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
Defined medium Eagle'sMEM Bicarbonate CO2 r - - ~ - - - ~ V NV 8"5xlo n
I ' 4 )< I07 5"9Io 4 4"7 I o n

r V

Cell line Human conjunctiva B H K 21 HeLa R K I3

TES ~--~ NV 1"81o 5

2. 5 x 105 2.oxIo n *

HEPES r - - ~ ' - - a V NV 8'5Io 6

I. 4 107 6-6Io 6 4"7 IOe

2"oIo 5
3.0 x I05 2'4xio 5 *

7"oxIo n
I-0 I07 5"5Ion 4"I IO 6

2'oIo 5
2" 5 x I05 2"5xio 5 *

Eagle's MEM
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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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
10"4

Titre (p.f.u./ml.) in media buffered with: , ~ , Bicarbonate + CO2 HEPES 0-9 x 1 0 7 o.6x io 7
1. 3 X 1 0 7 I'I X I0 7

I5"6
20.8

I'4x xo7 I "4x io 7

x'3x I07 1.4 x 1 0 7

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 RMK cells RMK cells RMK cells Human fibroblasts HeLa cells Defined medium used in growth of 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
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.

J.D. WILLIAMSON P. Cox

(Received 7 November I967)