Adapting VERO, MDBK, and MDCK Cells to a Serum-Free Environment Stephanie M. Carter and Joseph Granchelli Application Laboratory, Thermo Fisher Scientific, Rochester, New York

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Key WordsNunclon Delta, serum adaptation, HyClone, serum-free

AbstractGrowing cells with less serum may be desirable to reduce cost or to reduce the variables associated with using undefined reagents. However, most cell lines will not tolerate a rapid drop in serum concentration and must first be adapted. Three cell lines, VERO, MDBK, and MDCK, were adapted to a serum-free environment on the Thermo Scientific Nunclon Delta surface and were compared to a competitor’s surface. With each passage, the serum concentration of the media was decreased by 50% and allowed to stabilize until reaching 0.06% serum. At this point the cells could be maintained in a serum-free medium. When cells were adapted that way, the Nunclon™ Delta performance advantage was apparent.

IntroductionTraditional cell culture procedures require serum supplementation of media. More recently, the trend has shifted to serum-free conditions to reduce costs, limit variability observed between lots of serum, and the need for non-animal-derived components for some cell culture applications. There are many ways to adapt cell lines to serum-free media; the approach recommended here is the “Serum Halving Method”, adapted from Thermo Scientific HyClone. This method allows the culture to stabilize before gradually decreasing the serum concentration.

Although most assume that their cells are purely clonal, i.e. all behaving identically, the population of cells in culture is heterogeneous. This method gradually depletes the population of the most serum-dependant cells and allows the culture to be taken over by less serum-dependent cells. Therefore, during the first few passages cell viability is expected to drop and the remaining cells may become temporarily more sensitive to other factors such as pH.

The purpose of this paper is to provide a protocol for adapting cells to a serum-free environment. In addition,

the performance of VERO, MDBK, and MDCK cell growth on the Nunclon Delta surface will be compared to a competitor’s high oxygen, high binding surface.

MethodsVERO, MDBK, and MDCK cells were seeded with an initial seeding density of 10,000 cells/cm2 for VERO and MDBK cells and 20,000 cells/cm2 for MDCK cells. All cell lines were passaged with a seeding density of 10,000 cells/ cm2. VERO, MDBK, and MDCK cells were grown in media containing 1% FBS on Nunclon Delta for the control condition.

As a precaution, a fall-back culture of your cells should be maintained in “normal” serum conditions. As with any new process, commitment, careful observation, and patience are essential.

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Description Supplier Cat. No. Surface Area

Nunclon DeltaThermo Scientific Nunc

156499 75 cm2

High Oxygen/High Binding

Competitor xxxx 75 cm2

Fluid Supplier Cat. No

SMF4 MegaVir Thermo Scientific HyClone SH30552.02

Fetal Bovine Serum (FBS) Thermo Scientific HyClone SV30014.03

Antibiotic Antimycotic Solution (100×)

Sigma A5955

L-Glutamine Solution Sigma G7513

HyQTase Thermo Scientific HyClone SV30030.01

Experimental details

Surfaces Investigated

Fluid Source Information

Adaptation of VERO Cells to Serum-Free Medium

(0.5% FBS to 0% FBS)

Doubling Time (hours)

Passage #

Serum Concentration

(% FBS)

Control 1% FBS on Nunclon

Delta

Nunclon Delta

(75 cm2)

Competitor (75 cm2)

2 0.5% 25.3 21.7 22.8

3 0.25% 31.1 32.5 26.3

4 0.25% 43.4 29.9 27.5

5 0.125% 27.7 29.9 33.8

6 0.0625% 29.6 31.2 30

7 0% 40 42.8 34.8

Serum adaptation protocol

1. Grow cells in a basal medium supplemented with 10% FBS until the cells reach the peak of the linear log phase.

2. Subculture the cells at your normal subculture (split) ratio (usually 1mL cells/mL fresh medium) into serum-free medium containing 5% (v/v) FBS.

3. When the cells have reached saturation density, aspirate medium and subculture as above in the serum-free medium containing 1% FBS.

4. At each subsequent subculture, reduce the FBS by 50% until the FBS concentration is below 0.06%. At this point, the cells can be maintained in a serum-free medium.

5. If cell growth declines at any point during adaptation, return the serum concentration to that promoting cell growth. Allow the cell growth to stabilize at that serum concentration before proceeding with the serum reduction schedule.

6. During adaptation, cell density should not be allowed to fall below 2 x 105 cells/mL nor climb to more than 1.4 x 106 cells/mL

ResultsThe results shown above are typical of what is expected when adapting cell lines to serum-free environments. Comparable doubling times were maintained for VERO cells, adapted on Nunclon Delta and a competitor’s high oxygen, high binding surface (Table 1). The trends observed in serum adaptation of MDCK cells on Nunclon Delta were not equivalent to the competitor’s surface; however, at 0% doubling times were comparable on the two surfaces (Table 2).

Cells Adapting to Nunclon Delta

Control (1% FBS on Nunclon Delta)

Competitor

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g Ti

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(Hou

rs)

Passage #

10.02 3 4 5 6 7

0.5% 0.25% 0.25% 0.125% 0.0625% 0%

20.0

30.0

40.0

50.0

60.0

Serum Concentration

Table 1

When comparing MDBK cell adaptation to serum-free medium on the Nunclon Delta surface to the competitor’s surface, the Nunclon Delta product performance advantage was more dramatic. The results show that cells were able to adapt to a serum-free environment on the Nunclon Delta surface, while complete collapse of the culture occurred at Passage 7 on the competitor’s surface (Table 3).

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thermoscientific.com/bioproduction © 2012 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific Inc. and its subsidiaries.

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Conclusion•Under controlled conditions, VERO, MDBK, and

MDCK cells were able to adapt to a serum-free environment via the HyClone™ “Serum Halving Method” on the Nunclon Delta surface.

•The performance of Thermo Scientific Nunclon Delta surface was comparable to the competitor ’s high oxygen, high binding surface.

•MDBK cells were unable to adapt to the competitor’s high binding surface.

ReferencesThermo Scientific HyClone. Adapting Cells to a Serum-Free Environment. Protocol 8

Doubling Time (hours)

Passage #

Serum Concentration

(% FBS)

Control 1% FBS on Nunclon

Delta

Nunclon Delta

(75 cm2)

Competitor (75 cm2)

2 0.5% 20.8 19.6 23.9

3 0.5% 23.5 25 30

4 0.5% 20.4 20.6 25.1

5 0.5% 28.5 23.1 18.9

6 0.25% 23.8 20.9 36

7 0.25% 21.4 30.4 65.0*

8 0.125% 19.5 46 NA

9 0.125% 21.2 28.4 NA

10 0.0625% 17.8 35.7 NA

11 0% 20.4 36.1 NA

2 3 4 5 6 7 8 9 10 110.0

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

Dou

blin

g Ti

me

(Hou

rs)

Passage #

Cells Die

0.5% 0.5% 0.5% 0.5% 0.25% 0.25% 0.125% 0.125% 0.0625% 0%

Serum Concentration

Adaptation of MDBK Cells to Serum-Free Medium

Table 3 *Cell death occurs

Cells Adapting to Nunclon Delta

Control (1% FBS on Nunclon Delta)

Competitor

Dou

blin

g Ti

me

(Hou

rs)

Passage #

10.0

20.0

30.0

40.0

50.0

60.0

70.0

80.0

2 3 4 5 6 7 8

0.5% 0.25% 0.25% 0.125% 0.0625% 0.0625% 0%

Serum Concentration

Doubling Time (hours)

Passage #

Serum Concentration

(% FBS)

Control 1% FBS on Nunclon

Delta

Nunclon Delta

(75 cm2)

Competitor (75 cm2)

2 0.5% 51.2 46.8 27.3

3 0.25% 56.7 60.6 30.6

4 0.25% 51.3 45.8 26.8

5 0.125% 36.3 37.1 33.9

6 0.0625% 37.8 43.5 60.3

7 0.0625% 38.2 46.6 35.9

Adaptation of MDCK Cells to Serum-Free Medium

Table 2

Cells Adapting to Nunclon Delta

Control (1% FBS on Nunclon Delta)

Competitor