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[19] In vitro induction of interferon from guinea pig fibroblasts
[19]
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alence between the 50% cytopathic effect inhibition and the 50% plaque reduction (PRs0 VSV) endpoints. In our assay 1 PRs0 (VSV) unit is equivalent to about 10-25 units of the chick interferon MRC Research Standard A 62/4 provided by the National Institute of Allergy and Infectious Diseases Research Resources Branch. 2°-22 This means that the peak yield of 75,000 units o f l F N per 10 7 " a g e d " CEC shown in Fig. 2 would convert to an apparent yield of between 750,000 and 1,875,000 standard units, i.e., about 0.13 unit per cell. H o w e v e r , since IFN induction under these conditions results in a type r = 1 d o s e - r e s p o n s e c u r v e , 6A6 the actual yield per cell is 2.7 times higher (only 37% of the cells are producing IFNJ6), namely, 0.35 unit per cell. As an additional control, in each interferon assay we include our own chick interferon standard [a single batch of chick interferon containing 20,000 PRs0 (VSV) units/ml stored at - 2 0 °] in order to ensure that the different batches of chick embryo cell monolayers used for interferon assays throughout the course of our work respond uniformly to interferon treatment. Acknowledgments This work was supported by NIAID Grant AI-18381 and benefited from the use of a Cell Culture Facility supported in part by NCI Grant CA-14733 and by The University of Connecticut Research Foundation. 20N. B. Finter, in "Interferons and Interferon Inducers" (N. B. Finter, ed.), p. 161. Elsevier, Amsterdam, 1973. zl N. B. Finter, this series, Vol. 78 [2]. 22S. Pestka, this volume [2]. 23M. J. Sekellick and P. I. Marcus, J. Interferon Res. 5, 651 (1985).
[19] I n V i t r o I n d u c t i o n o f I n t e r f e r o n f r o m Guinea Pig Fibroblasts By TIMOTHY R. WINSHIP Introduction The guinea pig (Cavia cobaya, family Caviidae) is an attractive model for studies o f infectious disease since cavids are larger than most rodents but relatively easy to care for and manipulate. The production and use of biological response modifiers in guinea pigs, however, have been frustrating due in part to the difficulties associated with the in vitro culture of guinea pig cells. METHODS IN ENZYMOLOGY, VOL. 119
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Several investigators have reported attempts to produce guinea pig interferons, both in vitro and in vivo, with various levels of success. These efforts have been reviewed by Sonnenfeld in a previous volume of this series.l Interferon yields were usually low and difficult to assay. Our own efforts to produce guinea pig interferon in quantities sufficient for in vivo purposes centered around the use of fibroblasts originating from whole guinea pig embryos (GPE cells) for interferon induction and assay. We found that crude guinea pig fibroblast interferons do not share all the typical properties of classical fibroblast-derived interferons and require careful attention to induction and storage conditions. Cell Culture Suspensions of guinea pig embryo cells were prepared from minced and gently trypsinized whole guinea pig embryos of about 30 days gestation by a previously published technique. 2 Primary cells are seeded at a density of 3 x 104 cells per cm 2 of growth area in 0.25 ml of minimal essential medium with Hanks' salts (HMEM) plus 10% newborn calf serum (Flow Laboratories or Kansas City Biological, Inc.). This medium also included extra glutamine (tO 494 mg/liter) and nonessential amino acids. Primary cells seeded in this way generally achieve confluence after 48-72 hr incubation at 37°. The seeding density used is important since the interferon-producing capacity of GPE cells decreases with cell passage number. Confluent GPE monolayers can be maintained in MEM with Earle's salts (EMEM), extra glutamine (494 mg/ml), nonessential amino acids, and 5% newborn calf serum for several days. Optimal interferon induction conditions, which are somewhat different than those suitable for cell maintenance, are described in detail below. Monolayers of primary guinea pig embryo fibroblasts are useful for the production of relatively small volumes (1-2 liters) of interferon. Such monolayers often give the highest yields of interferons per milliliter of any guinea pig cell (up to 6000 PRs0 units/ml). Primary GPE cells do not, however, readily lend themselves to larger scale techniques. One can produce larger volumes of crude guinea pig fibroblast interferon at useful concentrations through the use of passaged guinea pig embryo fibroblasts. While GPE cells can be successfully passaged more than 10 times, the cells lose their ability to produce significant quantities of interferon after the fourth passage (counting the initial growth of cells from trypsinized G. Sonnenfeld, this series, Vol. 78, p. 162. 2 G. D. Hsiung, R. B. Tenser, and C. K. Y. Fong, Infect. lmmun. 13, 926 (1976).
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tissue as passage 1) when seeded at a density of 3 × 104 cells per cm 2 growth area. Passaged GPE cells can be adapted for growth on roller vessels. Particular attention must be paid to the degree of cleanliness of the vessels if glass roller bottles are used since the cells are intolerant of even minute quantities of detergent residue. Several brands of plastic disposable roller bottles were tried. We found both Corning and Falcon bottles equally suitable for GPE cell culture. Adaptation of GPE cells to roller culture is best achieved by the following conditions. The primary cell contents of two 120-cm2 culture flasks (32-oz glass prescription bottles) are transferred to a single 690-cm 2 glass roller bottle (Wheaton) with 175 ml of HMEM containing 10% newborn calf serum prepared as described above. The cells should achieve confluence in 48-72 hr at 37°. When these initial roller cultures become confluent they can be expanded to other roller flasks as long as a split ratio of 1 : 3 is not exceeded. The volume of medium added to any size roller flask should be 0.25 ml/ c m 2 of growth area to achieve reproducible cell growth and maintenance. It is important to initially seed these cells in medium whose pH will not change beyond the range of 6.8-7.2, particularly during the first 24 hr of culture. GPE cells used in interferon induction experiments showed extreme sensitivity to changes in pH. Interferon Induction Flasks or roller bottles of GPE cells are usually not suitable for interferon induction if the cells do not become confluent within 72 hr of seeding. When the cells reach confluence, the medium in each culture vessel is discarded and flesh EMEM plus 10% newborn bovine serum added. This medium is allowed to remain unchanged on the cells for an "aging" period of 7 days at 37°. Careful pH control of the medium during this period is essential for cell survival and optimal interferon production. We maintained medium pH between 6.8 and 7.2 by the periodic addition of small quantities of 7.5% sodium bicarbonate solution, but other buffering systems could be used for equal effect. We found an aging period to be essential for production of optimal interferon quantities. This requirement has also been observed in some other cell-virus systems, and was first noted by Carver and Marcus 3 (see also Sekellick and Marcus4). At the end of the aging period the medium is removed from each 3D. H. Carver and P. I. Marcus, Virology 32, 247 (1967). 4 M. J. Sekellickand P. I. Marcus, this volume [18].
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culture vessel and the monolayers washed with warmed Hanks' buffered salt solution (HBSS). The cells are then infected with an inducing virus. We found several viruses that would induce interferon from guinea pig embryo fibroblasts, including Newcastle disease virus (NDV), Sindbis virus, and vesicular stomatitis virus (VSV) T1026RI. Maximum interferon production with each inducing virus was very dependent on multiplicity of infection. Of the viruses we tested, VSV T1026R1 induced the greatest quantities of interferon. 5 VSV T1026R1 is a non-ts revertant of a mutant originated by C. P. Stanners. 6 It is best prepared in Vero cells since yields of this virus tend to be low when cell lines that will produce interferon are used. Wild type VSV did not induce measurable quantities of interferon from infected GPE cells. After an appropriate adsorption period at 37° (1 hr for VSV T1026RI or Sindbis virus, 2 hr for NDV) the viral inoculum is removed from the infected culture and the monolayers rinsed three times with warmed HBSS. The infected cells are then fed with EMEM containing 0.5% newborn bovine serum. We were not able to induce significant amounts of guinea pig interferon in totally serum-free media. The infected cultures are then incubated 24 hr at 37°. An incubation temperature below 36 or above 38° is detrimental to the yield of interferon. Harvest and Storage of Interferon Interferon-containing supernatant medium from infected monolayers can be harvested 18-24 hr after inoculation. Time of harvest is important since the period of maximum interferon production is brief and the crude product is unstable at 370.7 The crude material can be stored at - 7 0 ° for at least 6 months. It loses some activity after 2 weeks storage at - 2 0 ° or if held overnight at 4 °. Interferon Assay A standard plaque reduction assay was our method of choice for the quantitation of guinea pig interferon levels. Passaged guinea pig embryo cells were used for all such assays. The sensitivity of such cells to the antiviral effects of interferon was not affected by density of seeding, cell " a g e , " or passage number. We did note, however, that lots of passaged 5 T. R. Winship, C. K. Y. Fong, and G. D. Hsiung, J. Interferon Res. 3, 71 (1983). 6 C. P. Stanners, A. M. Francoeur, and T. Lam, Cell 11, 273 (1977). 7 T. R. Winship, C. K. Y. Fong, and G. D. Hsiung, J. Gen. Virol. 65, 843-847 (1984).
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guinea pig fibroblasts originating from different groups of embryos could vary significantly (up to 3-fold) in their sensitivity to a "standard" interferon preparation. This may have been due to the use of outbred Hartley strain guinea pigs as a source of cells. Therefore experiments dealing with guinea pig interferon need to be assayed on a single lot of GPE fibroblasts to avoid excessive variation of titers. Wild-type VSV was used as the challenge virus in all such assays. Results were expressed in 50% plaque reduction (PRs0) units per ml. The standard method of inactivating the inducing virus by treatment at pH 2 for 24 hr was inappropriate for guinea pig interferon since it resulted in the loss of more than half the antiviral activity of crude preparations. 5,7 Of the other methods available for the removal of inducing virus, irradiation of samples with shortwave ultraviolet light was rapid and reliable. One milliliter quantities of crude interferon were irradiated in 35-ram Petri dishes with a single 15-W germicidal lamp at a distance of 25 cm. An 8 min exposure under these conditions was used routinely to inactivate the virus in crude preparations. This exposure was approximately four times that needed to completely inactivate the inducing virus. VSV T1026R1, NDV, or Sindbis virus inactivated in this way could not induce interferon in guinea pig cells and did not interfere with wild-type VSV in interferon assays. Remarks Our experiments with the production of crude guinea pig interferon showed the critical role cell culture and type of viral inducers can have on interferon yields. It was clear from this work that not all antiviral substances produced by cell cultures behave as "classical" Type I interferons. 8 We strongly recommend that investigators attempting to induce interferon from uncharacterized animals or cell cultures initially avoid subjecting their preparation to heat, low pH, or other chemical and physical regimens to which interferons are " k n o w n " to be resistant.
s S. Pestka and S. Baron, this series, Vol. 78, p. 3.
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alence between the 50% cytopathic effect inhibition and the 50% plaque reduction (PRs0 VSV) endpoints. In our assay 1 PRs0 (VSV) unit is equivalent to about 10-25 units of the chick interferon MRC Research Standard A 62/4 provided by the National Institute of Allergy and Infectious Diseases Research Resources Branch. 2°-22 This means that the peak yield of 75,000 units o f l F N per 10 7 " a g e d " CEC shown in Fig. 2 would convert to an apparent yield of between 750,000 and 1,875,000 standard units, i.e., about 0.13 unit per cell. H o w e v e r , since IFN induction under these conditions results in a type r = 1 d o s e - r e s p o n s e c u r v e , 6A6 the actual yield per cell is 2.7 times higher (only 37% of the cells are producing IFNJ6), namely, 0.35 unit per cell. As an additional control, in each interferon assay we include our own chick interferon standard [a single batch of chick interferon containing 20,000 PRs0 (VSV) units/ml stored at - 2 0 °] in order to ensure that the different batches of chick embryo cell monolayers used for interferon assays throughout the course of our work respond uniformly to interferon treatment. Acknowledgments This work was supported by NIAID Grant AI-18381 and benefited from the use of a Cell Culture Facility supported in part by NCI Grant CA-14733 and by The University of Connecticut Research Foundation. 20N. B. Finter, in "Interferons and Interferon Inducers" (N. B. Finter, ed.), p. 161. Elsevier, Amsterdam, 1973. zl N. B. Finter, this series, Vol. 78 [2]. 22S. Pestka, this volume [2]. 23M. J. Sekellick and P. I. Marcus, J. Interferon Res. 5, 651 (1985).
[19] I n V i t r o I n d u c t i o n o f I n t e r f e r o n f r o m Guinea Pig Fibroblasts By TIMOTHY R. WINSHIP Introduction The guinea pig (Cavia cobaya, family Caviidae) is an attractive model for studies o f infectious disease since cavids are larger than most rodents but relatively easy to care for and manipulate. The production and use of biological response modifiers in guinea pigs, however, have been frustrating due in part to the difficulties associated with the in vitro culture of guinea pig cells. METHODS IN ENZYMOLOGY, VOL. 119
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
126
INDUCTION OF INTERFERONS
[19]
Several investigators have reported attempts to produce guinea pig interferons, both in vitro and in vivo, with various levels of success. These efforts have been reviewed by Sonnenfeld in a previous volume of this series.l Interferon yields were usually low and difficult to assay. Our own efforts to produce guinea pig interferon in quantities sufficient for in vivo purposes centered around the use of fibroblasts originating from whole guinea pig embryos (GPE cells) for interferon induction and assay. We found that crude guinea pig fibroblast interferons do not share all the typical properties of classical fibroblast-derived interferons and require careful attention to induction and storage conditions. Cell Culture Suspensions of guinea pig embryo cells were prepared from minced and gently trypsinized whole guinea pig embryos of about 30 days gestation by a previously published technique. 2 Primary cells are seeded at a density of 3 x 104 cells per cm 2 of growth area in 0.25 ml of minimal essential medium with Hanks' salts (HMEM) plus 10% newborn calf serum (Flow Laboratories or Kansas City Biological, Inc.). This medium also included extra glutamine (tO 494 mg/liter) and nonessential amino acids. Primary cells seeded in this way generally achieve confluence after 48-72 hr incubation at 37°. The seeding density used is important since the interferon-producing capacity of GPE cells decreases with cell passage number. Confluent GPE monolayers can be maintained in MEM with Earle's salts (EMEM), extra glutamine (494 mg/ml), nonessential amino acids, and 5% newborn calf serum for several days. Optimal interferon induction conditions, which are somewhat different than those suitable for cell maintenance, are described in detail below. Monolayers of primary guinea pig embryo fibroblasts are useful for the production of relatively small volumes (1-2 liters) of interferon. Such monolayers often give the highest yields of interferons per milliliter of any guinea pig cell (up to 6000 PRs0 units/ml). Primary GPE cells do not, however, readily lend themselves to larger scale techniques. One can produce larger volumes of crude guinea pig fibroblast interferon at useful concentrations through the use of passaged guinea pig embryo fibroblasts. While GPE cells can be successfully passaged more than 10 times, the cells lose their ability to produce significant quantities of interferon after the fourth passage (counting the initial growth of cells from trypsinized G. Sonnenfeld, this series, Vol. 78, p. 162. 2 G. D. Hsiung, R. B. Tenser, and C. K. Y. Fong, Infect. lmmun. 13, 926 (1976).
[19]
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tissue as passage 1) when seeded at a density of 3 × 104 cells per cm 2 growth area. Passaged GPE cells can be adapted for growth on roller vessels. Particular attention must be paid to the degree of cleanliness of the vessels if glass roller bottles are used since the cells are intolerant of even minute quantities of detergent residue. Several brands of plastic disposable roller bottles were tried. We found both Corning and Falcon bottles equally suitable for GPE cell culture. Adaptation of GPE cells to roller culture is best achieved by the following conditions. The primary cell contents of two 120-cm2 culture flasks (32-oz glass prescription bottles) are transferred to a single 690-cm 2 glass roller bottle (Wheaton) with 175 ml of HMEM containing 10% newborn calf serum prepared as described above. The cells should achieve confluence in 48-72 hr at 37°. When these initial roller cultures become confluent they can be expanded to other roller flasks as long as a split ratio of 1 : 3 is not exceeded. The volume of medium added to any size roller flask should be 0.25 ml/ c m 2 of growth area to achieve reproducible cell growth and maintenance. It is important to initially seed these cells in medium whose pH will not change beyond the range of 6.8-7.2, particularly during the first 24 hr of culture. GPE cells used in interferon induction experiments showed extreme sensitivity to changes in pH. Interferon Induction Flasks or roller bottles of GPE cells are usually not suitable for interferon induction if the cells do not become confluent within 72 hr of seeding. When the cells reach confluence, the medium in each culture vessel is discarded and flesh EMEM plus 10% newborn bovine serum added. This medium is allowed to remain unchanged on the cells for an "aging" period of 7 days at 37°. Careful pH control of the medium during this period is essential for cell survival and optimal interferon production. We maintained medium pH between 6.8 and 7.2 by the periodic addition of small quantities of 7.5% sodium bicarbonate solution, but other buffering systems could be used for equal effect. We found an aging period to be essential for production of optimal interferon quantities. This requirement has also been observed in some other cell-virus systems, and was first noted by Carver and Marcus 3 (see also Sekellick and Marcus4). At the end of the aging period the medium is removed from each 3D. H. Carver and P. I. Marcus, Virology 32, 247 (1967). 4 M. J. Sekellickand P. I. Marcus, this volume [18].
128
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[19]
culture vessel and the monolayers washed with warmed Hanks' buffered salt solution (HBSS). The cells are then infected with an inducing virus. We found several viruses that would induce interferon from guinea pig embryo fibroblasts, including Newcastle disease virus (NDV), Sindbis virus, and vesicular stomatitis virus (VSV) T1026RI. Maximum interferon production with each inducing virus was very dependent on multiplicity of infection. Of the viruses we tested, VSV T1026R1 induced the greatest quantities of interferon. 5 VSV T1026R1 is a non-ts revertant of a mutant originated by C. P. Stanners. 6 It is best prepared in Vero cells since yields of this virus tend to be low when cell lines that will produce interferon are used. Wild type VSV did not induce measurable quantities of interferon from infected GPE cells. After an appropriate adsorption period at 37° (1 hr for VSV T1026RI or Sindbis virus, 2 hr for NDV) the viral inoculum is removed from the infected culture and the monolayers rinsed three times with warmed HBSS. The infected cells are then fed with EMEM containing 0.5% newborn bovine serum. We were not able to induce significant amounts of guinea pig interferon in totally serum-free media. The infected cultures are then incubated 24 hr at 37°. An incubation temperature below 36 or above 38° is detrimental to the yield of interferon. Harvest and Storage of Interferon Interferon-containing supernatant medium from infected monolayers can be harvested 18-24 hr after inoculation. Time of harvest is important since the period of maximum interferon production is brief and the crude product is unstable at 370.7 The crude material can be stored at - 7 0 ° for at least 6 months. It loses some activity after 2 weeks storage at - 2 0 ° or if held overnight at 4 °. Interferon Assay A standard plaque reduction assay was our method of choice for the quantitation of guinea pig interferon levels. Passaged guinea pig embryo cells were used for all such assays. The sensitivity of such cells to the antiviral effects of interferon was not affected by density of seeding, cell " a g e , " or passage number. We did note, however, that lots of passaged 5 T. R. Winship, C. K. Y. Fong, and G. D. Hsiung, J. Interferon Res. 3, 71 (1983). 6 C. P. Stanners, A. M. Francoeur, and T. Lam, Cell 11, 273 (1977). 7 T. R. Winship, C. K. Y. Fong, and G. D. Hsiung, J. Gen. Virol. 65, 843-847 (1984).
[19]
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guinea pig fibroblasts originating from different groups of embryos could vary significantly (up to 3-fold) in their sensitivity to a "standard" interferon preparation. This may have been due to the use of outbred Hartley strain guinea pigs as a source of cells. Therefore experiments dealing with guinea pig interferon need to be assayed on a single lot of GPE fibroblasts to avoid excessive variation of titers. Wild-type VSV was used as the challenge virus in all such assays. Results were expressed in 50% plaque reduction (PRs0) units per ml. The standard method of inactivating the inducing virus by treatment at pH 2 for 24 hr was inappropriate for guinea pig interferon since it resulted in the loss of more than half the antiviral activity of crude preparations. 5,7 Of the other methods available for the removal of inducing virus, irradiation of samples with shortwave ultraviolet light was rapid and reliable. One milliliter quantities of crude interferon were irradiated in 35-ram Petri dishes with a single 15-W germicidal lamp at a distance of 25 cm. An 8 min exposure under these conditions was used routinely to inactivate the virus in crude preparations. This exposure was approximately four times that needed to completely inactivate the inducing virus. VSV T1026R1, NDV, or Sindbis virus inactivated in this way could not induce interferon in guinea pig cells and did not interfere with wild-type VSV in interferon assays. Remarks Our experiments with the production of crude guinea pig interferon showed the critical role cell culture and type of viral inducers can have on interferon yields. It was clear from this work that not all antiviral substances produced by cell cultures behave as "classical" Type I interferons. 8 We strongly recommend that investigators attempting to induce interferon from uncharacterized animals or cell cultures initially avoid subjecting their preparation to heat, low pH, or other chemical and physical regimens to which interferons are " k n o w n " to be resistant.
s S. Pestka and S. Baron, this series, Vol. 78, p. 3.