Archives of Virology
Archives of Virology 61, 69--85 (1979)
© by Springer-Verlag 1979
Clones of Cells From a Human Embryo Lung: Their Growth and Susceptibility to Respiratory Viruses By
D. A. J. TYt~RELL1, MARCIA MIKA-JoH~so~, and P. J. CI~APPI~E W. Alton Jones Cell Science Center, Lake Placid, New York, U.S.A. With 7 Figures Accepted March 5, 1979
Summary Colonies of cells were obtained from human fetal lung tissue and exposed to recently isolated respiratory viruses. There was a considerable variation in the number of rounded cells found in different colonies exposed to rhinovirus types 2 and 9 (RV2 and 9), human coronavirus 229E (HCV), adenovirus type 3 (Ad3) and respiratory syncytial virus (RSV). Smaller colonies had more rounded cells than larger colonies. Clones were established from 9 out of 11 colonies. They varied in their rate of growth and the pattern formed on a plastic surface. They varied also in their virus susceptibility particularly to "difficult" rhinoviruses such as RV 9 and SF 1340. One cell clone (HL 1/77 Clone 8), was highly susceptible to all these viruses. All cultures were more sensitive to ~ S V when maintained in F ] 2 K medium than in MEM, whereas there was no difference for rhinoviruses. Influenza A and B and parainfluenza 3 viruses sometimes produced cytopathie effect, and always produced haemadsorption, but unlike the previous strains could not be passed serially and presumably produced little infectious virus. All clones were rather insusceptible to Ad 3; but the virus could be passed, whereas coxsackie virus B 3 produced no CPE. Substantial yields of coronavirus and rhinovirus were obtained in gelatin sponge cultures. Two " v e r y difficult" respiratory viruses which had just been adapted to tissue culture; namely, HS rhinovirus and J K coronavirus grew in 7 of 9 and in 6 of 9 clones respectively.
i l~resent address: Clinical Research Centre, Division of Communicable Watford ]Road, Harrow, Middlesex HA 1 3U J, England. u Present address: Diagnostic Laboratory, Department of Preventive New York State College of Veterinary Medicine, Ithaca, NY 14853, U.S.A.
Diseases, Medicine,
0304-8608/79/0061/0069/$ 03.40
70
D . A . J . TYX~aELL, MAtCOIAMIKA-JOHNSO.W,and P. J. CI~APSLE:
Introduction H u m a n e m b r y o n i c l u n g is a complex tissue a n d contains the precursors of a v a r i e t y of cells r a n g i n g from fibrous tissue elements ~nd vascular e n d o t h e l i u m to bronchial epithelium, histiocytes, macrophages a n d p n e u m o c y t e s , including the type I I cells, l~ecently, type I I p n e u m o c y t e s h~ve been separated from the others b y cloning techniques, a n d their virus susceptibility has been studied (9). W e t h o u g h t t h a t it should also be possible to sepg:r~te out some of the other cells using the same basic techniques, a n d this report summarizes the results of our efforts. W e wished to d e t e r m i n e whether we could separate from a mixed culture of l u n g cells clones which were d e m o n s t r a b l y distinct, as j u d g e d b y their morphology, growth a n d o~her characteristics. More i m p o r t a n t l y , we wished to see if those clones were d e m o n s t r a b l y distinct in their susceptibility to infection with a range of respiratory viruses. Studies have shown t h a t organ cultures of bronchial epithelium are generMly susceptible to almost all k n o w n viruses, from rhinoviruses, a n d coronaviruses to influenza a n d p a r a i n f l u e n z a viruses (5). On the other hand, strains of fibroblasts obtMned b y mass culture of e m b r y o n i c lung, such us W I - 3 8 or MRC-5, are susceptible to some rhinoviruses a n d some coronaviruses b u t n o t to influenza or parMnfluenza viruses. Earlier studies showed t h a t mass cultures of fibroblasts obtMned from m a n y e m b r y o s are r e l a t i v e l y resistant to r h i n o v i r u s infection (1). We, therefore, hoped t h a t b y using cloning t e c h n i q u e s we would be able to detect, a n d cultivate virus-susceptible l u n g cells which would otherwise be lost if the usual m e t h o d s of mass culture were used. These susceptible cells m i g h t be useful as substratcs for producing virus vsccines or for c u l t i v a t i n g a n d s t u d y i n g viruses t h a t , ~t present, can be grown only in tracheal organ culture.
Materials and Methods Yledia Cells were, in general, grown in H a m ' s F 12K (modified) medium with 10 percent fetal bovine serum (FBS) (Flow Laboratories Lot ~00109t). They were maintained in the same medium with 2 percent FBS except where otherwise indicated. Tissue and Cell Cloning Lungs were obtained from fetuses up to 72 hours after hysterotomy. The tissue was chopped and dispersed with collagenase, trypsin and chicken serum as described elsewhere (9); and a first passage in mass culture was performed, and the resulting cells were stored in liquid nitrogen. The cells were then thawed, and established in viable culture. Cells from these cultures were counted using the erythrosin B dye exclusion technique. Four hundred viable eells were seeded in 10 ml of medium into a t00 m m petri dish and kept without disturbance in a humidified incubator at 37 ° C. Cultures were examined with an inverted microscope equipped with phase contrast optics. I n certain experiments, cells were classified as "fibroblast-like"--long, nalTow cells often in whorls, "endo- or epithelial-like"---pavements of polygonal cells or, " i n t e r m e d i a t e " - - a varied range of cells between these extremes. Selected colonies were isolated with a silicone-treated penicillin assay cylinder. They were washed twice with saline and then incubated with 0.25 percent trypsin: EDTA until the cells were released; they were then pipetted into a 35 m m dish and grown up until they filled a plastic flask with a surface area of 75 emK From t~his stock, cells were stored in liquid nitrogen in 5 percent dimethyl sutfoxide as a cryoprotectant. Stock cultures were trypsinized and passed at about five-day intervals, and each new flask was in-
V i r u s S u s c e p t i b i l i t y of L u n g Cell Clones
7t
oculaf~ed w i t h 1 × 106 cells i n 25 m l of m e d i u m c o n t a i n i n g 10 p e r c e n t F B S . A s r e q u i r e d , roller t u b e s w e r e p r e p a r e d u s i n g a b o u t 10 ~ cells in 1 m l of m e d i m n c o n t a i n i n g 10 p e r c e n t F B S a n d E a g l e ' s M E M b u f f e r e d w i t h H E P E S or in R a m ' s m o d i f i e d F 1 2 K . W h e n r e q u i r e d for v i r u s i n o c u l a t i o n , t u b e s w e r e c h a n g e d t o 1 m l of m e d i u m c o n t a i n i n g 2 p e r c e n t F B S , or w h e n u s i n g i n f l u e n z a or p a r a i n f l u e n z a v i r u s , t h e y were w a s h e d t h r e e t i m e s w i t h 1 mI a n d m a i n t a i n e d w i t h 1 m l of s e r u m - f r e e m e d i u m .
Approximate Cell Doubling Time T 75 flasks were seeded w i t h 106 cells, i n c u b a t e d for a b o u t five d a y s a n d t r y p s i n i z e d as t h e cell s h e e t w a s a p p r o a c h i n g confluence. T h e cell s h e e t w a s t r y p s i n i z e d a n d t h e n u m b e r of cells w a s m e a s u r e d w i t h a C o u l t e r c o u n t e r . T h e a p p a r e n t cell d o u b l i n g t i m e was computed from these numbers.
Viruses All t h e v i r u s e s h a d b e e n p a s s e d o n l y a few t i m e s since t h e i r i s o l a t i o n ~rom t h e h u m a n r e s p i r a t o r y t r a c t . D e t a i l s c o n c e r n i n g t h e m are s h o w n i n T a b l e 1. T h e y were e i t h e r k n o w n t o b e p a t h o g e n i c for m a n or could b e safely a s s u m e d to be.
Tests o/ Virus Sensitivity Serial t e n f o l d d i l u t i o n s were m a d e i n M o s c o n a ' s saline. E a c h t u b e r e c e i v e d 0.2 m l of i n o c u l u m a n d was t h e n rolled a t 33 o C. T u b e s were e x a m i n e d u n s t a i n e d b y o r d i n a r y l o w - p o w e r l i g h t m i c r o s c o p y . I n some e x p e r i m e n t s , viruses were " p a s s e d i n d i l u t i o n s " - in this, serial d i l u t i o n s of v i r u s were i n o c u l a t e d , a n d t h e t u b e w h i c h h a d r e c e i v e d t h e T a b l e 1. Respiratory viruses used/or testing cells
Virus type
Laboratory designation
Cultures passed in
Number of passages after recovery from man
Rhinovirus type 2 (RV2) ghinovirus type 9 (RV9) ~ Rhinovirus ? type Rhinovirus ? type R e s p i r a t o r y s y n c y t i a l v i r u s (RS) Coronavirus (HCVp
ItGP DC S F t340 HS 67/77 229E
Coronavirus
JK
Adenovirus type 3 (Ad 3) Influenza A Influenza B l~arainfluenza 3 Coxsaekie virus B 3
699/77 A/Viet/3/75 B/Mann/I/70 779/77 740/77
HeLa b HeLa WI-38 FT HeLa MRC-5 FT OC hil~C-C HeLa E m b r y o n a t e d egg Various-cloned MK HeLa
2 2 8 2 2 2 1 2 1 1 9 14 2 2
These viruses were passed in tissue culture after being isolated from patients, but were administered to volunteers and then reisolated b S ~ I z o v ~ - et al. (8) M R C - 5 - - s t r a i n of h u m a n e m b r y o l u n g f i b r o b l a s t s F T - - f e t a l t o n s i l l a r s t r a i n of h u m a n l u n g f i b r o b l a s t s (4) M K - - r h e s u s m o n k e y k i d n e y cells O C - - o r g a n c u l t u r e s of h u m a n r e s p i r a t o r y e p i t h e l i u m M R C - C - - c o r o n a v i r u s s e n s i t i v e c o n t i n u o u s line of h u m a n f i b r o b l a s t s
72
D.A.J.
TYRRELL, MARCIA M]:I
Archives of Virology 61, 69--85 (1979)
© by Springer-Verlag 1979
Clones of Cells From a Human Embryo Lung: Their Growth and Susceptibility to Respiratory Viruses By
D. A. J. TYt~RELL1, MARCIA MIKA-JoH~so~, and P. J. CI~APPI~E W. Alton Jones Cell Science Center, Lake Placid, New York, U.S.A. With 7 Figures Accepted March 5, 1979
Summary Colonies of cells were obtained from human fetal lung tissue and exposed to recently isolated respiratory viruses. There was a considerable variation in the number of rounded cells found in different colonies exposed to rhinovirus types 2 and 9 (RV2 and 9), human coronavirus 229E (HCV), adenovirus type 3 (Ad3) and respiratory syncytial virus (RSV). Smaller colonies had more rounded cells than larger colonies. Clones were established from 9 out of 11 colonies. They varied in their rate of growth and the pattern formed on a plastic surface. They varied also in their virus susceptibility particularly to "difficult" rhinoviruses such as RV 9 and SF 1340. One cell clone (HL 1/77 Clone 8), was highly susceptible to all these viruses. All cultures were more sensitive to ~ S V when maintained in F ] 2 K medium than in MEM, whereas there was no difference for rhinoviruses. Influenza A and B and parainfluenza 3 viruses sometimes produced cytopathie effect, and always produced haemadsorption, but unlike the previous strains could not be passed serially and presumably produced little infectious virus. All clones were rather insusceptible to Ad 3; but the virus could be passed, whereas coxsackie virus B 3 produced no CPE. Substantial yields of coronavirus and rhinovirus were obtained in gelatin sponge cultures. Two " v e r y difficult" respiratory viruses which had just been adapted to tissue culture; namely, HS rhinovirus and J K coronavirus grew in 7 of 9 and in 6 of 9 clones respectively.
i l~resent address: Clinical Research Centre, Division of Communicable Watford ]Road, Harrow, Middlesex HA 1 3U J, England. u Present address: Diagnostic Laboratory, Department of Preventive New York State College of Veterinary Medicine, Ithaca, NY 14853, U.S.A.
Diseases, Medicine,
0304-8608/79/0061/0069/$ 03.40
70
D . A . J . TYX~aELL, MAtCOIAMIKA-JOHNSO.W,and P. J. CI~APSLE:
Introduction H u m a n e m b r y o n i c l u n g is a complex tissue a n d contains the precursors of a v a r i e t y of cells r a n g i n g from fibrous tissue elements ~nd vascular e n d o t h e l i u m to bronchial epithelium, histiocytes, macrophages a n d p n e u m o c y t e s , including the type I I cells, l~ecently, type I I p n e u m o c y t e s h~ve been separated from the others b y cloning techniques, a n d their virus susceptibility has been studied (9). W e t h o u g h t t h a t it should also be possible to sepg:r~te out some of the other cells using the same basic techniques, a n d this report summarizes the results of our efforts. W e wished to d e t e r m i n e whether we could separate from a mixed culture of l u n g cells clones which were d e m o n s t r a b l y distinct, as j u d g e d b y their morphology, growth a n d o~her characteristics. More i m p o r t a n t l y , we wished to see if those clones were d e m o n s t r a b l y distinct in their susceptibility to infection with a range of respiratory viruses. Studies have shown t h a t organ cultures of bronchial epithelium are generMly susceptible to almost all k n o w n viruses, from rhinoviruses, a n d coronaviruses to influenza a n d p a r a i n f l u e n z a viruses (5). On the other hand, strains of fibroblasts obtMned b y mass culture of e m b r y o n i c lung, such us W I - 3 8 or MRC-5, are susceptible to some rhinoviruses a n d some coronaviruses b u t n o t to influenza or parMnfluenza viruses. Earlier studies showed t h a t mass cultures of fibroblasts obtMned from m a n y e m b r y o s are r e l a t i v e l y resistant to r h i n o v i r u s infection (1). We, therefore, hoped t h a t b y using cloning t e c h n i q u e s we would be able to detect, a n d cultivate virus-susceptible l u n g cells which would otherwise be lost if the usual m e t h o d s of mass culture were used. These susceptible cells m i g h t be useful as substratcs for producing virus vsccines or for c u l t i v a t i n g a n d s t u d y i n g viruses t h a t , ~t present, can be grown only in tracheal organ culture.
Materials and Methods Yledia Cells were, in general, grown in H a m ' s F 12K (modified) medium with 10 percent fetal bovine serum (FBS) (Flow Laboratories Lot ~00109t). They were maintained in the same medium with 2 percent FBS except where otherwise indicated. Tissue and Cell Cloning Lungs were obtained from fetuses up to 72 hours after hysterotomy. The tissue was chopped and dispersed with collagenase, trypsin and chicken serum as described elsewhere (9); and a first passage in mass culture was performed, and the resulting cells were stored in liquid nitrogen. The cells were then thawed, and established in viable culture. Cells from these cultures were counted using the erythrosin B dye exclusion technique. Four hundred viable eells were seeded in 10 ml of medium into a t00 m m petri dish and kept without disturbance in a humidified incubator at 37 ° C. Cultures were examined with an inverted microscope equipped with phase contrast optics. I n certain experiments, cells were classified as "fibroblast-like"--long, nalTow cells often in whorls, "endo- or epithelial-like"---pavements of polygonal cells or, " i n t e r m e d i a t e " - - a varied range of cells between these extremes. Selected colonies were isolated with a silicone-treated penicillin assay cylinder. They were washed twice with saline and then incubated with 0.25 percent trypsin: EDTA until the cells were released; they were then pipetted into a 35 m m dish and grown up until they filled a plastic flask with a surface area of 75 emK From t~his stock, cells were stored in liquid nitrogen in 5 percent dimethyl sutfoxide as a cryoprotectant. Stock cultures were trypsinized and passed at about five-day intervals, and each new flask was in-
V i r u s S u s c e p t i b i l i t y of L u n g Cell Clones
7t
oculaf~ed w i t h 1 × 106 cells i n 25 m l of m e d i u m c o n t a i n i n g 10 p e r c e n t F B S . A s r e q u i r e d , roller t u b e s w e r e p r e p a r e d u s i n g a b o u t 10 ~ cells in 1 m l of m e d i m n c o n t a i n i n g 10 p e r c e n t F B S a n d E a g l e ' s M E M b u f f e r e d w i t h H E P E S or in R a m ' s m o d i f i e d F 1 2 K . W h e n r e q u i r e d for v i r u s i n o c u l a t i o n , t u b e s w e r e c h a n g e d t o 1 m l of m e d i u m c o n t a i n i n g 2 p e r c e n t F B S , or w h e n u s i n g i n f l u e n z a or p a r a i n f l u e n z a v i r u s , t h e y were w a s h e d t h r e e t i m e s w i t h 1 mI a n d m a i n t a i n e d w i t h 1 m l of s e r u m - f r e e m e d i u m .
Approximate Cell Doubling Time T 75 flasks were seeded w i t h 106 cells, i n c u b a t e d for a b o u t five d a y s a n d t r y p s i n i z e d as t h e cell s h e e t w a s a p p r o a c h i n g confluence. T h e cell s h e e t w a s t r y p s i n i z e d a n d t h e n u m b e r of cells w a s m e a s u r e d w i t h a C o u l t e r c o u n t e r . T h e a p p a r e n t cell d o u b l i n g t i m e was computed from these numbers.
Viruses All t h e v i r u s e s h a d b e e n p a s s e d o n l y a few t i m e s since t h e i r i s o l a t i o n ~rom t h e h u m a n r e s p i r a t o r y t r a c t . D e t a i l s c o n c e r n i n g t h e m are s h o w n i n T a b l e 1. T h e y were e i t h e r k n o w n t o b e p a t h o g e n i c for m a n or could b e safely a s s u m e d to be.
Tests o/ Virus Sensitivity Serial t e n f o l d d i l u t i o n s were m a d e i n M o s c o n a ' s saline. E a c h t u b e r e c e i v e d 0.2 m l of i n o c u l u m a n d was t h e n rolled a t 33 o C. T u b e s were e x a m i n e d u n s t a i n e d b y o r d i n a r y l o w - p o w e r l i g h t m i c r o s c o p y . I n some e x p e r i m e n t s , viruses were " p a s s e d i n d i l u t i o n s " - in this, serial d i l u t i o n s of v i r u s were i n o c u l a t e d , a n d t h e t u b e w h i c h h a d r e c e i v e d t h e T a b l e 1. Respiratory viruses used/or testing cells
Virus type
Laboratory designation
Cultures passed in
Number of passages after recovery from man
Rhinovirus type 2 (RV2) ghinovirus type 9 (RV9) ~ Rhinovirus ? type Rhinovirus ? type R e s p i r a t o r y s y n c y t i a l v i r u s (RS) Coronavirus (HCVp
ItGP DC S F t340 HS 67/77 229E
Coronavirus
JK
Adenovirus type 3 (Ad 3) Influenza A Influenza B l~arainfluenza 3 Coxsaekie virus B 3
699/77 A/Viet/3/75 B/Mann/I/70 779/77 740/77
HeLa b HeLa WI-38 FT HeLa MRC-5 FT OC hil~C-C HeLa E m b r y o n a t e d egg Various-cloned MK HeLa
2 2 8 2 2 2 1 2 1 1 9 14 2 2
These viruses were passed in tissue culture after being isolated from patients, but were administered to volunteers and then reisolated b S ~ I z o v ~ - et al. (8) M R C - 5 - - s t r a i n of h u m a n e m b r y o l u n g f i b r o b l a s t s F T - - f e t a l t o n s i l l a r s t r a i n of h u m a n l u n g f i b r o b l a s t s (4) M K - - r h e s u s m o n k e y k i d n e y cells O C - - o r g a n c u l t u r e s of h u m a n r e s p i r a t o r y e p i t h e l i u m M R C - C - - c o r o n a v i r u s s e n s i t i v e c o n t i n u o u s line of h u m a n f i b r o b l a s t s
72
D.A.J.
TYRRELL, MARCIA M]:I
