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Growth of “pure” cervical epithelium in vitro
Growth of "pure" cervical epithelium in vitro RALPH M. RICHART, M.D. ,Vew York, Neu• York With the technical assistance o/
JEA:\f FREDERICK
T H E study of the cellular biology of cervical intraepithelial neoplasia (dysplasia and carcinoma in situ) in the human has been hampered by the inability to obtain functional cells of known histological type, either isolated or aggregate, in pure form. To overcome this difficulty a method was devised to grow cervical epithelium of known histological type in tissue culture. This paper reports on the method.
blue-positive. The biopsy is immediately placed in a test tube containing 2 c.c. of a wash solution consisting of glucose, potassium, and sodium ( G KN) containing 1,000 units of penicillin and 0.5 mg. of streptomycin per milliliter. The biopsy is then taken to the laboratory ;vhere it is further cleaned with five additional changes of wash solution. Following this the biopsy is placed in wash solution in a 60 mm. diameter watch glass and under 10 times magnification, using a dissecting microscope, the tissue is split in half longitudinally, using watchmaker's forceps and a No. 11 Bard-Parker blade. One half is retained for histology. The other half is transferred to a second ·watch glass containing Eagle's Minimal Essential Media iMEM) and 15 per cent fetal calf serum. In this media, again under the dissecting microscope. the epithelium is carefully separated from the stroma using a No. 15 Bard-Parker blade. The stroma is retained for histology I Fig. 1). The stripped epithelium is finely minced in the MEM using two No. ll BardParker blades. after which the fragments and media are aspirated '.vith a bent-tipped Pasteur pipette and transferred to specially designed microglass circular tissue culture chambers. I Fig. 2.) Precut pieces of perforated cellophane arc moistened in MEM and placed over the minced fragments, after which the chamber is placed in a humidified water-jacketed incubator at ~7° C. with 5 per cent co" and air as the gas phase. The culture plates are left undisturbed for 3 days: subsequently the medium is changed three times a week. When satis-
Method Cases are selected for the type of lesion that is desired through the use of the differential cell counting technique 3 and the cervix is stained with toluidine blue dye. 4 Only those patients are selected for study in whom the area of toluidine blue positivity, and hence of intraepithelial neoplasia, is large enough so that a punch biopsy can be taken to include only toluidine blue~posi tive epithelium. In these patients a photograph is made of the stained cervix and the patient is given a return appointment for biopsy. On the return visit the cervix is thoroughly cleaned with sterile saline and dried, after which a punch biopsy is made from the midst of the area which previously stained toluidine From the Departments of Pathology and Obstetrics and Gynecolog}', College of Physicians and Surgeons, Columbia University, and the Obstetrical and Gynecological Service (The Sloane Hospital) of the Presbyterian Hospital. Supported by United Stales Public Health Service Grants C-9800, CM-K3-IH975, RG-8165 and a General Research Support Grant from the National Institutes of Health.
710
Volume 88 Number 6
"Pure" cervical epithelium
711
Fig. 1. Stromal portion of biopsy from which epithelium ha s been stripped. A few fragments of ba sal cells remain.
factory growth is obtained the perforated cellophane is carefully removed with watchmaker's forceps and the nonviable fragments removed as the medium is changed. When adequate growth for subculture is obtained, the medium is removed and the cells are washed two times with GKN, which is allowed to remain on the cells for 5 minutes during the last wash. This fluid is then replaced by GKN containing 0.25 per cent trypsin and 0.2 per cent sodium versenate. The action of the trypsin versene mixture is monitored visually using an inverted Plankton microscope and, when the cells are noted to begin detaching from the glass, the plate is thoroughly scraped with a siliconized mbber policeman and the cells immediately harvested and placed in conical 15 mi. centrifuge tubes to which an excess
of complete medium is added. After centrifugation at 3000 x g, the supernatant is discarded, the cells resuspended m fresh medium and replated. Results
Fifty-two biopsies were prepared for tissue culture. Two cultures were found to be contaminated, 1 within 2 days after plating and the other after growth had occurred. In 9 biopsies no growth was obtained. The 22 cases which were successfully transferred were all taken from the 37 cases of "pure" culture. These biopsies included both normal epithelium and tissue from patients with dysplasia and carcinoma in situ. In 37 cases monolayers of "pure" cervical epithelium were obtained; in 5 biopsies the cervical epithelial growth was contaminated by fibro-
712
:\larch l'i. 1964 .\Ill.]. Obst. & Gynec.
Richart
l'lg. 2. The microglass circular culiure chamber consisting of a Corning No. 1 45 by 50 mm. microcoverglass attached with stopcock grease to 8 mm. length of standard wail glass tubing, 45 nnn. outside diameter and covered with a 40 mm. Bellco Petri dish cover.
blasts; 22 cases were successfully transferred for one or more generations. Comment
The technique described in this paper was developed over a period of 212 years, during which time a large number of different media and tissue culture vessels were used. In the initial studies the biopsies were placed in Petri dishes, plaque bottles, prescription bottles, and roller tubes, using Puck's tissue culture medium, both phosphate and bicarbonate buffered. Outgrowth was obtained from all the biopsies using these methods~ but it was always scanty and in only one instance was it possible to obtain a transfer generation. The usc of chick embryo extract-chick plasma clot produced adequate outgrowth, but was felt to be unsuitable due to the difficulty in obtaining good morphological detail and an inability to transfer the cells. After changing to Eagle's MEM with 15 per cent fetal calf serum and the microglass culture chambers the percentage of successful cultures was equivalent to that using other methods, the outgrowth was excellent, and large numbers of cells were obtained. We initially attempted to transfer the cells by using a rubber policeman alone and by using trypsin in concentrations ranging from 0.05 to 1 per cent, either alone or with a rubber policeman. In both instances the
percentage of cells survrvmg transfer was low and it was difficult, or impossible, to establish transfer generations. Several different combinations of trypsin and versene were used, but the one presently used has given the most satisfactory results. Initially the trypsin-versene mixture was used alone and the cells wPre exposed to the solution until they detached from the glass. It was found that a large number of cells were damaged by this procedure, however, and the rubber policeman was used to remove the cells after they had been loosened by the trypsin-versene solution. Using this method, virtually all cultures with an adequate monolayer can be transferred for one or more generations. The cells which grow from lesions of different histological types and from normal cprvical Ppithelium differ strikingly in their pattern of growth and morphology. As seen in Figs. ~~ and 4, the growth is monolayer in type and the cells have an epithelial appearance. The appearance of the cells from different patients is so characteristic that if a limited number of cases are being grown simultaneously one can identify each patient's culture plates through cellular morphology, except for the outgrowth of normal epitheliunt \vhich is similar in ali the patients.
This data will be reported in detail sub.tO.:PrllJPntlu ._ '1"' •••• , .
Through the use of the toluidine blue staining procedure and by choosing the biopsy site so that only neoplastic epithelium or normal epithelium is included in the tissue removed, it is possible to obtain monolayers of cells that are derived from a known cell type. The ability to consistently grow cells derived from a single tissue of known morphological type, uncontaminated by cells of origins other than the desired type, comprises a system which has obvious advantages. A number of previous authors have reported methods through which both normal and neoplastic cervical epithelium was grown in tissue culture. In most of these methods
the biopsies were not obtained from areas known to be "pure" neoplasia and it would appear from the photographs that many of
Volume 88 ~umb~r f}
··pure" cervical epithelium
713
Fig. 3. Photomicrograph of outgrowth from normal cervical epithelium. Ex· plant is in lower left-hand corner. CeBs grow as a confluent epithelioid sheet. Note the cell completing mitosis in the upper portion of the field. (Phase-contrast. x260.)
Fig. 4. Photomicrograph of outgrowth from cervical biopsy containing mod· erate dysplasia. There is more varia· tion in the size and shape of the cells and a loss of the confluent growth pattern seen in normal epithelium, but the individual cells still have an epithelial appearance. (Phase-contrast. x260.)
714
Richart
the cultures were of mixed type, which may acount for the failure to observe the characteristic differences that are apparent between cases in our cultures. The most recent authors describing cervical tissue culture are Mellgren, Boeryd, and Hagman 1 who successfully grew carcinoma in situ, dysplasia, and normal epithelium, both in vitro and in the hamster cheek pouch. While these authors succeeded in obtaining excellent growth in
REFERENCES
1. Mellgren, }., Boeryd, B., and Hagman, M.: Cancer Res. 22: 139, 1962.
2. Merchant, D. ]., Kahn, R. H., and Murphy. W. H., Jr.: Minneapolis, Minn., 1961, B:.ugpss Publishing Co.
.\m.
J.
\farch I'•. !964 Obst. & ~ :ynec.
the majority of instances they were unable to obtain transfer generations and had mixed types of epithelium in culture. Summary
A method IS described through which "pure" cultures of normal or neoplastic cervical epithelium can be grown in vitro and transferred for one or more generations after monolayer growth is obtained.
:l. Okagaki, T., Lerch, V., Younge, P. A., McKay. D. G., and Kevorkian, A. Y.: Acta cytol. 6: 343, 1962. +. Richart, R. M.: AM. J OBsT. & GYNEc. 86: 703, 196:'1.
6:10 West 168 Street New York .'12. New York
JEA:\f FREDERICK
T H E study of the cellular biology of cervical intraepithelial neoplasia (dysplasia and carcinoma in situ) in the human has been hampered by the inability to obtain functional cells of known histological type, either isolated or aggregate, in pure form. To overcome this difficulty a method was devised to grow cervical epithelium of known histological type in tissue culture. This paper reports on the method.
blue-positive. The biopsy is immediately placed in a test tube containing 2 c.c. of a wash solution consisting of glucose, potassium, and sodium ( G KN) containing 1,000 units of penicillin and 0.5 mg. of streptomycin per milliliter. The biopsy is then taken to the laboratory ;vhere it is further cleaned with five additional changes of wash solution. Following this the biopsy is placed in wash solution in a 60 mm. diameter watch glass and under 10 times magnification, using a dissecting microscope, the tissue is split in half longitudinally, using watchmaker's forceps and a No. 11 Bard-Parker blade. One half is retained for histology. The other half is transferred to a second ·watch glass containing Eagle's Minimal Essential Media iMEM) and 15 per cent fetal calf serum. In this media, again under the dissecting microscope. the epithelium is carefully separated from the stroma using a No. 15 Bard-Parker blade. The stroma is retained for histology I Fig. 1). The stripped epithelium is finely minced in the MEM using two No. ll BardParker blades. after which the fragments and media are aspirated '.vith a bent-tipped Pasteur pipette and transferred to specially designed microglass circular tissue culture chambers. I Fig. 2.) Precut pieces of perforated cellophane arc moistened in MEM and placed over the minced fragments, after which the chamber is placed in a humidified water-jacketed incubator at ~7° C. with 5 per cent co" and air as the gas phase. The culture plates are left undisturbed for 3 days: subsequently the medium is changed three times a week. When satis-
Method Cases are selected for the type of lesion that is desired through the use of the differential cell counting technique 3 and the cervix is stained with toluidine blue dye. 4 Only those patients are selected for study in whom the area of toluidine blue positivity, and hence of intraepithelial neoplasia, is large enough so that a punch biopsy can be taken to include only toluidine blue~posi tive epithelium. In these patients a photograph is made of the stained cervix and the patient is given a return appointment for biopsy. On the return visit the cervix is thoroughly cleaned with sterile saline and dried, after which a punch biopsy is made from the midst of the area which previously stained toluidine From the Departments of Pathology and Obstetrics and Gynecolog}', College of Physicians and Surgeons, Columbia University, and the Obstetrical and Gynecological Service (The Sloane Hospital) of the Presbyterian Hospital. Supported by United Stales Public Health Service Grants C-9800, CM-K3-IH975, RG-8165 and a General Research Support Grant from the National Institutes of Health.
710
Volume 88 Number 6
"Pure" cervical epithelium
711
Fig. 1. Stromal portion of biopsy from which epithelium ha s been stripped. A few fragments of ba sal cells remain.
factory growth is obtained the perforated cellophane is carefully removed with watchmaker's forceps and the nonviable fragments removed as the medium is changed. When adequate growth for subculture is obtained, the medium is removed and the cells are washed two times with GKN, which is allowed to remain on the cells for 5 minutes during the last wash. This fluid is then replaced by GKN containing 0.25 per cent trypsin and 0.2 per cent sodium versenate. The action of the trypsin versene mixture is monitored visually using an inverted Plankton microscope and, when the cells are noted to begin detaching from the glass, the plate is thoroughly scraped with a siliconized mbber policeman and the cells immediately harvested and placed in conical 15 mi. centrifuge tubes to which an excess
of complete medium is added. After centrifugation at 3000 x g, the supernatant is discarded, the cells resuspended m fresh medium and replated. Results
Fifty-two biopsies were prepared for tissue culture. Two cultures were found to be contaminated, 1 within 2 days after plating and the other after growth had occurred. In 9 biopsies no growth was obtained. The 22 cases which were successfully transferred were all taken from the 37 cases of "pure" culture. These biopsies included both normal epithelium and tissue from patients with dysplasia and carcinoma in situ. In 37 cases monolayers of "pure" cervical epithelium were obtained; in 5 biopsies the cervical epithelial growth was contaminated by fibro-
712
:\larch l'i. 1964 .\Ill.]. Obst. & Gynec.
Richart
l'lg. 2. The microglass circular culiure chamber consisting of a Corning No. 1 45 by 50 mm. microcoverglass attached with stopcock grease to 8 mm. length of standard wail glass tubing, 45 nnn. outside diameter and covered with a 40 mm. Bellco Petri dish cover.
blasts; 22 cases were successfully transferred for one or more generations. Comment
The technique described in this paper was developed over a period of 212 years, during which time a large number of different media and tissue culture vessels were used. In the initial studies the biopsies were placed in Petri dishes, plaque bottles, prescription bottles, and roller tubes, using Puck's tissue culture medium, both phosphate and bicarbonate buffered. Outgrowth was obtained from all the biopsies using these methods~ but it was always scanty and in only one instance was it possible to obtain a transfer generation. The usc of chick embryo extract-chick plasma clot produced adequate outgrowth, but was felt to be unsuitable due to the difficulty in obtaining good morphological detail and an inability to transfer the cells. After changing to Eagle's MEM with 15 per cent fetal calf serum and the microglass culture chambers the percentage of successful cultures was equivalent to that using other methods, the outgrowth was excellent, and large numbers of cells were obtained. We initially attempted to transfer the cells by using a rubber policeman alone and by using trypsin in concentrations ranging from 0.05 to 1 per cent, either alone or with a rubber policeman. In both instances the
percentage of cells survrvmg transfer was low and it was difficult, or impossible, to establish transfer generations. Several different combinations of trypsin and versene were used, but the one presently used has given the most satisfactory results. Initially the trypsin-versene mixture was used alone and the cells wPre exposed to the solution until they detached from the glass. It was found that a large number of cells were damaged by this procedure, however, and the rubber policeman was used to remove the cells after they had been loosened by the trypsin-versene solution. Using this method, virtually all cultures with an adequate monolayer can be transferred for one or more generations. The cells which grow from lesions of different histological types and from normal cprvical Ppithelium differ strikingly in their pattern of growth and morphology. As seen in Figs. ~~ and 4, the growth is monolayer in type and the cells have an epithelial appearance. The appearance of the cells from different patients is so characteristic that if a limited number of cases are being grown simultaneously one can identify each patient's culture plates through cellular morphology, except for the outgrowth of normal epitheliunt \vhich is similar in ali the patients.
This data will be reported in detail sub.tO.:PrllJPntlu ._ '1"' •••• , .
Through the use of the toluidine blue staining procedure and by choosing the biopsy site so that only neoplastic epithelium or normal epithelium is included in the tissue removed, it is possible to obtain monolayers of cells that are derived from a known cell type. The ability to consistently grow cells derived from a single tissue of known morphological type, uncontaminated by cells of origins other than the desired type, comprises a system which has obvious advantages. A number of previous authors have reported methods through which both normal and neoplastic cervical epithelium was grown in tissue culture. In most of these methods
the biopsies were not obtained from areas known to be "pure" neoplasia and it would appear from the photographs that many of
Volume 88 ~umb~r f}
··pure" cervical epithelium
713
Fig. 3. Photomicrograph of outgrowth from normal cervical epithelium. Ex· plant is in lower left-hand corner. CeBs grow as a confluent epithelioid sheet. Note the cell completing mitosis in the upper portion of the field. (Phase-contrast. x260.)
Fig. 4. Photomicrograph of outgrowth from cervical biopsy containing mod· erate dysplasia. There is more varia· tion in the size and shape of the cells and a loss of the confluent growth pattern seen in normal epithelium, but the individual cells still have an epithelial appearance. (Phase-contrast. x260.)
714
Richart
the cultures were of mixed type, which may acount for the failure to observe the characteristic differences that are apparent between cases in our cultures. The most recent authors describing cervical tissue culture are Mellgren, Boeryd, and Hagman 1 who successfully grew carcinoma in situ, dysplasia, and normal epithelium, both in vitro and in the hamster cheek pouch. While these authors succeeded in obtaining excellent growth in
REFERENCES
1. Mellgren, }., Boeryd, B., and Hagman, M.: Cancer Res. 22: 139, 1962.
2. Merchant, D. ]., Kahn, R. H., and Murphy. W. H., Jr.: Minneapolis, Minn., 1961, B:.ugpss Publishing Co.
.\m.
J.
\farch I'•. !964 Obst. & ~ :ynec.
the majority of instances they were unable to obtain transfer generations and had mixed types of epithelium in culture. Summary
A method IS described through which "pure" cultures of normal or neoplastic cervical epithelium can be grown in vitro and transferred for one or more generations after monolayer growth is obtained.
:l. Okagaki, T., Lerch, V., Younge, P. A., McKay. D. G., and Kevorkian, A. Y.: Acta cytol. 6: 343, 1962. +. Richart, R. M.: AM. J OBsT. & GYNEc. 86: 703, 196:'1.
6:10 West 168 Street New York .'12. New York