Analysis Of CHO Cell Requirements For Assay Miniaturization In ...

THE HTS FORUM
Corning Incorporated • Science Products Division Volume 8 • Fall 1999
ANALYSIS OF CHO CELL REQUIREMENTS FOR ASSAY
MINIATURIZATION IN HIGH-THROUGHPUT SCREENING
THIS
ISSUE ...
Analysis of
CHO Cell
Requirements
for Assay
Miniaturization
in High-
Throughput
Screening
Products for
every phase
of the Drug
Development Cycle
From storage to
genomics to HTS and
Test Kit Components,
Corning offers the
most comprehensive
line of products. With
a worldwide distribution
network and
unsurpassed levels of
quality, support and
service, Corning has
you covered no matter
where you are in the
drug development
cycle.
Catherine Ennis, M.S. and
Allison Tanner, Ph.D.
Corning Incorporated
Science Products Division
Advanced Life Science Products
Portsmouth, NH 03801
Summary
Culture conditions for Chinese
hamster ovary (CHO) cells were
varied to analyze cellular requirements
in assay miniaturization for
high-throughput screening. CHO
cells were seeded into Corning ®
96 well, 96 well half-area and
384 well tissue culture treated
microplates in media volumes
from 25 to 200 µL per well. The
medium was sampled for glucose
and pH measurement. A tetrazolium
reagent was used to evaluate
cellular proliferation, and cells
were harvested for counting. After
24 hours, the number of cells in all
wells had doubled from the initial
seeded quantity. The percentage of
glucose in all wells and volumes
remained above 50% of the value
for the control medium that was
incubated without cells, and pH
remained constant at 7.4. Metabolic
activity as indicated by the
reduction of the tetrazolium
reagent to a soluble, colored formazan
product was similar for
cells in all volumes and formats.
The lower limit of detection was
calculated to be less than 3,000
cells per well in all formats and
volumes, indicating that there
was no decrease in the sensitivity
of the cell proliferation assay
with miniaturization.
Introduction
Cell culture has become a valuable
and necessary tool for numerous
research laboratories, and many
more groups are adding cell-based
assays to their arsenal of methods
for drug discovery. While some
are analyzing the consequences of
compound addition utilizing cells
that have been genetically manipulated
to announce these effects
through the linkage of a gene of
interest to a reporter molecule,
others concerned about the biological
relevance of these responses,
are using probes that fluoresce
upon activation of signaling pathways
or other changes within cells
(1). Although there are obvious
differences in cells cultured in vitro
from their counterparts in vivo, the
use of cells in culture enhances the
ability to identify and optimize
potential candidates for therapeutic
intervention with more clinical
relevance than those drug leads
provided by assays operating
outside the cellular context.
While non-cell based assay miniaturization
presents many difficulties
in dispensing and handling minuscule
quantities of reagents, as well
as determining the appropriate
proportions of reactive components
to provide the signal intensity necessary
to discern potential drug
candidates, cell-based assay miniaturization
poses all of these problems
and more. The entire range
of environmental requirements to
culture cells with specialized functions
lie outside the scope of this
article, but basic to all cells in
monolayer culture are the need to
maintain asepsis, a receptive substrate
for adhesion, proper incubation
temperature, medium of the
appropriate constitution and volume
to support cell establishment
and the necessary gas phase. Since
CHO cells are commonly used in
high-throughput screening for the
ease with which they can be manipulated
and maintained, this article
analyzes culture conditions for
CHO cells in assay miniaturization.
Materials and Methods
Cell Culture
CHO K1 cells from American
Type Culture Collection (Rockville,
MD) (ATCC No. CRL 9618) were
cultured in T-225 flasks (Corning
Cat. No. 3001) to confluence in
Hams F-12 basal medium, pH 7.4
(Cat. No. 12615F, BioWhittacker,
Walkersville, MD), supplemented
with 10% (v/v) FBS (Cat. No.
14501F, BioWhittaker, Walkersville,
MD) containing 1% (v/v) sodium
pyruvate (Cat. No. 13115E,
BioWhittaker, Walkersville, MD)
and 1% (v/v) L-Glutamine (Cat.
No. 17605E, BioWhittaker,
Walkersville, MD) in an atmosphere
of humidified 5% CO 2 in
air at 37°C. The confluent cells
were harvested and dispensed into
clear, tissue culture treated 96 well
(Corning Cat. No. 3596), 96 wellhalf
area (Corning Cat. No. 3696),
and 384 well microplates (Corning
Cat. No. 3701) using a LabSystems
Multidrop 384 dispensing unit
(Cat. No. 1507010, Helsinki,
Finland) at varying densities and
media volumes.
Culture Conditions, Cellular
Adherence, Metabolic Activity
Culture conditions, cellular metabolism
and adherence were analyzed
( in quadruplicate) following
timed incubations in microplates.
Medium of interior and perimeter
Continued inside

The HTS Forum
wells was examined for pH changes
using indicator strips (Cat. No. 9582,
EM Science, Gibbstown, NJ). Glucose
concentrations were determined enzymatically
(Cat. No. 510A, Sigma
Diagnostics, St. Louis, MO) according
to manufacturer’s instructions (Procedure
No. 510) following collection
of conditioned media and media incubated
without cells, at timed intervals.
Glucose concentrations were measured
on a SpectraMax 250 (Molecular
Devices, Sunnyvale, CA) at 450 nm in
clear 96 well microplates (Corning ®
Cat. No. 3370). Cellular adherence was
examined following removal of conditioned
media and washing of nonadherent
cells from microplate wells
with Dulbecco’s Phosphate Buffered
Saline (DPBS) (Cat. No. 17512F,
BioWhittaker, Walkersville, MD). For
3 and 24-hour incubations, adherent
cells were photo-documented at 400X
using an Olympus microscope with an
Olympus OEV 142 monitor and OEP
color video printer (Optical Analysis
Corp, Nashua, NH). Adherent cells
were washed once with DPBS and
enzymatically detached from microplate
wells with 50 µL of trypsin/ versene
(Cat. No.17161E, BioWhittaker,
Walkersville, MD) and incubation at
37°C for five minutes. Detached cells
were collected and resuspended in
950 µL of media and counted manually
using a hemocytometer. Metabolic
activity of CHO K1 cells was examined
with Cell Titer 96 ® AQ ueous One Reagent
MTS Assays (Cat. No. G358B, Promega,
Madison, WI). Reactions were conducted
according to manufacturer’s
instructions as well as modified by
increasing the proportion of MTS
added according to the number of cells
seeded, to compare the addition of
MTS reagent based upon cell numbers.
At timed intervals following incubations
at 37°C, optical densities were
determined at 490 nm on the Wallac
Victor 2 1420 Multilabel Counter
(EG&G Wallac, Turku, Finland). The
adhesion of CHO K1 cells was also
examined in black opaque 384 well
(Corning ® Cat. No. 3713) and 96 well
(Corning Cat. No. 3603) microplates
Fluorescence intensity (cps x 10 6 )
50
40
30
20
10
0
50,000 25,000 12,500 6,250 12,500 6,250 1,325 1,562
Figure 2. Percent Glucose Remaining. Glucose concentrations were determined enzymatically and
measured spectrophotometrically at 450 nm in 96 well microplates following collection of conditioned
media and media incubated without cells, at timed intervals. The percent glucose remaining was calculated
from the glucose concentration in the media sample incubated without cells at each timepoint. Error bars
indicate (+/-) the standard deviation of four replicates.
over time, using Vybrant Cell Adhesion
Assay Kits (Cat. No. V-13181, Molecular
Probes, Eugene, OR). CHO K1 cells
Number of cells seeded per well
96 wells
384 wells
Figure 1. Flourescence intensity after 90 minutes. The adhesion of CHO K1 cells was examined over
time using Vybrant( Cell Adhesion Assay Kits (Cat. No. V-13181, Molecular Probes, Eugene, OR). CHO
K1 cells were harvested and manually dispensed at 50,000, 25,000, 12,500, 6,250 cells per 100 µL and
12,500, 6,250, 3,125, 1,562 cells per 25 µL into 96 well black opaque and 384 well microplates, respectively.
Fluorescence intensity was measured (as stated in Materials and Methods) and increased as the
number of cells initially seeded increased. Representative data using the 90 minute timepoint is shown.
Percent Glucose Remaining
120
100
80
60
40
20
0
96/100 µL 96/200 µL 96 half/50 µL 96 half/100 µL 384/25 µL 384/50 µL
Format/volume
0
2 hr
4 hr
24 hr
were harvested as stated previously and
manually dispensed at 50,000, 25,000,
12,500, 6,250 cells per 100 µL and

Table 1. MTS Measure of Metabolic Activity
Number of Standard Standard Standard
Plate cells seeded Volume MTS 2 hr. deviation 4 hr. deviation 24 hr. deviation
96 well 50,000 100 µL 20 µL 0.340 0.04 0.519 0.06 0.602 0.01
40 µL 0.483 0.04 0.573 0.09 0.677 0.04
96 well 50,000 200 µL 20 µL 0.237 0.03 0.238 0.02 0.633 0.04
40 µL 0.320 0.04 0.389 0.04 0.937 0.09
half-area 25,000 50 µL 10 µL 0.405 0.10 0.542 0.12 0.740 0.05
20 µL 0.492 0.10 0.850 0.04 0.800 0.07
half-area 25,000 100 µL 10 µL 0.228 0.04 0.350 0.02 0.745 0.05
20 µL 0.266 0.10 0.361 0.15 0.864 0.21
384 well 12,500 25 µL 5 µL 0.355 0.02 0.369 0.09 0.527 0.06
10 µL 0.466 0.06 0.567 0.10 0.671 0.02
384 well 12,500 50 µL 5 µL 0.196 0.05 0.285 0.14 0.570 0.06
10 µL 0.219 0.04 0.391 0.11 0.664 0.05
Comparison of metabolic activity in CHO K1 cells seeded in different volumes in microplate wells of the same surface area for 96 well, 96 well half-area,
and 384 well microplates using Cell Titer 96 ® AQ ueous One Reagent MTS Assays (Cat. No. G358B, Promega, Madison, WI). Reactions were conducted according
to manufacturer’s instructions as well as modified to compare the addition of MTS reagent based upon cell numbers. At timed intervals, optical densities were
determined at 490 nm on the Wallac Victor 2 1420 Multilabel Counter (EG&G Wallac, Turku, Finland).
12,500, 6,250, 3,125, 1,562 cells per
25 µL into 96 well and 384 well microplates,
respectively. All reactions were
carried out according to manufacturer’s
instructions and fluorescence intensity
was detected using an LJL Biosystems
“Analyst” (LJL Biosystems, Sunnyvale,
CA) as follows: comparator conversion,
attenuator = medium, units = cps, plate
= Costar ® 96 well solid, 384 well solid,
polarizer settling time = 150ms, z-height
= 2.2 mm, integration time = 100,000
µs, excitation filter 485 nm, emission
filter 615 nm.
Assay Miniaturization and Sensitivity
The sensitivity after miniaturizing the
Cell Titer 96 ® AQ ueous One Reagent MTS
Assay (Cat. No. G358B, Promega,
Madison, WI) was compared in clear,
tissue culture treated 96 well (Corning
Cat. No. 3596), 96 well-half area
(Corning Cat. No. 3696), and 384 well
microplates (Corning Cat. No. 3701)
was compared. CHO K1 cells were
harvested as stated previously and manually
dispensed at 100,000, 50,000,
25,000, 12,500, 6,250, 3,125, 1,562
cells per 100 µL and 200 µL into 96
well microplates, 50 µL and 100 µL
into 96 well-half area microplates, and
25 µL and 50 µL into 384 well microplates.
All reactions were carried out
according to manufacturer’s instructions.
Following 3 hour incubations at
37°C, optical densities were determined
at 490 nm on the Wallac Victor 2 1420
Multilabel Counter (EG&G Wallac,
Turku, Finland).
Results and Discussion
The conditions for CHO cell-based assay
miniaturization were examined in 96
well, 96 well half-area, and 384 well
tissue culture treated microplates in an
effort to determine rapid, cost effective
methods to identify potential therapeutic
compounds. To produce the number
of cells necessary to seed a large number
of microplates, cells were first grown to
confluence in T-225 flasks. After harvesting,
dilutions of cells were seeded in
volumes of 100 µL and 25 µL per 96 and
384 wells, respectively. The Vybrant
Cell Adhesion Assay was used to assess
the effect of the quantity of cells seeded
on the number of adherent cells at incubation
times up to three hours. As
anticipated, an increase in fluorescence
intensity correlated with an increase in
the number of cells seeded. This appears
to indicate that increasing the number
of cells seeded leads to an increased
number of adherent cells (Figure 1). It
was also observed that the 384 well
plates gave a higher fluorescent signal
for the same densities (12,500 and
6,250) plated in 96 well microplates.
This was presumably a result of higher
plating density per unit area for the
same number of cells. Furthermore, the
relative fluorescence obtained with just
1,325 cells per well in a 384 well microplate
was comparable to 6,250 per well
in a 96 well microplate. This indicates
that higher plating densities obtained in
384 well microplates should be beneficial
for cell-based analysis by enabling
higher signals to be obtained with
fewer cells. Analysis of culture conditions
in decreasing volumes of media
were compared across the three microplate
formats after increasing times of
incubation, using a combination of pH
measurement, glucose utilization, cell
proliferation and metabolic assessment.
Cells were seeded into the microplate
wells so that a confluent monolayer
would be achieved after 24 hours of
incubation. Two different volumes of
media were used per microplate format.
To retain the appropriate oxygen and
carbon dioxide tensions for monolayer
cells in culture, it is recommended that
the depth of the medium be in the
range of 2 to 5 mm, depending on specific
cell type and growth conditions
(2). Since cells in culture use glycolysis
as the primary pathway to provide
metabolic energy (2), the amount of
glucose remaining in the medium pro-

Fall 1999
vides a measure of the adequacy of
media volume to support cells. The
amount of glucose in the medium
appeared to be utilized gradually over
time in all microplate wells containing
cells, while the pH remained constant
at 7.4. The percentage of glucose in all
wells remained above 50% of the value
for the control medium that was incubated
in absence of cells (Figure 2). The
rate of glucose depletion seemed within
reasonable limits of experimental variation
for all the microplates with different
volumes of media. This suggests
that in spite of miniaturizing, the metabolic
rates of the cells was unaltered.
Equal numbers of cells were seeded in
different volumes of media to determine
if it took longer for cells to settle
and adhere in a greater depth of media.
While the initial cell counts at one and
two hours seemed to support this, by
three hours cell numbers equivalent to
the amount seeded were counted in all
wells. At 24 hours, the number of cells
in all wells had doubled from the initial
seeded quantity and there were no differences
visible microscopically (Figure
3). MTT, is a tetrazolium compound
that has long been used as an indicator
of cell proliferation, viability and cytotoxicity
(3). MTT is reduced by metabolically
active cells to an insoluble
formazan product, the absorbance of
which is directly proportional to the
number of living cells in culture (4).
Cell Titer 96 ® AQ ueous One Reagent
(Promega) combines MTS, a related
tetrazolium compound, with phenazine
methosulfate (PMS), an electron coupling
reagent, to produce a water-soluble
formazan product after reduction
by living cells (5). This MTS reagent
was used to assess the viability of cells
incubated in reduced media volumes as
well as providing a measure of assay
miniaturization sensitivity upon miniaturization.
Instructions included with
the reagent suggest that the proportion
of the MTS reagent should be kept constant
with the volume of media in the
assay, but to better gauge whether the
cells were influenced by media volume,
the addition of the MTS reagent was
also varied based on the number of
cells seeded. Since the additional media
diluted the absorbance of the soluble
formazan product, the differences in
absorbance as a function of media volume
were more pronounced at the early
timepoints (2 and 4 hours). However,
incubation for 24 hours seemed to indicate
that there was little difference in
metabolic activity between cells seeded
into microplate wells with the same
growth surface area in different media
volumes. Lower media volumes with a
higher proportion of MTS generally
lead to greater absorbance (Table 1). To
determine assay sensitivity upon miniaturization,
the MTS reagent was used
for another experiment in the proportions
suggested by the manufacturer.
Equal numbers of cells were seeded
regardless of the surface area available
A
B
Figure 3. At 3-hours (a) and 24-hours (b) post-seeding, adherent CHO K1 cells in 25 and 50 µL per 384 microplate well, 50 and 100 µL per 96 half-area microplate
well, and 100 and 200 µL per 96 microplate well were photo-documented at 400x using an Olympus microscope with an Olympus OEV 142 monitor and OEP
color video printer (Optical Analysis Corp, Nashua, NH). Upon visual inspection, there were no obvious differences in cells seeded in wells of different surface
areas, in any of the assessed volumes, at 3 or 24 hours.

The HTS Forum Fall 1999
for attachment, into wells of all three
microplate formats. The greatest
absorbance for the lowest cell number
occurred in Corning ® 384 well microplates
irrespective of the volume of media
used for growth, with the 96 well and
96 well half-area microplates in close
approximation (Figure 4). These results
strongly support assay miniaturization
for reagent and cost reduction.
Conclusions
As evidenced by the amount of glucose
remaining in the wells, cell proliferation
and metabolic activity, reduction of
media volumes to 25 and 50 µL per
well in 384 well microplates, 50 and
100 µL per well in 96 half-area microplates,
is suitable for maintaining CHO
cells in culture for 24 hours. If time is
of the essence and cells are abundant,
increasing the number of cells seeded
will lead to an increased number of
adherent cells up to times of less than
three hours of incubation. Results
demonstrate that assay sensitivity is
maintained down to the detection of
less than 3,000 cells in Corning 384
and 96 well half-area microplates.
Enhancement of signal intensity upon
miniaturization of these cell-based assays
into 384 well microplates appears to be
due to the increased plating density per
unit area for the same number of cells.
The benefits of assay miniaturization
for pharmaceutical discovery include
conservation of biological reagents and
chemical compounds, reduction in the
waste stream, and more rapid screening
of potential therapeutic candidates.
Corning is supporting these efforts with
an array of microplates for cell-based
and non-cell based assays used in highthroughput
screening.
• Corning 96 well half-area microplates
provide a novel way to decrease the
consumption of biological reagents
and chemical compounds without
costly automation format retooling.
Absorbance at 490 nm
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
384/25 µL
384/50 µL
half-area/50 µL
half-area/100 µL
96/100 µL
96/200 µL
Media 1,562 3,125 6,250 12,500 25,000 50,000 100,000
Number of cells seeded
Figure 4. MTS assay miniaturization. The sensitivity of the Cell Titer 96 ® AQ ueous One Reagent MTS Assay
(Cat. No. G358B, Promega, Madison, WI) in 96 well, 96 well half-area, and 384 well microplates was
compared. CHO K1 cells were harvested as stated (in Materials and Methods), and dispensed at 100,000,
50,000, 25,000, 12,500, 6,250, 3,125, 1,562 cells per 100 µL and 200 µL into 96 well microplates, 50 µL
and 100 µL into 96 well-half area microplates, and 25 µL and 50 µL into 384 well microplates. All reactions
were carried out according to manufacturer’s instructions. Measurements were spectrophotometric at
490 nm, following three hour incubations at 37°C.
• Corning offers a multitude of 384
well microplates to augment throughput
and conserve biological reagents
and cehmical compounds in laboratories
that have incorporated the 384
well automation format.
• Corning 96 well half-area and 384
well microplates help to reduce the
waste stream and overall cost of
high-throughput screening.
• Corning 96 well half-area and 384
well microplates enable assay miniaturization
without loss of sensitivity
to help prevent missing potential candidates
for therapeutic intervention.
References
1. Okun, I. and Veerpandian, P. 1997.
Nature Biotechnology 15:287-288.
2. Freshney, I.R. 1994. Culture of Animal
Cells, A Manual of Basic Technique,
third edition., Wiley-Liss, Inc. New
York, NY.
3. Mosmann, T. 1983. J. Immunol.
Methods 65:55-63.
4. Celis, J.E. 1998. Cell Biology, A
Laboratory Handbook, second edition,
Academic Press, San Diego, CA.
5. Promega Technical Bulletin. 1996.
No.169, CellTiter 96 AQ ueous Non-
Radioactive Cell Proliferation Assay,
Promega, Madison, WI.
We would like to thank Elizabeth
Martin and Jennifer Brackett of
Corning Incorporated in Portsmouth,
NH, for their assistance with the completion
of experiments for this article.