N° 17

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TECHNICAL NOTES & APPLICATIONS FOR LABORATORY WORKCONTENT
1. Key Facts
2. Introduction
3. Culture of spheroids andstem cell aggregates

CELLSTAR® CELL CULTURE
VESSELS WITH
CELL-REPELLENT SURFACE
1/ KEY FACTS/

Effectively prevents the process of cell attachment/

For suspension culture of semi-adherent and adherent cell lines/

Ideal surface for spheroid formation/

Perfect for the formation of stem cell aggregates/

Non-cytotoxic/

Free of detectable endotoxins/

Free of detectable DNase / RNase and human DNA/Availble in various dish, flask and plate formats/Sterile, individually wrapped, easy to open

4. Inhibition of cellattachment of semiadherent and adherent
cell lines in vessels withcell-repellent surface
5. Ordering Information
6. Literature

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N° 172/ INTRODUCTION
For most applications in cell culture, the properties of thevessels to be used are optimised to enhance conditions
for cell attachment. With platforms made of polystyrene,
this is achieved by using a physical method to treat thesurface of the vessels. This treatment leads to the incorporation of polar carboxyl and hydroxyl groups to thehydrophobic polystyrene surface resulting in improved
and very regular cell attachment. For fastidious cell lines
or applications that will stress the cells, protein-coatedsurfaces or advanced non-biological surfaces are applied.
Nevertheless, for some applications a surface that prevents
cell attachment is required. These applications include:/

Spheroid culture/

Aggregation of stem cells/

Suspension culture of semi-adherent and adherent cell lines

The CELLSTAR® cell-repellent surface from Greiner BioOne fulfils these demands. Achieved through an innovative
chemical surface modification, Greiner Bio-One’s cellrepellent surface is an ideal substrate for cell culture
as it does not degrade or ‘leach out’ under standard cellculture conditions.

AAll cell culture vessels with a cell-repellent surface aresterilised by irradiation (SAL of 10 -3). They are quality
controlled for the absence of detectable endotoxins,
DNase/RNase and human DNA and show no cytotoxiceffects. Evaluation of cytotoxicity is done in accordance
to EN ISO 10993-5 with mammalian cell lines.
More detailed information concerning general quality
aspects can be found on the Greiner Bio-One website
www.gbo.com/bioscience and in our product catalogue.
To control the performance of the surface of cell-repellent
cell culture vessels, attachment of cells is compared to thestandard tissue culture CELLSTAR® surface. CaCo-2 cells
are seeded at a density of 18,000 cells/cm2 and incubated for 24 hours at 37 °C in an incubator with a 5 % CO2

environment.

After 24 hours of incubation, the medium is removed,
the vessels are washed with PBS and EtOH is added to fix
adherent cells, if present. The sample vessels are thenexamined and compared under a microscope with 10-foldmagnification (Fig. 1).

BFigure 1: No adherent cells are present on vessels with cell-repellent surface
A: Cell culture dish with cell-repellent surface after incubation with EtOH.
B: Cell culture dish with CELLSTAR® TC surface after incubation with EtOH.

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3/ CULTURE OF SPHEROIDS AND
STEM CELL AGGREGATESIn pharmaceutical and basic research, two-dimensional(2D) cultures are still predominant. Nevertheless, 2D cultures can only mimic to a limited extent the conditions inphysiological tissue where cells are able to interact in athree-dimensional network. Therefore, results generated
from 2D cultures have often limited relevance for clinicaleffectiveness i.

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Pluripotent stem cells with their ability to differentiate
into cell types of all germ layers (endoderm, mesodermand ectoderm) iv hold great promise for drug development,
therapeutic applications as well as for basic research.
A key step for the cultivation of stem cells is the formationof non-adherent cell aggregates, called embryoid bodies v.
Pluripotent cell types which are able to form embryoid
bodies comprise both embryonic stem cells and induced
pluripotent stem cells.

The employment of spheroid cultures plays an importantrole as an alternative approach to better mimic physiological conditions, especially in cancer research.

OxygenSpheroids are self-assembled spherical cell clusters withdifferent zones (Fig. 2). These zones are characterisedby metabolic and proliferative gradients resembling thephysical conditions in avascular tumours or micrometastases ii,iii. As a consequence, experimental data obtainedwith spheroids are often more significant than data from
2D cell culture experiments.

Metabolinio
ratlifeProKatabolite

teNutri

Necrotic core

men

ts

Quiescent viable cell zone
Proliferating zone

Figure 2: Schematic description of a tumour spheroid

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N° 17

For the formation of spheroids and embryoid bodies cellcell interaction has to be dominant over the interactionof the cells with the surface of the culture vessel used.
Standard cultivation approaches comprise the hanging
drop method, spinner flask or rotary cell culture forspheroid cultivation i and static suspension culture forembryoid bodies. However, these approaches have only
limited compatibility with automation and high-throughput screening. On the other hand, ‘classic’ cell culture
vessels like microplates fulfil all the necessary demands
for automated handling and imaging. If equipped with asurface effectively preventing cell-surface interactions
like the Greiner Bio-One cell-repellent surface these vessels represent a perfect platform for cultivating spheroids(Fig. 3) and stem cell aggregates (Fig. 4, 5).

CULTIVATION OF SPHEROIDS

Number of cells per well
500 cells

1,000 cells

3,000 cells

6,000 cells

Day 1Day 3

Day 7Day 14

Figure 3: LNCaP cells form single spheroids in 96 well U-bottom plates with cell-repellent surface.
Their size is dependent on the number of cells used for seeding.

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10,000 cells

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Tested cell lines for spheroid and aggregate formation are listed in Table 1.
Tested Cell Lines

Application

VeroMDCKII
alveolar Macrophages (rat)Cell Adhesion

CaCo-2
JurkatCHO
SK-N-MCSTO-DA

Stem Cell AggregateFormation
Figure 4: Aggregates of murine ES-D3 cells grown for 10 daysin a 96 well F-bottom microplate with cell-repellent surface.
Before the transfer to the cell-repellent surface, cells were
cultivated on feeder cells and detached with accutase.

mES-D3
human iPSCs
HeLa

Spheroid Culture

HepG2
LNCaP
HEK-239

Table 1:

Tested cell lines

Figure 5: Aggregate formation of human induced
pluripotent stem cells (iPSCs)
cultured in a 96 well U-bottom microplate with cell-repellent
surface. Before the transfer to the cell-repellent surface, cells
were cultivated on Matrigel TM and detached with accutase.

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N° 17

4/ INHIBITION OF CELL ATTACHMENTOF SEMI-ADHERENT AND
ADHERENT CELL LINES IN VESSELSWITH CELL-REPELLENT SURFACE
For the culture of suspension cells, surfaces of a stronghydrophobic nature are generally used. With semi-adher-ent cell lines like macrophages or even adherent cell lines
like Vero or CaCo-2 cells, this kind of surface does notreliably prevent cell attachment.
However, if cultivated in vessels with a cell-repellent surface, these cell lines exhibit an almost total inhibition ofattachment (Fig. 6).

INHIBITION OF CELL ATTACHMENT ON THE CELL-REPELLENT SURFACEOD 590 nm

A0, 50, 5

Vero0, 4Makrophages

0, 4

CaCo-20, 30, 30,20,20,10,10, 0
Cell-Repellent
Surface

Competitor 1

Competitor 2

CELLSTAR®
Suspension

Blank

B

Figure 6: The cell-repellent surface inhibits cell attachment effectively.
A) Spectroscopic analysis of cell attachment of Vero, CaCo-2 cells and macrophages. Cells were seeded in F-bottom 96 well
microplates, incubated at 37 °C and 5 % CO2. After 24 hours the media were discarded. Cell attachment was analysed by crystal violet
staining. After dissolving the crystal violet dye in the cells attached to the well surface, optical density was measured at 590 nm.
B) Microscopic analysis with 10 x magnification after crystal violet staining (Left: CELLSTAR® suspension; Right: cell-repellent surface).

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5/ ORDERING INFORMATIONCELLSTAR® CELL CULTURE VESSELS WITH CELL-REPELLENT SURFACE
Cell Culture Dishes
Cell-Repellent Surface
Raw material: PS, Surface treatment: cell-repellent, Vent nock: yes
Item No.

HeightØ nominal size

Working volume

Total volume

SterileQty. inner / outer

627979

10 mm

35 mm

≤3 ml

10 ml

+

10 / 40628979

15 mm

60 mm

6 ml - 7 ml

28 ml

+

10 / 20664970

20 mm

100 mm

16 ml - 17 ml

100 ml

+1 /5

Total volume

Cap design

SterileQty. inner / outer

Cell Culture Flasks
Cell-Repellent Surface
Raw material: PS, Surface treatment: cell-repellentItem No.

Flask design

Cap Colour

690980

flat

white

50 ml

screw cap+

10 / 20690985

flat

white

50 ml

filter screw cap+

10 / 20658980

flat

white

250 mlscrew cap+

5 / 15658985

flat

white

250 mlfilter screw cap+

5 / 15660980

flat

white

550 mlscrew cap+

5 /5660985

flat

white

550 mlfilter screw cap+

5 /5661980

high

white

650 mlscrew cap+

4 /4661985

high

white

650 mlfilter screw cap

+

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N° 17

Multiwell Plates / Microplates
Cell-Repellent SurfaceItem No.

Well format

Well Profile

BottomProductColour

Total volume(Well)

Workingvolume (well)

LidSterile

Qty. inner /outer657970

6F-bottom

solidclear

16.1 ml

2 ml - 5 ml

condensation
rings+

1 /5

66597012F-bottom

solidclear

6.5 ml2 ml - 4 ml

condensation
rings+

1 /5

66297024

F-bottomsolidclear

3.3 ml

0.5 ml - 1 ml

condensation
rings

+

1 /5

67797048

F-bottomsolidclear

1.7 ml

0.5 ml - 1 ml

condensation
rings

+

1 /5

65097096

U-bottomsolidclear

323 ml

40 μl - 280 μl

condensation
rings

+

1 /6650979

96

U-bottomsolidclear

323 ml

40 μl - 280 μl

condensation
rings

+

8 / 32651970

96

V-bottomsolidclear

234 μl

40 μl - 200 μl

condensation
rings

+

1 /6655970

96

F-bottom /Chimney Well

solidclear

392 μl

25 μl - 340 μl

condensation
rings

+

1 /6655976

96

F-bottom /Chimney Well

μClear®

black

392 μl

25 μl - 340 μl

condensation
rings

+

8 / 32655976-SIN

96

F-bottom /Chimney Well

μClear®

black

392 μl

25 μl - 340 μl

condensation
rings+

1 / 32781970384

F-bottomsolidclear

131 μl

15 μl - 110 μl

yes+

1 / 60781974

384

F-bottom

μClear®

white

131 μl

15 μl - 110 μl

yes+

8 / 32781976

384

F-bottom

μClear®

black

131 μl

15 μl - 110 μl

yes+

8 / 32781976-SIN

384

F-bottom

μClear®

black

131 μl

15 μl - 110 μl

yes+

1 / 32787979

384

U-bottomsolidclear

122 μl

10 μl - 90 μl

yes+

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6/ LITERATURE
iii
iii
ivv

Friedrich J. et al. ( 2007). Experimental anti-tumour therapy in 3-D: Spheroids - old hat or new challenge? Int J Rad Biol. 83(11-12):849-871.
Friedrich J. et al. (2009). Spheroid-based drug screen: considerations and practical approach. Nat Protoc. 4(3):309-24C.
Kunz-Schughart L. A. et al. (2004). The use of 3-D cultures for high-throughput screening: the multicellular spheroid model. J. Biomol Screen. 9(4):273–285.
Itskovitz-Eldor J. et al. (2000). Differentiation of human embryonic stem cells into embryoid bodies comprising the three embryonic germ layers. Mol Med. 6:88-95.
Höpfl G. et al. (2004). Differentiating embryonic stem cells into embryoid bodies. Methods Mol boil. 254:79-98.

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Bio-One GmbH is expressly prohibited. Media owner: Greiner Bio-One GmbH / Represented by Managing Directors Jakob Breuer and Heinz Schmid.
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F073777 [rev. 04.2022]

Raw material: PS, Surface treatment: cell-repellent