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SUMMARY - The use of a chemically defined medium supplemented with serum has proved most suitable for the growth of the isolated embryonic otocyst in vitro. Complete differentiation of the cochlear duct and of the vestibular apparatus ensued and is described. The system is reliable for organ cultures of both the avian and mammalian otocyst and for the study of various lesions affecting the inner ear and middle ear.

Ordered, controlled growth is essential for the normal development of tissues in culture and the organ culture system offers an excellent method for the study of the cytology of the developing inner ear. Studies on the isolated embryonic otocyst in tissue culture began with the pioneering work of Fell' in 1928. The original watchglass technique of Fell and Robison- has been successfully employed in the study of the differentiation of the neuroepithelial elements of the inner ear,":" and in the evaluation of various ototoxic agents as shown by Friedmann and Bird," in 1961 and Friedmann" in 1969. In all these studies the authors have utilized the otocyst of 3)f-day-old chick embryos and it has been confirmed that the neuroepithelial structures differentiated in tissue culture, were almost identical with those differentiated in vivo at both the light and electron microscopic level. Orr? investigated the behavior of dissociated and reaggregated cells of the chick embryo otocyst and emphasized the importance of the influence of neural tissue on sensory cell differentiation. This is supported by the evidence presented by Friedmann? and by Sher-? that nerves exert an inductive influence on the undifferentiated epithelium of the otocyst and that full func-

tional differentiation required the establishment of neuroepithelial contact at the synaptic level. Notwithstanding earlier attempts at culturing the mammalian otocyst by Maximowl! in 1925 and by Lawrence and Merchant'< in 1953, the first successful organ cultures of the isolated mouse embryo otocyst were achieved and reported on by Van De Water and Ruben'" in 1971. They have succeeded in making "the organ culture system for the development of the mammalian inner ear into a tool with which some forms of inner ear deafness may be studied."14·15 Yamashita and Vosteen-" have cultured the organ of Corti and the isolated outer and inner hair cells of the newborn guinea-pig which have been maintained in vitro for more than 20 days. There was no difference in their sensitivity to toxic agents. The present paper is based on the technique that has proved reliable in the study of another complex tissue, i.e., mandibular explants 17.18 reaching full differentiation when grown on a chemically defined medium. METHODS AND :MATERIALS

Culture Technique. The otocysts of 3Mday-old chick embryos and of 12-day-old mouse embryos, age being established by the

From the Imperial Cancer Research Fund Laboratories, London, and Northwick Park Hospital and Clinical Research Centre, Harrow, England.


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Fig. 1. A) Survey picture of 12-day gestation period mouse embryo otocyst grown in organ culture for ten days. Cochlea (left) vestibular region (center) and cross-sections of the semicircular canals (right); note cartilaginous otic capsule and developing stapes (between cochlea and vestibule). (X60). B) Detail showing scala media with the developing organ of Corti and tectorial membrane. Scalae vestibuli and tympani are developed. Note neurones of the spiral ganglion forming a small group (bottom right) . ( X225 ) vaginal plug method, were cultured on cellophane strips previously sterilized in 70% ethanol, washed twice in Hank's saline solution over a period of 60 minutes and equilibrated in culture medium. Each explant assembly was laid over a 3-4 mm hole punched in a stainless steel wire mesh square lying flat on the bottom of an optical flat glass Petri dish. ( Anumbra®)." The amount of medium added ( 1.5 ml ) to each Petri dish came to the level of the cellophane substrate. The cultures were incubated at 37 C in sealed, humidified PerspeX®"" chambers under an atmosphere of 5% CO, in air. Some of the cultures were filmed and some of the illustrations are frames from the Iilm. Culture Medium. The culture medium con-

sisted of antibiotic-free medium]', supplemented with 0.45 iLg/ml ferrous sulphate, 300 iLg!ml ascorbic acid, 1 iLg/ml cortisone and with 5% newborn calf serum.j ] RESULTS

The otocysts were well maintained in an atmosphere of 95% air and 5% CO 2 • Light Microscopy. Figures 1 and 2 are survey pictures of fully matured mouse embryo otocysts grown in vitro for ten days. Figure 2 is a frame from a time-lapse film and shows the curved cochlear duct on the left and a compli-

" Glass Export, Bohemia, Czechoslovakia, imported by Smith, Glassware and China Ltd, London. "" Imperial Chemical Industries, England. t Waymouth's MB 752/1, Wellcome Reagents Ltds., England. tt Flow Laboratories, Inc., Rockville, MD.

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Fig. 2. Frame of the time lapse film showing the cultured mouse embryo otocyst of 12 days with the curved c~chlear duct clearly ~isible above and the semicircular canals below. (X40)

cated system of tubular structures on the right. Figure 1 shows a section through thc main diameter of such an organ culture: thcre are cross sections of the different coils of the cochlea on the left and the organ of Corti is visible. The vestibular area with the saccull'S is located in the central area and cross sections of the semicircular canals can be recognized in the right third of the picture. Figures 3 to 5 show details of the neuroepithelial structures of various organ cultures of the isolated embryo mouse otocyst. Figure 3 shows the three scalae of a cochlear coil with the developing organ of Corti in the middle or scala media. It is composed of a stratified layer of epithelial cells resting on the basilary membrane. Figure 3 shows additional details, i.e., the tectorial membrane covering the whole surface and a vascular stria. Figure 4 shows a macula lined bv tall columnar cells resting on a layer of cuboidal basal supporting cells. Ciliary

Fig. 3. Mouse embryo otocyst of 12 days. Scala media in another culture. Maintained for eight days. The organ of Corti rests on the basilar membrane and is covered by the tectorial membrane. There is a developing vascular stria ( above) attached to cartilage (otic capsule). (X225)

tufts are visible. Figure 5 shows the crista nearest to the macula depicted in the previous picture. It is surmounted by a cupula-like structure. All cultures had a well-developed cartilaginous capsule and the stapes was present in some of the cultures. Squamous epithelium occurred on the surface and formed implantation-cysts with considerable keratinization resembling a cholesteatoma (Fig. 6). Neurones could be seen in the avian otocyst cultures and in some of the organ cultures of the mouse embryo otocysts.

Electron Microscopic Observations. There was advanced differentiation of the sensory neuroepithelial structures of both the isolated chick and mouse embryo otocysts. Both the epithelial structures, the ciliary apparatus and

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Fig. 4. Culture as in Fig. 3. Detail of macula formed by columnar (ciliated) epithelium resting on a layer of cuboidal basal cells. The crista is just visible (top right, Fig. 5). Neural elements (confirmed electron microscopically) in fibrous tissue covering cartilage. (X595)

organelles, have developed in a normal fashion. Figures 7 to 10 show the innervation of the chick embryo otocyst developing in vitro. The present observations confirm that nonmyelinated nerves emerge from their nonmyelinated neurones (Fig. 7) and may form large structures composed of many axons surrounded by a single Schwarm cell. Figure 8 shows large structures which may be mistaken under the light microscope for neurones. Eventually the axons lose their myelin sheaths and, penetrating the basal lamina, they spread along the basal cells (Fig. 9) and between the supporting cell, protected and nourished by them, before reaching the synaptic area of the sensory hair cells. The ciliary apparatus (Fig. 10) and other cellular features may have been formed as suggested by Hilding!? but functional dif-

Fig. 5. Cultures as Figs. 3 and 4. Crista-like structure with cupula and neural elements (confirmed electron microscopically) in the underlying stroma. (X900).

ferentiation cannot be completed before synaptic linkage between hair cell and nerve has been established." The cells are connected by well developed desmosomes and tight junetions." Similar features can be seen in the developing mouse embryo otocyst as shown by Van De Water, Heywood and Ruben."?

Effect of Ototoxic Antibiotics. The effect of streptomycin sulphate has been studied. The antibiotic was added to the medium and two experimental schemes were used. 1) 1000 mgs/ml streptomycin: Otocyst explant exposed for 30 minutes and then transferred onto fresh medium. Time lapse cinematography and light microscopy revealed no major inhibitory effect on in vitro development. 2) Explanted otocyst exposed in vitro to 100 mgs/ml for 72

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microscope level when grown in vitro using fresh chick embryo extract and cockerel plasma, the original media having been used by Fell in her pioneering studies leading to the present high standard of tissue culture methods. This method, although ill-defined, seems to provide the necessary growth and differentiating factors. Melcher and Hodges-? have shown that complicated embryonic rudiments containing various epithelial and connective tissue elements, such as mouse embryo mandible, can be maintained in vitro on a chemically defined medium supplement with 3% calf serum. This method has formed the basis of the present organ culture system and has proved successful for the growth and differentiation of both the isolated avian and mammalian embryo otocysts.

Fig. 6. Squamous epithelium with keratin cyst growing into the cultured mouse embryo otocyst surrounded by cartilage and secretory epithelium. (X190)

hours and then transferred onto fresh medium. Time lapse cinematography shows considerable inhibition of the in vitro development of the explanted mouse embryo otocyst (Fig. 11). Light microscopy shows degeneration of the cochlear region, leading to a gradual disintegration of the neuroepithelium. These results are to be confirmed but are in keeping with our previous observations on the chick embryo otocyst." DISCUSSION

The present investigations have confirmed the excellent results of Van De 'Vater and Ruben14 ,1 5 and their conclusion that "organ culture now allows an investigator to directly manipulate the development of the ear either surgically and/or chemically." It has been shown that the isolated chick embryo otocyst reached full differentiation at both light and electron

The growth of normal cells is probably controlled by the interaction of cells with various polypeptide hormones or hormone-like growth factors present in the surrounding Huids.21 In our organ culture some of these factors may have been supplied by the explanted tissue or by the addition of serum factors, which include various growth promoting factors or factors contributing to the differentiation of specific cells and tissues, e.g., nerves and sensory cells. The mechanism that seems to control mammalian cell growth is responsive to a complex interaction among various factors as pointed out by Holley." The growth of cells under experimental conditions may be controlled by certain substances, e.g., hydrocortisone, calcium ions, and glucose, as has been used by various authors and in the present experiments. The role of the developing innervation in the differentiation of the sensory cells is of great significance and the supporting cells appear to be protecting and nourishing the nonmyelinated

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nerve fibers passing via the basal lamina across the epithelium toward their ultimate destination at the synaptic base of the hair cells. In this connection it is interesting to note that Yamashita

and Vosteen-" have cultured the organ of Corti and isolated hair cells of the newborn guinea pig. Although there was no outgrowth of hair cell origin there was an extensive outgrowth of

Fig. 7. Neurone of spiral ganglion - chick embryo otocyst. Twelve days in vitro. (X22.300)

Fig. (single) Fig. lamina).

8. Nerve bundle composed of multiaxonal fibres; occasional myelinated axons present; chick embryo otocyst 12 days in vitro. (X32.300) 9. Non-myelinated nerves spreading into the neuroepithelium (near basal Chick embryo otocyst 12 days in vitro. (X32.300)

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Fig. 8

Fig. 9

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Fig. 10. Ciliated surface of hair cell kino cilium and basal corpuscle stereocilia contain microtubules. Chick embryo otocyst - 12 days in vitro. (X22.300)

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Fig. 11. Mouse embryo otocyst exposed in vitro to 100,u/ml of streptomycin sulphate and grown on fresh culture media for another ten days. Note small shrunken size when compared with Fig. 2. (X40)

supporting cells. It has been concluded by these authors that intact supporting cells are essential for the maintenance and normal metabolism of the hair cells. The presence of stratified squamous epithelium raised some questions of interest in the pathology of the so-called cholesteatoma of the ear and its associ-


ation with the development of squamous cell carcinoma of the middle ear cleft. Cholesteatomas are basically epidermoid cysts but the origin of the squamous epithelium has remained the subject of heated arguments in the otological literature. In the majority of the cases it migrates from the skin lining, the external auditory meatus and the tympanic membrane through a manifest or sometimes latent defect of the membrane, into the middle ear. Alternatively it may be of metaplastic origin and rarely it may have arisen from activated embryonic cell nests. These theoretical considerations could be tested on organ cultures of the embryonic otocyst and may also throw light on the development of squamous carcinoma of the ear. The effect of ototoxic agents has been widely studied and in the present paper we have limited our observations on the persistence of streptomycin. Whereas exposure at 1000 mcg/ml for 30 minutes seemed to have no deleterious effect on the developing otocysts, exposure to only 100 mcg/ml, for 72 hours has inhibited their growth and differentiation.

REFERENCES 1. Fell HB: Development "in vitro" of the isolated otocyst of the embryonic fowl. Arch Exp Zellforschung 7 :69, 1928 2. Fell HB, Robison R: The growth and development and phosphatase activity of embryonic avian femora and limb buds cultivated in vitro. Biochem J 23:767, 1929 3. Friedmann I: In vitro culture of the isolated otocyst of the embryonic fowL Ann Otol Rhinol Laryngol 65:98, 1956 4. Friedmann I: Electron microscopic observations on in vitro cultures of the isolated fowl embryo otocyst. J Biophys Biochem Cytol 5:263, 1959 5. Friedmann 1: Attachment zones of cells in organ cultures of the fowl embryo otocyst. J Ultrastruct Res 5 :44, 1961 6. Friedmann I: Innervation of the developing fowl embryo otocyst. Acta Otolaryngol (Stockh) 67 :224, 1969 7. Friedmann I: The Pathology of the Ear. Blackwell, London, 1974, p 509 8. Friedmann I, Bird ES: The effect of ototoxic antibiotics on the isolated chick embryo otocyst. J Pathol 81 :81, 1961

9. Orr MF: Histogenesis of sensory epithelium in reaggregates of dissociated embryonic chick otocysts. Dev Btol 17 :39, 1968 10. Sher AE: The embryonic and postnatal development of the inner ear of the mouse. Acta Otolaryngol (Stockh) Suppl 285, 1971 11. Maximow A: Tissue culture of young mammalian embryos. Embryology 6:80, 1925 12. Lawrence M, Merchant DI: Tissue culture techniques for the study of the isolated otic vesicle. Ann Otol Rhinol Laryngol 62: 770, 1953 13. Van De Water TR, Ruben RJ: Organ culture of the mammalian inner ear. Acta Otolaryngol (Stockh) 71 :303,1971 14. Van De Water TR, Ruben RJ: Organ culture of the mammalian inner ear. A tool to study inner ear deafness. Laryngoscope 84: 738, 1974 15. Van De Water TR, Ruben RJ: Growth of the inner ear in organ culture. Ann Otol Rhinol Laryngol83 (SuppI14:1-16) 1974 16. Yamashita T, Vosteen KM: Tissue culture of the organ of Corti and the isolated hair cells from the newborn guinea pig. Acta Otolaryngol (Stockh) Suppl 330, 1975

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17. Melcher AH, Hodges GM: In vitro culture of an organ containing mixed epithelial and connective tissues on a chemically defined medium. Nature 219:301, 1968 18. Hodges GM, Melcher AM: Chemicallydefined medium for growth and differentiation of mixed epithelial and connective tissues in organ culture. In vitro. In press 19. Hilding DA: Electron microscopy of the

developing hearing organ. Laryngoscope 7~): 1991, 1969 20. Van De Water TR, Heywood P, Ruben RJ: Development of sensory structures in organ cultures of the 12th and 13th gestation day mouse embryo inner ears. Ann Otol Rhinol Laryngol82 (SuppI4:3-17) 1973 21. Holley RW: Control of growth of mammalian cells in cell culture. Nature 258:487, 197,5

ACKKOWLEDG:\IENTS-Our thanks are due to Dr. L. M. Franks for his encouraging interest and to Dr. R. Dourmashkin. Mrs. Thelma Barnes and Miss S. Luckett prepared the sections for light and electron microscopy. REPRINTS-I. Friedmann, MD, DSc, Department of Histopathology, Northwick Park Hospital and Clinical Research Centre, Watford Road, Harrow, Middlesex HAl 3UJ, England.

GUIDELINES OF GRANTS TRUSTEES OF MvIERICAN OTOLOGIC SOCIETY A matter of continual concern to the members of the Board of Trustees of the American Otological Society is the determination of guidelines for the funding of grants from its research fund. The following is our current formulation of policy. Our objective is clearly before us as the problem of otosclerosis: its diagnosis and management, the possible causes and underlying conditions (such as the hereditary background), and an understanding of the disorder that may lead eventually to its prevention and cure. Our present lack of understanding of the disorder and the absence, as far as known, of any similar condition in animals below man, makes research in this field particularly difficult. A direct attack on the disease is hardly feasible; therefore it seems to be sound strategy to try circu.tous approaches, such as the investigation of bone development, growth, and repair under various conditions, and the study of other diseases that show suggestive correspondence. It is possible that directions of investigation that may seem initially somewhat remote from otosclerosis itself may provide insights that will eventually lead to success in reaching our goal. The applicant should describe correlations between proposed research with the clinical patho'ogical entity of otosclerosis. Therefore, our policy is to aid and encourage research in the immediate field of otosclerosis and also in adjoining fields if in our considered opinion there is a substantial promise that the results obtained will be of assistance in the ways just indicated, and may lead to the attainment of our objective. To stimulate the attainment of this objective, the Board of Trustees wishes to general otolaryngologic community that funds are still available for suitable grants. forms may be obtained from the office of the secretary, David A. Hilding, MD, Taylor Street, Chicago, IL 60612. Grant requests should be submitted by January

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remind the Application 1855 West 31.

Organ culture of the mammalian and avian embryo otocyst.

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