Results of a survey of BGM cell culture practices

Environment International, Vol. 10, pp. 309-313, 1984

0160-4120/84 $3.00 + .00 1985 Pergamon Press Ltd.

Printed in the USA. All rights reserved.

RESULTS OF A SURVEY OF BGM CELL CULTURE PRACTICES Daniel R. Dahling, Robert S. Safferman, and Betty A. Wright Virology Section, Biological Methods Branch, Office of Research and Development, U.S. Environmental Protection Agency, Cincinnati, Ohio 45268, USA (Received 25 June 1984; Accepted 4 September 1984) Ninety-eight laboratories in 16 countries were surveyed in 1979 to determine the uniformity of methods for the assay of human viruses in BGM ceils. None of the 58 responding laboratories applied identical methodology. A number of these practices were sufficiently different to assure a significant variance in titer with the assay of standardized virus samples. The results of this survey indicate a definite need for implementing uniform cell culture practices for the enumeration and identification of viruses in the environment.

Introduction

because tissue culture techniques are often practiced more as an art than a science. Thus it was deemed important to determine the degree of compatibility of existing methodology among the nearly 100 laboratories that we had previously provided with the BGM cell line. The BGM cell line, developed by Barron et al. (1970) ten years earlier, is susceptible to a wide range of viruses. Barron et al.'s original primary cultures were propagated in Medium 199 (Hanks' salt base) with 2070 newborn calf serum. We first obtained the BGM cell line from Barron in its 155th passage and initially cultured the cells in Medium 199 (Hanks' salt base) supplemented with 10% fetal calf serum and 0.22% NaHCO3 (Dahling et aL, 1974). Better cell growth and virus sensitivity was obtained when the cell line was subsequently adapted to a medium consisting of equal parts of Eagle's minimum essential medium (Hanks' salt base) and L-15 medium (Leibovitz), supplemented with 1007o calf serum and 0.075070 NaHCO3 (Berman et al., 1981). We later secured from Barron other passages of the BGM cell line, the lowest of which was passage 69. In 1970, Barron et aL reported a diploid number of 60 chromosomes per cell from a karyological study of 190 cells at passages 158 and 178. Six years later a limited chromosomal analysis was performed by S. Soukup (Children's Hospital, Cincinnati, OH) on cells that we had been culturing from an earlier passage. Of the 20 ceils studied at passage 144, no chromosome aberrations (such as chromosome breaks) and no contamination with other cell lines was revealed; however, the G-banding karyology showed the 2 n number had gone

Our laboratory adopted the Buffalo African green monkey kidney (BGM) cell line for recovery of waterborne human enteroviruses in 1972 after our studies showed this cell line to be more sensitive than primary rhesus and primary African green monkey kidney ceils for the isolation of indigenous viruses in environmental samples (Dahling et al., 1974). Today this cell line is the most often used system to monitor routinely for enteroviruses in ground and surface waters, sewage, wastewater effluents, and sludges. Developing procedures for detection of waterborne human enteric viruses embodies the problem of dealing with small numbers of highly infectious virus panicles from waters differing in quality. Quantification of these viruses requires the collection of large water samples and the concentration of widely dispersed viruses into a small volume, from which they can be detected by assaying against a sensitive host. Environmental virus research during the last decade has focused its efforts almost exclusively on development of virus concentration techniques (Singh et aL, 1983) and has paid little attention to cell culture procedures. Not only have a variety of different cell lines been used, but even with the use of the BGM cell line there has been a total lack of uniformity in cell growth procedures and viral assay techniques. Therefore the reliability of the results reported by individual laboratories would be difficult to duplicate, especially when viruses are isolated from environmental samples. This is 309

310

D. R. Dahling, R. S. Safferman, and B. A. Wright Table 2. Cell line history.

f r o m 60 to 120, indicating a heteroploid alteration in the cell line. Such changes f r o m the normal diploid chromosomal configuration almost invariably arise in established cell lines, but are o f no practical significance if the performance of the cell line remains unchanged. To date, no attempt has been made to achieve uniformity within the culture techniques used with the BGM cell line. This led us to examine the extent to which c o m m o n technology has been practiced.

Response

Data Requested

Responding Lowest Highest Average Laboratories(%)

Length of time cell line used 1 month Passage number of cell line received 20 Passage number at which cell line begins losing sensitivity 125

Methods Information on the BGM cell line was gathered in 1979 by surveying the institutes that had previously received, from our laboratory, the cell line along with the publication describing cultivation o f the cells (Dahling et al., 1974). Table 1 lists the location of the 98 laboratories contacted and the number o f those responding. I n f o r m a tion requesting on the B G M cell line was divided into three categories: (1) cell line history; (2) propagation, maintenance, and harvesting o f the cells; and (3) virus susceptibility and assay procedures.

9 yr

3.6 yr

88

362

146

72

403

220

53

showed considerable variations in experiences with the BGM cell line. In several instances it was evident that adequate measures had not been taken to document accurately the history of the cell line. More than onequarter of the participating laboratories did not indicate the passage number at which the cell line was received, presumably due to their failure to record it. Cases o f improper record keeping were also evident when passage numbers as low as 20 were indicated, for as already noted, passage 69 was the lowest we secured f r o m Barron. One can only surmise that upon receipt of the cell line, some investigators simply began recording their first passage number as one, leading not only to errors in their own laboratory, but also in other laboratories to which they m a y have distributed the cell line. At high passage numbers, we have observed a reduction in the sensitivity o f the B G M cell line. This was also evident in data gathered f r o m participating laboratories, in which they included the highest passage number at

Results Cell Line History Table 2 provides data on the length of time each responding laboratory had been working with the cell line, the passage level o f the cells when first received, and the highest passage at which the cells remained sensitive enough for their particular research. Responses

Table 1. Laboratories reporting on the BGM cell line. Number of Surveys Completed

Number of Surveys Uncompleted a

Number Not Returned

Location

Number Surveyed

Australia Brazil Canada Czechoslovakia Denmark England Hungary Israel Italy Japan Mexico Portugal Scotland Switzerland South Africa United States

3 l 5 1 1 4 2 5 1 1 1 1 1 1 3 67

3 36

14

17

Totals

98

58

15

25

3 3 1 1 4 l 3 1 1 1 1

aSurvey not completed because of insufficient experience with the cell line.

BGM cell culture practices

311

which cells still maintained the necessary sensitivity for their work. Several investigators stated that there was apparently a relationship between cell passage number and sensitivity to viruses. Failure of some laboratories to maintain proper records of cell passage numbers undoubtedly contributed to the wide variance noted in the passage numbers at which the cell line was no longer suitable for virus assays. Regardless of which cell culture system is used, procedures should include careful documentation of cell identification, origin, and passage number.

Propagation, Maintenance, and Harvesting o f BGM Cells In this section are grouped the results obtained from a systematic examination of the media, sera, culture vessels, planting densities, cultivation conditions, and suspending cell procedures used by participating laboratories. Table 3 lists the media used to culture the BGM cells. The responses showed negligible agreement as to a single preferred media composition. Serum was the one additive in the growth media which was used the most consistently. Fetal calf serum was used by 91°70 of the laboratories, 83070 of which used it at a 10070 concentration and 8°70 of which used it at a 5°7o concentration. The remaining respondents reported the use of either newborn calf serum, or calf serum alone or in combination with fetal calf serum. In response to other additives used, we were unable to establish a basis for agreement. The most striking variance was seen in the concentrations of NaHCO3 used to buffer the growth media. The concentrations ranged from a low of 0.02070 NaHCO3 to a high of 0.3%, with 0.075°70 and 0.11 °/0 most frequently listed. Other additives included HEPES buffer and L-glutamine, which were used by 15°70 and 27°70 of the laboratories, respectively. Nearly one-half the laboratories (4507o) indicated that they did not change the growth medium between each

Table 3. Growth media used for the BGM cell line by reporting laboratories.

Media Minimum Essential Medium (Eagle)a Minimum Essential Medium (Eagle) and Leibovitz's L-15 Minimum Essential Medium (Eagle) and Medium 199 Medium 199 Basal Medium Eagle RPMI Medium 1640 Hanks' Basic Salt Solution with lactalbumin hydrolysate Dulbecco's Modified Eagle Medium Dulbecco's Modified Eagle Medium and Basal Medium Eagle (Diploid)

Reporting Laboratories (%) 34 31 12 8 5 3

a35% of the reporting laboratories used autoclavable MEM.

passage of the cell line. Of those who did change to a maintenance medium, all listed the same medium as was used for cell growth, but with a lower concentration of serum. The interval between planting and medium change varied, with 82°70 changing between the second and fourth day after planting and the remaining 18% changing between the fifth and seventh day after planting. Temperatures at which cell cultures were grown and maintained ranged from 35-37°C, with the majority (6507o) at the higher temperature. The information submitted on the types of culture vessels used showed no significant degree of preference for glass or plastic. Nearly 4007o of the responding laboratories reported the use of both glass and plastic vessels, whereas the remaining laboratories were about equally divided between the use of either glass or plastic. The cell densities reported for planting culture vessels did not correlate to the vessel sizes used. At the extremes, planting densities ranged 500-fold (1 x 10" to 5 × 106 cells per mL) between the responding laboratories. With few exceptions, either a combination of trypsin and EDTA or trypsin alone was used to release cells attached to culture vessels. On the other hand, the time period allotted for the proteolytic enzyme to remain in contact with the cells varied from as little as 30 sec to as long as 30 min. Ten minutes or less was the time most frequently reported, although a significant number of the responding laboratories (33%) indicated a higher contact time. The frequency at which cell cultures were split also varied. Approximately one-half the laboratories subcultured the cell line at 7-day intervals. The others transferred the cell line at lesser time intervals, the least being 3 days. The great variety of techniques used only further illustrates the lack of uniformity in cell culture practices used with the BGM cell line.

Virus Susceptibility and Assay Procedures Results compiled on the viral detection methodology pertains solely to virus enumeration by the plaque technique. The marked variations we encountered in culturing procedures among the participating laboratories were also prevalent in those procedures applied for virus infection of this host cell line. This multitude of practices reported demonstrates the individualism exercised by monitoring laboratories. It further emphasizes the prevailing need for uniform methodology in the monitoring for these pathogens in environmental samples. Of the many variances noted, the most striking were the time intervals allowed between exposure of the cell monolayer to viruses and subsequent introduction of the agar overlay. As seen in Table 4, the time interval varied from as little as 10 min to the 120 min first used for virus determination with this cell line (Dahling et al., 1974). There were also deviations from the 22-24 °C incubation temperatures originally reported for this viral infection period. Incubation at 37 °C was used by 52% of the respondents; 36 °C by 18%; 35 °C by 10%; 32 °C by 2%, while 20-28 °C was used by only 18%. The

D. R. Dahling, R. S. Safferman, and B. A. Wright

312 Table 4. Time between BGM cell line exposure to virus sample and introduction of the agar overlay.

Exposure Time (min)

Reporting Laboratories (%)

10 15 30-45 60 90 120

4 2 15 43 12 24

time lapsing from the introduction of the agar overlay to final plaque count ranged from 4 days to more than 20 days, and in some laboratories as long as 30 days (Table 5). To provide a basis by which to evaluate the virus types that can be detected with this cell line, participating laboratories were requested to list the viruses that they could and could not plaque assay with BGM. Their responses indicated that the following enteroviruses could be plaque assayed: three types of polioviruses, six types of coxsackie B viruses, types 3, 5, 7, 9, and 16 of the coxsackie A viruses, and the 31 types of echoviruses. Viruses of other genera included the three reovirus types, adenovirus (not identified as to types) as well as parainfluenza, mumps, measles, and herpes viruses. Among the nonplaquable viruses listed by some respondents were the same viral types other respondents reported that they had plaque assayed. It is quite possible that some laboratories were able to plaque assay certain viruses that others could not because of differences in overlay media and/or growth media. Similarly, reactions of the respondents varied as to their experiences working with the BGM cell line. Additional comments on personal experiences with the BGM cell line were provided by 24 of the responding laboratories. For 56°7o of them, this cell line was sensitive to more viruses and/or yielded more viral isolates than other primary or continuous cell lines with which it had been compared. For 12o70, the cell line was no better or worse than other cell lines. The remaining 32°7o indicated other cell lines were better for isolation of viruses than the BGM cell line.

Table 5. Maximum incubation time for observing plaque formation in quantitative virus testing. Time (days)

Reporting Laboratories (%)

4 5-9 10-14 15-19 20-29 30

7.5 50 20 10 7.5 5

Focusing on the apparent disparity among laboratories were comments by some participants on differences in BGM cells when grown in different media under different overlay and on findings that reactions deviated from normal patterns when supplements were added at different stages of culture. They noted that these changes affected not only cell growth, but also sensitivity to viruses. Especially interesting was the fact that the majority of laboratories using the BGM cell line for both clinical and environmental work found these ceils to be less sensitive when evaluating clinical sampies. Also pertinent to these inconsistencies in cell line sensitivity was the discovery that one-half of those who used an autoclavable medium claimed reduced virus recoveries with the cell line. Discussion The widespread application of the BGM cell line has been the result of its superior sensitivity to many human enteric viruses contaminating water, wastewater, and soil environments. In the absence of guidelines clearly detailing step-by-step procedures, personal bias in an individual laboratory negates production of analytical data of known quality. It thus becomes increasingly important that a consistent system of virus detection and enumeration be established among virus monitoring laboratories. Unlike detection technology which has become standardized in other fields, environmental virology is only now reaching a stage of development which is sufficiently refined to allow attention to uniformity in the methods of detecting and quantifying viruses in environmental samples. Our first step toward this goal of a uniform viral assay procedure has been to evaluate the currently used practices that exist with the BGM cell line. The result of this evaluation raised an awareness of the need for conformity. The marked disparity that we observed in the methods used by the laboratories contacted indicated serious discrepancies in virus monitoring. No two respondent laboratories employed identical methodology for viral detection with the BGM cell line. More important, the methods used by some of these laboratories would ensure an appreciable reduction in viral sensitivity. This variation in methods will continue until reliable information and guidelines to upgrade and optimize laboratory performance are established and cooperation achieved. It has often been noted that one of the concerns in virus monitoring is negative results arising not from the absence of virus but from the lack of sensitivity due to procedures (Committee Report, 1970; Keswick et al., 1970; Mahdy, 1973; and Sobsey, 1982). Data resulting from the use of insensitive methodology may lead to a false sense of security. The key issue of this report is the variation in the materials and methods used by the participating laboratories. The environmental viral laboratories are faced with the problem of validating the

BGM cell culture practices

methods used in virus monitoring activities. On the basis of the data gathered from this study, we are developing a comparative testing program to maximize the BGM cell line sensitivity to human enteric viruses, the aim of which is to establish and promulgate a single uniform methodology for waterborne virus monitoring. Results of this program will be discussed in a forthcoming paper.

References Barron, A. L., Olshevsky, C., and Cohen, M. M. (1970) Characteristics of the BGM line of cells from African green monkey kidney. Archiv. Gesamte Virusforsch. 32, 389-392.

313 Berman, D., Berg, G., and Safferman, R. S. (1981) A method for recovering viruses from sludges. J. ViroL Methods 3, 283-291. Committee on Environmental Quality Management (1970) Engineering evaluation of virus hazards in water. J. San. Eng. Div. Proc. Am. Soc. Chem. Eng. 96, 111-161. Dahling, D.R., Berg, G., and Berman, D. (1974) BGM, a continuous cell line more sensitive than primary rhesus and African green kidney cells for the recovery of viruses from water. Health Lab. Sci. 11,275-282. Keswick, B. H. and Gerba, C. P. (1980) Viruses in groundwater. Environ. Sci. TechnoL 14, 1290-1297. Mahdy, M. S. (1973) Viruses in the environment and their significances, in Viruses in the environment and their potential hazards, M. S. Mahdy and B. J. Dutka, eds., pp. 2-10. Canada Centre for Inland Waters, Burlington, ON. Singh, S. N., Rose, J. B., and Gerha, C. P. (1983) Concentration of viruses from tap water and sewage with a charge-modified filter aid. J. ViroL Methods 6, 329-336. Sobsey, M. D. (1982) Quality of currently available methodology for monitoring viruses in the environment. Environ. Int. 7, 39-51.