. The images in this figure were collected at 100 x using an EVOS® LED imaging system; scale bars represent 200 μm.

A b C d

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in cell culture labs. They feature complex,

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* Performance Plans (also for other Eppendorf products) are available in selected countries only.

9

A Great Team for Cell Culture Applications

BRIGITTE kLOSE, EPPENDORF AG

A GREAT TEAM FOR CELL CuLTuRE APPLICATIONS · NEws

A completely new series of serological pipettes is expanding our portfolio

of liquid handling tools for cell culture applications. the new Eppendorf

serological Pipets are a perfect complement to the Easypet® 3 pipet con-

troller. the new team allows you to tackle your cell culture liquid handling

tasks with speed, safety, precision and comfort.

Premium performance

The new Eppendorf Serological Pipets work in complete harmony with your Easypet 3. They have clear and precise graduations for fast and easy volume determination. A color-coding system ensures easy identification of the desired pipet volume.

Premium material

The pipets are made from ultra-clear virgin polystyrene, complying with uSP class VI plastic tests. Each pipet has a sterility assurance level of 10-6. Eppendorf Sero-logical Pipets are certified non-cytotoxic and free from detectable pyrogens, DNA, RNase and DNase.

Premium packaging

Eppendorf Serological Pipets are available in 1 mL, 2 mL, 5 mL, 10 mL, 25 mL and 50 mL versions. They are individually wrapped and packaged in a robust dis-penser box to ensure consistent premium quality.

For more information on our new great team for your cell culture liquid handling, please visit www.eppendorf.com.

Or order the current brochure using the reference number denoted below.

Easypet® 3 & Eppendorf Serological Pipets • Ref. no. 250

Eppendorf Serological Pipets: available in 6 sizes; color-coding system allows easy identification of the pipet size.

Robust packaging with dispenser function to ensure convenient storage and reliable usage

Easypet 3: it has never been easier to combine speed, safety, precision and comfort!

10

epMotion® – Automated Liquid Handling at its Best

CARSTEN BuHLMANN, EPPENDORF AG

strAIght from thE lAb · epMotion® – AuTOMATED LIQuID HANDLING AT ITS BEST

Whether it is molecular biology applica-tions, like PCR set-up, SNP detection, next generation sequencing, or nucleic acid purification, complex assay set-up or cell culture applications, scientists worldwide love Eppendorf's epMotion systems! With an epMotion system all your routine pipetting tasks can be automated with more accuracy and reproducibility than you might have experienced with manual pipetting.

epmotion® 5070

The most compact solution for accurate and reproducible automated pipetting. A perfect match for any routine application such as serial dilutions, reagent distribu-tion, sample transfer from tubes to plates, and sample normalization.

Product features epMotion 5070 / 5070f

> 4-position worktable and 3 virtual positions

> Small footprint of 65 × 48 cm > Space saving touch screen tablet > Automatic tool exchange for 2 tools > Optical sensor (verification of set-up before the run)

epmotion® 5073

uniquely tailored for PCR setup and nu-cleic acid purification, yet flexible enough to be used as open platforms for diverse automated liquid handling applications. These systems automate and simplify traditionally complex, labor intensive pipetting tasks, saving time and improving the reliability and reproduc-ibility of results.

Product features epMotion P5073 / M5073

> 6-position worktable > Application specific software assistants, tools and accessories included

> Option for gripper, 1 thermal module* or Eppendorf MagSep™ module**

> CleanCap option for uV decontamination and HEPA air filter

* Available only with P5073 **Already included with M5073

epmotion® 5075

With 12 to 15 worktable positions and many additional features the epMotion 5075 versions have a higher application flexibility.

Perfectly suited for demanding, small-volume applications such as real-time PCR set-up, magnetic bead based nucle-ic acid purification, next generation se-quencing as well as cell assays or any routine pipetting task.

Product features epMotion 5075l / 5075t / 5075v /5075vt / 5075m

> up to 15 worktable positions > Eppendorf MultiCon PC controller with simulation, network and software upgrade options

> Automatic exchange of 4 dispensing tools

> Option for gripper and 1–3 thermal modules

> System control by touch screen, mouse, keyboard or network

> Thermomixer®, Vacuum manifold and magnetic separation options

> Available as CleanCap versions

Automation means precision and reduced workload in one – discover the possibili-ties now for your lab, too!

The new epMotion family brochure »Friends for Life« (Ref. no. 254) includes all details.

epMotion® Family brochure • Ref. no. 254

11

New Systems for Micromanipulation of Cells

NICOLE LESSNER, EPPENDORF AG

NEW SYSTEMS FOR MICROMANIPuLATION OF CELLS · INNovAtIoN

since 1984 Eppendorf's automated microinjectors and micromanipulators have been synonymous with reliable

systems solutions which, thanks to their co-operative interaction, guarantee an ergonomic workflow. the innovative

youngest generation of products, comprising the micromanipulators Injectman® 4 and transferman® 4r, as well as

the microinjectors femtoJet® 4i and femtoJet® 4x, allow many experiments, e. g. in the areas of suspension cell

manipulation, adherent cell injection and developmental biology, to be carried out more precisely, as well as in a

safer and simpler manner than ever before.

Micromanipulation of cells has retained its place in the laboratory routine. On the other hand, increasingly sophisticated questions are being addressed, and the work environment is becoming more complex.

As a result, increasing demands are placed on the micromanipulation work station. Work processes are to be optimally sup-ported by the micromanipulation system; at the same time the workspace has to be ergonomic and safe, ensuring rapid work which is gentle on the cells.

Ergonomic and gentle

In accordance with the Eppendorf Physio-Care Concept® the TransferMan 4r and InjectMan 4 not only satisfy by means of their excellent stability and wide range of functionalities. They also support your

application through an innovative user interface and pre-programmed methods. The ergonomically designed operating console with its clearly structured dis-play shows all important parameters at one glance. Their easily legible scaling allows the compact motor modules to offer rapid, reproducible adaptation to a variety of microscope setups.

Clearly laid out operating elements allow quick selection of the injection angle be-tween 0 ° and 90 °. Changing the sample is accomplished by simply swinging out the motor module, supported by the de-vices' home function. Furthermore, the TransferMan 4r and InjectMan 4, as well as the injectors FemtoJet 4i and FemtoJet 4x convince through their very low noise emission, thus contributing considerably to a stress-free laboratory environment.

Invincible as a team

In combination with the FemtoJet 4i or FemtoJet 4x the TransferMan 4r and InjectMan 4 offer an ideal systems solution. Injection may be triggered on the injec-tor, as well as on the manipulator. Newly introduced: the electronic connection between TransferMan 4r and FemtoJet, which optimally supports pronucleus in-jection for the purpose of generating transgenic animals.

In order to be able to work independent-ly from external pressure sources, the FemtoJet 4i is equipped with an internal compressor. It is especially well suited for precise injection of smallest volumes up to approximately 100 pL. The FemtoJet 4x, in combination with an external pressure source, is ideally suited for serial injections of volumes of approximately 100 pL, up to 1 µL or more.

TransferMan® 4m/r and InjectMan® 4 • Ref. no. 266 FemtoJet® 4i/4x • Ref. no. 271

Swinging out the motor module for easy capillary exchange

tip

Go Farther with Us!

In our newly equipped seminar rooms at

our Hamburg location, in the modern

Training Center in kuala Lumpur (Malaysia),

at the renowned European Molecular

Biology Laboratory (EMBL) in Heidelberg,

Germany, as well as at local Eppendorf

organizations, we offer a diverse training

program.

Product specialists with broad experience

support you

In small groups, experienced instructors

convey basics from everyday laboratory

routine, as well as new technologies, in

theory and practice. They also support

refreshment of existing knowledge in the

areas of liquid handling, laboratory automa-

tion, cell technology and PCR and detection.

In addition to liquid handling customer

trainings, certain Eppendorf organizations

also offer individual training in the areas of

cell technology for specialized techniques

in human and animal reproductive medi-

cine upon request.

Profit from the high competence of the

Eppendorf training Center

Detailed information about course selec-

tion, as well as the application form, is

available on the local Eppendorf websites.

For individual training, please contact your

local organization directly.

12

Perfect Asymmetry: Mastercycler® nexus X2

kAY köRNER, EPPENDORF AG

INNovAtIoN · PERFECT ASYMMETRY: MASTERCYCLER® NEXuS X2

flexible in many respects

The Mastercycler nexus X2 features two asymmetric blocks, consisting of 64 and 32 wells respectively, which can be pro-grammed and run completely indepen-dently. Smaller assays fit nicely on the 32-well block; larger assays can exceed 48 samples and run on the 64-well block.

The larger block is also available with a gradient function for PCR optimization. Both blocks accommodate 0.1 mL and 0.2 mL PCR strips, 0.2 mL and 0.5 mL PCR tubes as well as divisible Eppendorf twin.tec® PCR Plates. No matter what type of consumable is used, the flexlid® concept enables an automatic height ad-justment of the lid.

Ideal networker

The Mastercycler nexus X2 can be com-bined with other Mastercycler nexus models in a network of up to three units. This way you can increase your through-put and benefit from the same intuitive software on all your PCR cyclers. Like the Mastercycler nexus, the X2 is able to send a status e-mail to you.

Comfortable to work with

With reduced noise emission (<40 dB), low power consumption and a small, well-designed footprint, the Mastercycler nexus X2 is perfectly suited for use in laboratories with many users. In com-parison to other dual block cyclers, it provides an elegant solution for users wishing to run procedures with a large number of samples, without taking up a large amount of bench space.

In nature, functional asymmetry is a well known phenomenon. We are confident that it will also work in your lab – in the form of the new Mastercycler nexus X2!

For more information, please order the current brochure or visit www.eppen-dorf.com/mastercycler.

many of our customers have expressed a need for a multi-block cycler that

can accommodate more than 48 samples in a single block. In response to

this, we developed the mastercycler nexus X2 with its asymmetric blocks.

the new mastercycler nexus X2 is ideal for researchers who want to carry

out two separate PCr runs simultaneously, without any compromise on the

number of samples.

Perfect asymmetry: The new Mastercycler nexus X2 features a 64-well and a 32-well block – the ideal multi-block solution for simultaneous PCR runs.

Mastercycler® nexus X2 • Ref. no. 272

13

Eppendorf Research Prizes: Winners 2013

BERRIT HOFF AND CAROLYN TAuBERT, EPPENDORF AG

EPPENDORF RESEARCH PRIZES: WINNERS 2013 · NEws

The Israeli scientist, Michael Yartsev, Ph.D., CV Starr Postdoctoral Research Fellow of the Princeton Neuro-science Institute at Princeton university, uSA, has won the 2013 Eppendorf & Science Prize for Neurobiology endowed with uS $ 25,000.

Michael Yartsev uses an unusual animal model, the bat, to study the underlying neural mechanisms of spatial memory and navigation in the mammalian brain. The use of the bat, along with the development of techniques to record the activity of single cells in brains of these flying mammals, allowed him to conduct a comparative exami-nation of current hypotheses as well as to provide novel insights into the neural codes underlying the representation of three-dimensional space in the brain. His work also underscores the potential benefits of using new animal models in neuroscience.

More information at www.eppendorf.com/prize

The British scientist Ben Lehner, Ph.D., ICREA Research Professor, EMBL-CRG Systems Biology unit at the Centro de Regulación Genómica, in Barcelona, Spain, has won the 2013 Eppendorf Award for Young European Investigators. Ben Lehner received the 15,000 Euro Eppendorf Award for his discoveries concerning the fundamental question why mutations in the genome result in variable phenotypes.

His work has contributed to the understanding how the function of genes is modulated during development by environmental factors and by the interplay with other gene products. The results obtained by Ben Lehner are ground-breaking and offer novel approaches towards the understanding of genetic predisposition for diseases, particularly cancer. The insights obtained by his work are likely to lead to the development of novel therapeutic strategies.

More information at www.eppendorf.com/award

with the aim of encouraging and supporting outstanding work of promising young scientists, Eppendorf awards

two research prizes: the international Eppendorf & Science Prize for Neurobiology (since 2002) and the Eppendorf

Award for Young European Investigators (established 1995). over the years, both prizes have been awarded to an

impressive list of winners. the prize winners 2013 are michael Yartsev and ben lehner.

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Michael Yartsev Ben Lehner

Prize Competition

sErvICE · PRIZE COMPETITION14

1 2 3 4 5 6 7

8 9 10

11 12 13

14 15

16

17 18 19

20 21 22 23

24 25 26 27

28 29 30

31 32 33 34

35 36

37 38 39

40 41 42

ACross1 A person one likes and enjoys being

with4 Part of an airplane wing8 Roman goddess of the hunt

11 Chemical symbol for bromine13 Postal abbreviation of Montana 14 Primate species mainly living in

Madagascar (sing.) 15 Felis catus (sing.) 16 O2

17 Chemical symbol for rhenium18 Versus (abbrev.)19 Small constellation21 Irish for Ireland24 Female given name of Greek origin

25 Apparatus/organelle found in eukaryotic cells

29 Single Nucleotide Polymorphism (abbrev.)

30 Musical drama31 Prefix denoting one billionth32 Spanish aunt34 ante meridiem (abbrev.)35 Lacking motion or activity37 Ring-shaped coral reef encircling a

lagoon 39 Chemical symbol for calcium40 Chemical symbol for lutetium41 Folk hero of Switzerland (family name)42 French street

dowN1 Chemical symbol for iron2 Postal abbreviation of North Dakota 3 The length, width, height, or depth

of something 5 Releasable uV-absorbing substance 6 Valuable Moroccan oil 7 Vessel used for cell cultures9 Atmosphere (abbrev.)

10 Term from DNA chip technology11 Muhammad Ali was one12 Beam, line of light14 Specific height (pl.) 16 Made or produced first19 French male article (sing.)20 Something shocking, upsetting or

unacceptable

22 General term for a snake, lizard or alligator (sing.)

23 Oriental cat25 Target, objective26 Abbreviation for opus27 Inventor of a differentiating staining

method for bacteria28 Short form of Pamela 33 Postal abbreviation of Illinois35 ISO country code of Somalia 36 Solid piece, can be made of e.g. gold

or candy 38 French personal pronoun

disclaimer and trademarks Information

The solution of the prize competition of BioNews No. 39 was »Eppendorf Thermomixer«. Walter Gilbert (Small Animal Clinic at the university of Giessen, Germany) won the first prize.

have fun in our new crossword!

How to find out the solution: Simply arrange the letters in the light grey boxes of the crossword in the correct order. Send us the solution until 30th November 2014.

You can either use the reply fax (p. 15), send us an e-mail to [email protected], or participate online at www.eppendorf.com/bn-service.

All correct answers will be considered for a prize. Winners will be notified in writing. Cash payment of the prize is not possible. No recourse to legal action. The judges' decision is final. Eppendorf employees and their families may not participate. The winner of the first prize will be published in BioNews No. 43.

1st Prize:1 Multipette® M4* with 2 sets of Combitips advanced® of your choice *(u.S./CAN: Repeater® M4)

2nd to 5th Prize:1 Amazon® Voucher worth 50 Euros

6th to 15th Prize:400 bonus epPoints® each

epMotion® M5073 and 5075m: This product and its use may be covered by one or more patents owned by Gen-Probe Incorporated. The purchase price for this product includes only limited, nontransferable rights under certain claims of certain patents owned by Gen-Probe Incorporated to use this product for research purposes only. No other rights are conveyed. Purchaser is not granted any rights under patents of Gen-Probe Incorporated to use this product for any commercial use. Further information regarding purchasing a license under patents of Gen-Probe Incorporated to use this product for any other purposes, including, with-out limitation, for commercial use, may be obtained by contacting Gen-Probe Incorporated, Attn: Business Development Department, 10210 Genetic Center Drive, San Diego, California 92121-4362, u.S.A.

Amazon® is a registered trademark of Amazon Tech., Inc., uSA. B. Braun® and Meliseptol® are regis-tered trademarks of B. Braun Melsungen AG, Germany. Cevec® and CAP® are registered trademarks of CEVEC Pharmaceuticals GmbH, Germany. Corning® is a registered trademark of Corning, Inc., uSA. Eurogentec® is a registered trademark of Eurogentec S.A., Belgium. Hellma® and TrayCell® are regis-tered trademarks of Hellma Analytics, Germany. Infors® and Multitron® are registered trademarks of Infors AG, Switzerland. EVOS® is a registered trademark of Life Technologies Corp., uSA.

NucleoSpin® is a registered trademark of MACHEREY-NAGEL, Germany. GoTaq® and Promega® are registered trademarks of Promega Corp., uSA. QIAGEN® is a registered trademark of QIAGEN Group, The Netherlands. Cedex®, Innovatis® and Roche® are registered trademarks of Roche Diagnostics GmbH, Germany. YSI® is a registered trademark of Xylem, Inc., uSA. CAP-T™ is a trademark of CEVEC Pharma-ceuticals GmbH, Germany.

Eppendorf®, the Eppendorf logo, BioBlu®, Biopur®, Combitips advanced®, Easypet®, epMotion®, Eppendorf BioPhotometer®, Eppendorf LoBind®, Eppendorf PhysioCare Concept®, Eppendorf Reference®, Eppendorf Thermomixer®, Eppendorf ThermoMixer®, Eppendorf Tubes®, Eppendorf twin.tec®, epPoints®, the epServices® logo, epT.I.P.S.®, FemtoJet®, flexlid®, InjectMan®, Mastercycler®, Multipette®, OptiTrack®, Repeater®, Thermo-mixer® and TransferMan® are registered trademarks of Eppendorf AG, Germany. ConvexAccess™, Eppendorf MagSep™, Eppendorf Plates™, Eppendorf Quality™, New Brunswick™ and SplashProtect™ are trademarks of Eppendorf AG, Germany. DASbox®, DASGIP® and DASware® are registered trademarks of DASGIP Information and Process Technology GmbH, Germany. BioCommand® and Galaxy® are registered trademarks of Eppendorf, Inc., uSA. u.S. Design Patents are listed on www.eppendorf.com/ip. Copyright © July 2014.

Please send me information on the following products: Kindly help us to update your subscription details:

250 Easypet® 3 and Eppendorf Serological Pipets Please change my details (see above).

254 epMotion® M5073 / P5073 I am a new subscriber! Please send me Eppendorf BioNews on a regular basis (2 issues per year, free of charge).264 Eppendorf Tubes® 5.0 mL

266 TransferMan® 4m/r and InjectMan® 4 I have comments on bioNews No. 41:

269 Eppendorf Reference® 2

270 Eppendorf Cell Culture Consumables

271 FemtoJet® 4i /4x

272 Mastercycler® nexus X2

273 New Brunswick™ Galaxy® 170 R CO2 Incubator

274 Brochure »We know Bioprocessing«

Eppendorf Catalog 2014

Catalog 2014: Bioprocess products

Catalog 2014Bioprocess products

We know Bioprocessing

15

E R C

REPLY FAX / READERS' SERVICE · sErvICE

solution of bioNews No. 41 prize competition:

To Eppendorf AG, Hamburg, Germany, Attn. Carolyn Taubert, Fax (+49) 40 - 5 38 01- 8 40(Please – print or type, no private address)

Company / university / Clinic Title / Name

Institute Address

Department / Function Address

E-Mail Telephone

Closing date: 30th November 2014 Visit www.eppendorf.com/bn-service to participate online.

visit www.eppendorf.com/bn-service to subscribe or to request literature online!

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The New Eppendorf Cell Culture Consumables

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