J. gen. ViroL 0968), ~, 3o9-3IZ
Printed in Great Britain
309
U s e o f a N e w Buffer in the Culture o f A n i m a l Cells
(Accepted 20 November I967)
Growth of animal cells in a nutritionally complete tissue culture medium is usually
optimal when the medium is buffered at a pH in the range 7"2 to 7"4. To function most
efficiently the pKa of the chosen buffer should be as close to the required pH as
possible. The most commonly used buffer in tissue culture media is the bicarbonate +
carbon dioxide buffer but phosphate or tris buffers have also been used. Each of these
buffers has particular disadvantages for use with in vitro biological systems. The
bicarbonate+ CO2 buffer, pKa of 6"3 at 37 °, has the obvious limitation that one component is in the gaseous phase and tissue cultures must be maintained in a closed
system. Although the phosphate buffer has a more suitable pKa, 6"9 at 37 °, phosphates
form insoluble complexes with essential divalent cations when used at concentrations
necessary for a suitable buffering capacity. In addition, the components of both
bicarbonate+ COs and phosphate buffers are utilized in biochemical reactions of the
cell cultures. Tris buffer has a pKa of 7"9 at 37 ° and is often cytotoxic except at very
low concentrations.
Recently a new range of zwitterion buffers has been described which was specifically
developed to meet the particular requirements of biological systems (Good et al.
I966). These buffers, covering a pKa range from 6 to 8, are very soluble, have low
binding capacities for divalent cations and are stable. Two of these zwitterion buffers,
N-tris (hydroxymethyl) methyl-2-aminoethanesulphonic acid (TES), pKa = 7"I4 at
37 °, and N-2-hydroxyethylpiperazine-N'-ethanesulphonicacid (HEPES), pKa = 7"3 I
at 37 °, proved superior to conventional buffers in comparative biochemical assays
with cell-free preparations (Good et al. r966).
We have tested the efficacy of these two zwitterion buffers in the serial passage of
continuous lines of cells. One of these buffers, HEPES, was also used in quantitative
studies of the growth of viruses in cells grown or maintained in tissue culture media
containing this buffer.
Growth o f continuous cell lines. Continuous lines of cells were grown in complete
media based either on Eagle's minimum essential medium or on medium 199. The
test media were modified only by the further addition of TES or HEPES (Calbiochem)
to a final concentration of 28 mM. Cells were grown as monolayers in 4 oz medical
fiats in either unmodified or TES- or HEPES-buffered growth media starting from
identical inocula of viable cells. When the monolayers were confluent they were
examined microscopically for cytological appearance before total and viable counts
were made by counting the resuspended cells in the presence of o.I % trypan blue. The
results of cell counts at the end of the third passage in unmodified and modified media
are shown in Table I. There was no difference in the rates of cell growth as determined
by the time taken for the monolayers to become confluent. Cells grown in unmodified
and both modified media were of identical cytological appearance. The buffering
capacity of both TES- and HEPES-buffered media, as indicated by the pH values of
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the media when the cell monolayers became confluent, was equal to or better than that
of unmodified media. The yield of cells grown in HEPES-buffered media determined
by both total and viable counts was the same as in unmodified media, or even slightly
greater with certain lines of cells. However, although the proportion of non-viable
cells was not higher, the total yield of cells growing in TES-buffered media was less
than that of the other two media. Since better results were obtained with HEPESbuffered media, and in view of the very similar pKa values of the two buffers, use of
TES-buffered media was discontinued.
All results presented in Table ~ were obtained with cells grown in sealed medical
flats. This is essential when bicarbonate + CO2 buffer is used unless special precautions
are taken to maintain the partial pressure of CO2 in the gaseous phase at the level
necessary to obtain the pH required. To investigate the suitability of HEPES as a
buffer of open tissue culture systems, BHK 2I and RK ~3 cell lines were grown in
HEPES-buffered media in 4 oz. medical flats closed only with sterile cotton wool
plugs. The yield of cells from both cell lines under these conditions was identical with
that from cells grown in closed systems.
Table I. Growth of continuous cell lines after third passage in tissue culture
media buffered with bicarbonate + COs, TES or HEPES buffers
Cell line
Human
conjunctiva
B H K 21
HeLa
R K I3
Defined
medium
Bicarbonate CO2
r - - ~ - - - ~
V
NV
r
V
TES
~--~
NV
HEPES
r - - ~ ' - - a
V
NV
Eagle'sMEM
8"5xlo n
2"o×Io 5
7"oxIo n
1"8×1o 5
8'5×Io 6
2'o×Io 5
Eagle's MEM
I ' 4 )< I07
5"9×Io 4
4"7 × I o n
3.0 x I05
2'4xio 5
*
I-0 × I07
5"5×Ion
4"I × IO 6
2. 5 x 105
2.oxIo n
*
I. 4 × 107
6-6×Io 6
4"7 × IOe
2" 5 x I05
2"5xio 5
*
Eagle'sMEM
I99
V = v i a b l e cells.
N V = cells s t a i n i n g w i t h o. i ~ t r y p a n b l u e .
• Not significant.
Virus infectivity titrations. Consequent on these results, preliminary virus infectivity
titrations in cell cultures in HEPES-buffered media were made by plaque titrations of
vaccinia virus in RK I3 cells grown in Petri dishes. The efficiency of this system was
assessed by comparison with titres obtained by identical procedures but using media
buffered by bicarbonate with 5 % CO2 in air. Preliminary experiments indicated a
significant reduction in the efficiency of titrations using HEPES-buffered medium.
Similar reductions in titre in the presence of HEPES buffer compared with controls
were also observed in a study of haemagglutinin production by variola virus in human
fibroblasts (Wells, 1967). Haemagglutinin production in this system has been shown
to be dependent on bicarbonate availability. Using a medium based on Eagle's basal
medium with the addition of z8 mM HEPES and containing I3"I mM bicarbonate,
haemagglutinin production was almost fully suppressed. This was largely reversed by
further addition of bicarbonate to I5. 7 raM. Direct interaction between HEPES and
bicarbonate in solution could not be demonstrated by physical methods. In the present
study the efficiency of vaccinia virus titrations in RK I3 cells could be increased to
90% of that of the control by reducing the level of HEPES to I4 mM and using
bicarbonate at a concentration of Io'4 mM. Higher concentrations of bicarbonate
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31 i
exhausted the buffering capacity of HEPES when used at this lower concentration.
Results from a typical experiment are shown in Table 2.
Further infectivity titrations were made in rhesus monkey kidney cells, human
fibroblasts and HeLa cells grown in test-tubes in complete media based on either
medium I99 or Eagle's minimum essential medium. Bicarbonate was added to
medium I99 to a final concentration of I5.6 mM and to Eagle's medium to Io'4 mM.
Herpes simplex virus, a type I parainfluenza virus and E C H O virus type 3 were
titrated in monkey kidney cells, cytomegalovirus in human fibroblasts and adenovirus
type 5 in HeLa cells in both umodified media and HEPES-buffered media with HEPES
at 14 mM. Growth of the parainfluenza virus was detected by haemadsorption and of
the other virus by development of characteristic cytopathic effects. There were no
Table 2. Plaque titrations of vaccinia virus in R K x3 cells in media containing various
amounts of sodium bicarbonate and buffered with either bicarbonate + CO~ or H E P E S
buffe~
Concentration
of sodium
bicarbonate
(raM)
5"2
Titre (p.f.u./ml.) in media buffered with:
,
~
,
Bicarbonate+
CO2
HEPES
0-9 x 1 0 7
o.6x io 7
10"4
1. 3 X 1 0 7
I'I X I0 7
I5"6
I'4x xo7
I "4x io 7
x'3x I07
1.4 x 1 0 7
20.8
Table 3- Virus infectivity titration systems with which identical titres were obtained
in cell monolayers grown in both unmodified and HEPES-buffered tissue culture media
Virus
Herpes simplex virus
Parainfluenza virus type I
ECHO virus type 3
Cytomegalovirus
Adenovirus type 5
Cells used for
infectivity titrations
Defined medium used
in growth of cells
RMK cells
RMK cells
RMK cells
Human fibroblasts
HeLa cells
Medium I99
Medium I99
Medium I99
Medium 199
Eagle's MEM
Parainfluenza virus type t was detected by haemadsorption; other infective virus was detected by
development of characteristic cytopathic effect.
significant differences between final titres in unmodified and HEPES-buffered media
with any of the viruses used in these experiments. Rates of growth of these viruses, as
measured by the criteria indicated, were similar in the different media except with
adenovirus type 5 which developed cytopathic effects earlier in HEPES-buffered
medium. Final titres with adenovirus type 5 could be read 24 hr earlier in HEPESbuffered medium compared with the unmodified medium. Results of these experiments
are summarized in Table 3.
HEPES buffer may thus be used in animal cell cultures without apparent cytotoxic
effects. The efficiency of virus infectivity titrations in these cell cultures compares very
favourably with titrafions in cell cultures grown in media using conventional buffers.
The slight effect described of HEPES on plaque titrations is very probably due to the
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more exacting bicarbonate requirement of vaccinia virus compared with other viruses
(Chang, I959). We concluded, therefore, that HEPES buffer could be very profitably
utilized in tissue culture media.
Virology Department
St Mary's Hospital Medical School
London, W. 2
J.D. WILLIAMSON
P. Cox
REFERENCES
CHANG,R. S. (I959). Participation of bicarbonate in RNA and protein syntheses as indicated by virus
propagation in human cells. J. exp. Med. lO9, 229.
GOOD, N. E., WINGET,G. D., WINTER,W., CONNOLLY,T. N., [ZAWA,S. • SINGH,R. M. M. (1966).
Hydrogen ion buffers for biological research. Biochemistry 5, 467.
WELLS, n . G. T. (1967). Studies on variola virus. Ph.D. thesis, University of London.
(Received 7 November I967)
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