Hope E. Hopps

PROBLEMS CONCERNING “NONCULTIVABLE” MYCOPLASMA CONTAMINANTS IN TISSUE CULTURES

The inadvertent contamination of cell cultures by a variety of agents-bacteria, fungi, bacteriophages, viruses, and mycoplasmas-has long plagued the cell biologist.’,* Contamination of cultures with mycoplasmas is a particularly difficult problem because many of these agents exert no obvious effect on the appearance of the culture and hence go undetected unless the investigator makes a special effort to look for them. Indeed, experience has taught many that good cell husbandry can be effective in the elimination of these ubiquitous pests.3 Propagation of cell cultures in the absence of antibiotics, good handling techniques, and frequent monitoring are three of the major practices that can be used to eliminate contamination. Even under these conditions, however, one occasionally encounters mycoplasma contaminants that cannot be readily isolated by conventional techniques.4~5 The characterization and subsequent isolation of one such contaminant constitutes the subject of this report.

THE IDENTIFICATION AND SOURCES OF MYCOPLASMAS ISOLATED FROM CONTAMINATED CELL CULTURES

Mycoplasmas have been established as common contaminants of cell cult u r e ~ . ~ In 1962, the Bureau of Biologics* instituted a requirement to test for the presence of mycoplasmas in viral vaccines for human use that were produccd in cell cultures. Accordingly, we have maintained a continuing study in order to examine and establish the sources of mycoplasma contamination of cell cultures and of biologics for clinical use. The results of this continuing study will be summarized in this report.

Microbiological Methods and Fluorescent Microscopy for the Direct Demonstration of Mycoplasma Infection of Cell Cultures

Our principal experience with immunofluorescence is in its application to the identification of mycoplasma colonies growing on agar medium (5). The immunofluorescent identification results of mycoplasmas isolated from cell cultures over an 11-year period are summarized herein. Most recently we have used immunofluorescence to identify M. hyorhinis in infected cell cultures. These results will be presented with special reference to auxotrophic variants of M. hyorhinis. The auxotrophs are strains that are noncultivable on media that support the growth of the type strain of M. hyorhinis (BTS-7) and other similar strains. The term “noncultivable mycoplasma” (7) is used in this sense to describe a strain (DBS 1050 = ATCC 29052) that grew in cell cultures but not on agar medium. This specific connotation is obscured by the current use of the term in reference to apparent isolation failures.

Development and characerization of cell lines from subhuman primates

SummarySeven epithelial cell lines derived from kidney and 20 fibroblastic cell lines deriving from lung, heart, muscle, kidney, and skin tissue of five rhesus and six African green monkey fetuses have been established and propagated in culture. Four epithelial cell two fibroblastic cell lines resumed cell multiplication after a period of growth decline, and these lines developed cytogenetic changes and growth characteristics of cells capable of unlimited growth in vitro.Sixteen of the fibroblastic lines derived from lung, heart, muscle, or skin were characterized by a finite life consisting of a period of active cell multiplication, followed by growth decline, senescence, and cell death. Fibroblasts derived from lung appeared to have the greatest growth potential in terms of total population doublings, and fibroblastic lines from rhesus monkeys were usually capable of more doublings than similar lines from African green monkeys. All fibroblastic lines were predominantly diploid during active growth from passages 1 to 30, but several lines developed karyological changes preceding or during growth decline and senescence.All lines tested were found sensitive to a number of human viruses. All tests on these cells for microbial agents and for tumorigenicity have been negative, and the have been preserved by freezing without loss of properties.These cell lines may be useful as standardized substrates in studies requiring nonhuman primate cells.

Cell growth optimization in microcarrier culture.

SummaryThree monkey kidney cell lines and primary chicken embryo cells were grown in microcarrier culture. The carrier support was DEAE-Sephadex gel beads at low anion exchange capacity prepared according to a protocol developed at the Massachusetts Institute of Technology.The growth rate of the cells and the final cell density in microcarrier culture was dependent on the concentration of the beads in culture and on the size of the initial cell inoculum. A bead concentration of 1.0 to 2.0 mg of beads/ml of tissue culture medium and a cell inoculum of 20,000 cells/cm2 of bead surface appeared to be optimal. The efficiency of the microcarrier culture system was compared to that of stationary and roller bottle cultures. Stationary flasks gave cell densities about twofold higher than maximal densities in roller bottles and about threefold and twofold higher than cell densities in microcarrier culture at a bead concentration of 2.5 and 1.0 mg/ml, respectively.In terms of cell yield per millitier of tissue culture medium, the microcarrier culture was superior to roller bottle and stationary cultures. An advantage of the microcarrier culture system is its suitability for a scale up into large volume production units.

Cultivation of formerly noncultivable strains ofMycoplasma hyorhinis

Growth on axenic agar medium is one of several characters by which mycoplasmas are defined. In apparent contradiction of the definition, DBS 1050 and other noncultivable strains ofMycoplasma hyorhinis do not grow on axenic medium but grow in cell culture. Our results show that BHK-21 cell extracts support DBS 1050 growth in appropriate medium. An inhibition assay, based on a virus neutralization format, shows that a variety of common medium ingredients inhibit DBS 1050 growth. The most potent activity was found in yeast extract. All other noncultivable strains ofM. hyorhinis tested have a yeast extract sensitivity, while cultivable strains do not. The apparent cell dependence of DBS 1050 can be attributed to growth inhibition due to factors present in a wide variety of peptones and extracts commonly used in medium; preferential growth in cell cultures is due to the absence of effective levels of these factors. Data are not available to determine if cell cultures provide growth factors not found in standard medium. The infraspecific taxon,M. hyorhinis cultivar α, is proposed for formerly noncultivable strains ofM. hyorhinis.

Development of a diploid cell line from fetal rhesus monkey lung for virus vaccine production

SummaryThe primary goal of this study was to develop and characterize diploid cell lines from fetal tissues of subhuman primates for use in virus vaccine production. Cell lines have been established from fetal tissues of rhesus and African green monkeys, and these have been characterized according to the general criteria recommended by the International Cell Committee for Microbiological Standardization. Of these cell lines, DBS-FRhL-2, developed from lung tissue of a rhesus monkey fetus, has been found to meet the requirements of populations proposed as substrates for virus vaccines.Characterization studies show that DBS-FRhL-2 cells have a finite life of more than 50 population doublings in vitro and maintain the diploid karyotype through an active growth phase. The cells are nontumorigenic, and tests have not revealed the presence of adventitious agents. They are susceptible to a number of human viruses and can be preserved by freezing without change in virus susceptibility, cytogenetic, or growth characteristics. These results indicate the need for further evaluation of this rhesus monkey diploid cell line for acceptability as an alternate substrate in the manufacture of human virus vaccines.

Incidence and Sources of Mycoplasma Contamination: A Brief Review

In 1956, Robinson and colleagues (1) reported the first isolation of a mycoplasma from a contaminated cell culture. Subsequently, mycoplasmas have been shown to be common and bothersome contaminants capable of altering the activity of cells and affecting the results of study. Because many of the vaccines prepared for human use are produced in cell cultures and are subject to mycoplasma contamination, the Bureau of Biologics has maintained a continuing study for the past 18 years to examine various aspects of mycoplasma contamination. This report will review some of our findings and present a brief, updated status report on the incidence, prevalence and sources of mycoplasma contamination. The subject has been reviewed in detail elsewhere (2–5).

Propagation of poliovirus inmicrocarrier cultures of three monkey kidney cell lines

The microcarrier culture system was applied to the propagation of polioviruses in three established monkey kidney cell lines, Vero, LLC-MK 2 and CV-1, and the yields were compared with that obtained in roller bottle and stationary cultures. The yield of the three types of polioviruses in the various cell lines and culture systems were similar. However, the use of the microcarrier culture system for anchorage-dependent cells together with the use of continuously propagating cell lines offer great advantage for large-scale cultivation of poliovirus for the preparation of killed poliovirus vaccines.

Report of Ad Hoc Committee on Karyological Controls of Human Cell Substrates

A conference on the Standardization of Cell Substrates for the Production of Virus Vaccines was jointly sponsored by the World Health Organization and by the International Association of Biological Standardization at Geneva in 1976. Participants at the meeting agreed that recommendations then current regarding karyological control of human cell substrates should be reviewed and revised. Extensive experience using human diploid cells in vaccine manufacture as documented at that symposium indicated that certain karyological requirements of existing standards should be eliminated or modified. An Ad Hoc committee appointed by the chairman of the conference, Dr Frank Perkins, was charged with this task; however, since opinions varied widely regarding modification this committee requested the organization of a karyology workshop.