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GROWTH OF THE INNER EAR IN ORGAN CULTURE

'THOMAS VAN DE WATER,M.A: R. J. RUBEN, M.D.

SUPPLEMENT 14 - VOL. 83, SEPT.-OCT., 1974, NO. 5, PART 2

THE ANNALS OF O T O L O G Y , R H I N O L O G Y

&

LARYNGOLOGY

THE ANNALS PUBLISHING COMPANY 4949

FOREST PARK BLVD.

ST. LOUIS, MISSOURI63108 COPYRIGHT, 1974, ANNALS PUBLISHING COMPANY

GROWTH O F T H E INNER EAR I N ORGAN CULTURE THOMAS VAN DE WATER,M.A. R. J. RUBEN,M.D. BRONX,NEW YORK SUMMARY - An organ culture system is presented for the mammalian inner ear. The development of the inner ear is recorded with time lapse cinematography and conventional histological techniques. The system allows for morphological development of differentiation of the neurosensory structures of the inner ear. The use of the organ culture system as a research tool for the understanding of normal and abnormal development of the inner ear is discussed.

There have been numerous reports of the organ culture of the avian inner ear by Friedmax~n,~-I~ Lawrence and Merchant,I3 Orr14J5and Sugahara.16 There have been three reports concerning organ culture of the mammalian inner ear by Maximow,I7 Lawrence and MerchantI3 and Sobkowicz et al.Is This laboratory reported an organ culture system for the mouse ear in 1969.19 Since then, several other reports concerning quantification, development, ultrastructure and development of a genetic congenital anomaly have been r e ~ o r t e d . ~ OThis - ~ ~ report will present the present technique and some of the morphological developmental changes which occur with the present system. TECHNIQUES

Experimental Animal. The CBA-J/ CBA-J strain of normal mouse obtained from Jackson Memorial Laboratories was used to supply the timed Pregnancies. The gestation period was timed by the vaginal PI% method. The day of ~bservation of a mucoid plug obstrutting the vaginal orifice was designated gestation day one. Dissection Procedure. The pregnant mice were sacrificed by cervical dislocation on the twelfth or thirteenth

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day of gestation. The gravid uteri were dissected free and placed in a petri dish with prewarmed Hank's balanced salt solution (HBSS). The embryos were dissected free of the uterus one at a time and placed in a petri dish containing HBSS without phenol red indicator. The otocysts were dissected free of the embryo with the aid of a Leitz dissecting scope with transillumination, No. 3 and No. 5 Dumont watchmaker's forceps that have been sharpened on an Arkansas oil stone and fine fire-polished tungsten probes. The excised otocysts were placed immediately into the organ culture system and then into an incubator.

Organ Culture System. Falcon plastic organ culture dishes, 60 x 15 mm, with an outer absorbent ring were used as the organ culture receptacle. The outer ring with its absorbent material was filled with approximate~y3 ml of sterile, demineralized, distilled water. ~h~ inner organ culture chamber was filled with 0.15 ml of organ culture medium. The inner surface of the top of the organ culture dish was ringed with a bead of sterile, unbleached vaseline so that a sterile, gas tight closure could be made when the dish was closed. The organ culture medium used

From the Department of Otorhinolaryngology, Albert Einstein College of Medicine, Bronx, New York. This work has been supported by the Deafness Research Foundation; National Institute of Neurological Diseases and Stroke; Children's Hearing, Education and Research; the Manheimer Fund and the Nebur Fund. Presented at the meeting of the American Otological Society, Palm Beach, Florida, April 21-22, 1974.

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was Neumann and Tytell's serumless medium23that was supplemented with 20% fetal calf serum and penicillin at a concentration of 100 units/ml of media. The organ culture medium was changed every fourth day. The cultures were incubated at 34.5' C in an ambient air atmosphere. India Ink Drawings. The India ink drawings of the living organ culture specimen were made by projecting the image of the organ culture upon the surface of a sheet of white paper. The outline of the developing endolymphatic area was traced over with a drawing pen. A Bausch & Lomb TriSimplex projection scope with a medium power objective was used to project the cultures. A Corning glass heat shield was placed over the culture dishes to protect the cultures from excessive heat. Time Lapse Cinematography. A Nikon inverted microscope with a built-in stage incubator was used for observation of the developing culture. A Nikon AFM side-arm connected to a Bolex 16 mm, motor-driven, time lapse movie camera provided the time lapse which was run at one frame per minute. The film used was Kodak 16 mm plus X reversal, and the exposure was determined automatically by the AFM shutter control box. A blue green filter was used for contrast and the exposure control box was compensated for the filter factor. The specimen was fine focused three times each day and the film was changed every three days. Histology. The organ culture specimens were fixed by withdrawal of the organ culture medium and the addition of 2 ml of 5% glutaraldehyde fixative in 0.1 molar cacodylic acid-sucrose buffer for one hour at 4' C. The specimen was then washed for one hour in two changes of 0.1 molar cacodylic acid-sucrose buffer. Dehydration was accomplished by two one-half hour washes of a 1 : l mixture of methanol and ethylene glycol monomethyl ether ( M&M) . The specimen was then infiltrated by a 9 : l mixture of polyethylene glycol 400 disterate and cetyl alcohol. The tissue was embedded so that the

plane of section was approximately parallel to the plane of the tissue that was parallel to the bottom surface of the plastic organ culture dish. The embedded tissue was allowed to stand overnight at room temperature and then stored at 4' C until sectioned. The specimen was sectioned at 7 mp on a Leitz rotary microtome. The cut ribbons of specimen were serially mounted on slides and exposed in a formalin vapor chamber overnight. The slides were then dewaxed and stained by a standard Harris hematoxylin and phloxin B procedure. RESULTS

Figures 1-3 demonstrate the morphology of the in vitro development of the inner ear. Figure 1 shows freshly explanted 12-day mouse otocyst in organ culture. Superiorly an endolymphatic duct is noted. The triangular shape of the endolymphatic portion of the otic cyst is apparent. The statoacoustic ganglion complex is seen on the left side of the endolymphatic portion of the otocyst. Figure 2 is a 12-day otocyst which has been in the organ culture system for seven days. The endolymphatic duct and sac are noted in this photomicrograph. Two semicircular canals join in a crus commune. A utriculosaccular space and the cochlear duct are seen. Figure 3 shows a 13-day explanted otocyst in culture for nine days. The superior semicircular canal with its ampulla is present. The statoacoustic ganglion mass is apparent. The cochlear duct has extended and begun to coil. Figures 4-6 are India ink tracings of actual morphological changes that occurred in explanted otocysts of twelfth and thirteenth gestation day mice. Figure 4 is a 12-day otocyst1314A4 that was followed for seven days in vitro. The tracings represent 1 ) day of explantation, 3/11; 2 ) two days in vitro, 3/13; and 3) seven days in vitro, 3/18. The otocyst on day two has the primary fold of the developing semicircular canals, has formed a utriculosaccular area, and the cochlear duct has grown and started to enlarge on its distal end. The oto-

l N N E R EAR IN ORGAN C U L T U R E

Fig. 1. An explanted twelfth gestation day otocyst, zero hours i n oitro. The statoacoustico ganglion complex and endolymphatic duct are identified. ( X 100). Fig. 2. A twelfth gestation day otocyst, seven days in uitro. A cleveloping semicircular canal, endolymphatic duct and cochlear duct are identified. ( X 100). Fig. 3. A thirteenth gestation day otocyst, nine days in uitro. A developing semicircular canal with its ampulla and a developing cochlear duct are indicated. ( X 100). Abbreviations: A - Ampulla; Cart. - Cartilaginous otic capsule; CC - Crus cornmune; C D - Cochlear duct; ED - Endolymphatic duct; ES - Endolymphatic sac; N Stato-acoustico ganglion complex; PC - Pigmented cell; PI - Pars inferior; PL - Perilymphatic space; PS - Pars superior; SH - Sensory hair tuft; SL - Spiral limbus; SSC Semicircular canal; SV - Stria vascularis.

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4

- SSC N

3/11 0 day

Fig. 4. A series of India ink tracings of the in uitro morphogenesis of an explanted twelfth gestation day otocyst (Specimen 1314AI).

3/18 Oday

3/19 lday

Fig. 5. A series of India ink tracings of the in uitro morphogenesis of an explanted thirteenth gestation day otocyst (Specimen 1316A2).

CD

3/18 Oday

3/19 lday

3/25 'days

Fig. 6. A series of India ink tracings of the in uitro morphogenesis of an explanted thirteenth gestation day otocyst (Specimen 1316Bl).

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Fig. 7. Prints taken at %day intervals from a 1 6 mm time-lapse movie recording the in uitro development of a twelfth gestation day otocyst (Specimen 1312A2): A ) 0.5 days in uitro; B ) 2.5 days in uitro; C ) 4.5 days in uitro; D ) 6.5 days in vitro. ( X 100).

cyst on the seventh day of in vitro development has formed two distinct semicircular canals that communicate via a crus commune. The outline of the lateral semicircular canal is also evident. The utriculosaccular area is more defined and the cochlear duct has extended and coiled. An area of endolymphatic sac is present that has the morphological appearance of pars rugosa. Figures 5 and 6 represent the morphological changes that occurred in two thirteenth gestation day otocysts ( 1316A, and 1316B, ) that developed for seven days in vitro. Tracings made on the day of explantation are 3/18 and on one day in vitro, 3/19. The final tracings made after seven days in vitro development are labeled 3/25. The tracings of the explanted 13-day otocyst show the beginning folds of the semi-

circular canals. There is no defined utriculosaccular area and the cochlear duct has just begun to elongate with a slight flexure in a rostrad direction. After 24 hours of in vitro development the semicircular canal folds are more prominent, forming rudimentary semicircular canals but without defined ampullar enlargements. The cochlear duct has elongated and begun to have a definite hook. The final tracings done on the completion of the seventh day of in vitro development demonstrate the marked changes that have occurred. The cochlear duct has extended to three times its length and coiling of this duct is evident in both of these specimens. A defined utriculosaccular area is evident in both specimens and the semicircular canals are present with ampullar enlargements. Histological examination of all of the above specimens confirmed

VAN DE WATER - R U B E N

SSC

CD

2/28 2.5days

Fig. 8. A series of India ink tracings taken every two days of specimen 1312A? of Figure 7. The tracings taken from the time-lapse movie correspond to the stages of development of the microphotographs of Figure 7.

the presence of well defined sensory structures located in the appropriate morphological positions. Figure 7 represents prints of a twelfth gestation day mouse otocyst (1312A2) as it develops i n vitro taken from a 16 mm time-lapse movie. A total developmental period of six days i n vitro was covered by the movie. Figure 7A represents the 12-day otocyst after twelve hours of i n vitro development because the explanted otocyst was allowed to attach to the surface of the organ culture dish overnight, prior to the start of the time-lapse movie. Figures 7B, C and D represent two-day intervals of in vitro development. The morphological changes that occurred are best visualized in Figure 8 by the

India ink tracing taken from the film at the same stages of development illustrated by Figure 7. At the start of the film recording, at one-half day i n uitro, the cochlear duct has begun to extend and a slight flexure is noted. There is no clearly defined utriculosaccular area and there is just a beginning of the primary folds of the semicircular canals. After two and one-half days of in vitro development, there is a marked flexure of the cochlear duct and considerable extension of this developing sensory structure. The folds of the semicircular canals are prominent and rudimentary canals are being formed but no ampullar enlargement is noted at this stage of development. During the next two days of in vitro development, consider-

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Fig. 9. A microphotograph of a twelfth gestation day otocyst (Specimen 1304) taken from a 16 mm time-lapse movie at the time of explantation, zero hours in uitro. (X80). Fig. 10. A microphotograph of the twelfth gestation day otocyst (Specimen 1304) seen in Figure 9 after eight days of in oitro development. This photograph represents only the cochlear duct portion of the developing organ culture with an associated bundle of nerve fibers developing on the surface of the stato-acoustico ganglion complex. ( X 100).

Fig. 11. A medium power photomicrograph of a cross section of a twelfth gestation day otocyst in vivo prior to extirpation. The endolymphatic duct is present entering into the pars superior portion of the otocyst. ( X 200).

able morphological change occurs, and at four and one-half days of in vitro development the cochlear duct has extended for a greater length and has a prominent hook. A defined utriculosaccular area can now be identified and the two semicircular canals that can be visualized join in a crus commune. The ampullar enlargements of the semicircular canals are identified. During the next two days of in vitro development no new structures were seen to-form. The structures that had formed by 4.5

days in vitro appear to undergo additional development. At 6.5 days in vitro the ampullar enlargements of the semicircular canals appear to be more prominent than they were at 4.5 days. The cochlear duct has continued to extend but now appears to be limited by its cartilaginous capsule which also prevents additional flexure of this structure. Figures 9 and 10 are prints taken from a 16 mm time-lapse movie. A 12-day otocyst (1304) developed for ten days in uitro. Figure 9 represents

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Figs. 12 to 25. A series of serial sections of a twelfth gestation day otocyst (1312A2) that has developed for ten days in uitro. Specimen 1312A2 is the otocyst whose in zjitro development was recorded on a time-lapse movie and is represented by Figures 7 and 8. The cartilaginous otic capsule is noted in Figure 12. The developing cochlear duct portion of the organ culture specimen is indicated in Figure 14. In Figure 1 5 an asterisk marks the perilymphatic space of a semicircular canal. Arrow 1 of Figure 16 denotes a developing organ of Corti formation and arrow 2 indicates a macular formation. The opening of the endolymphatic duct into the utriculosaccular space is indicated by an arrow in Figure 17. In Figure 18 the endolymphatic duct is separated from the utricnlosaccular area and is indicated by an arrow. Two arrows in Figure 19 point out two cristae. An arrow in Figure 20 notes the endolymphatic duct as it enlarges to form an endolymphatic sac. In Figure 22 a folded area of the endolymphatic sac which resembles the pars rugosa is indicated by an arrow. ( X 65).

the 12-day otocyst on its day of explantation into the organ culture system. The endolymphatic projection is identified; there is also a prominent pars superior with the beginnings of a primary fold of a semicircular canal. The pars inferior has begun to form a cochlear duct. Figure 10 was taken at approximately the same magnification as Figure 9. The organ cultured otocyst has undergone such extensive enlargement and development in the preceding eight days of in vitro development that only the area of the developing cochlear duct (pars inferior) is represented in the print. A large bundle of nerve fibers is seen sending processes to the area of the developing cochlear duct. Figure 11 is a cross section of a twelfth gestation day mouse otocyst at the time of explantation. This section is at the level of the entrance of the

endolymphatic d u c t into t h e pars superior. Figures 12 to 25 are a series of serial sections from specimen 1312A2 whose morphological development is noted in the frames taken from the time lapse studies (Figs. 7 and 8). This otocyst was placed in organ culture at 12 days of age and remained in the culture for ten days. Figure 12 shows the cross section through the cartilaginous capsule of the organ culture. Figure 13 is a further section into the specimen showing the endolymphatic and perilymphatic areas. A further serial section is demonstrated in Figure 14 showing the developing cochlear duct and the utriculosaccular area. Figure 15 is a continuation of the serial sections and shows the cochlear duct cut in two planes with the developing organ of u s limbus, stria Corti, internal s ~ ~ l c cells, vascularis and tectorial membrane. A

INNER EAR IAJ (3RCAN C U L T U R E

perilymphatic area of a semicircular canal is noted superiorly on the right. Figure 16 is a further serial section with the same elements as seen in Figure 15, plus a macula and another semicircular canal. Figure 26 is a higher magnification of one of the maculae. Tufts of sensory hair are seen. Figure 27 is a higher magnification of the developing organ of Corti. The developing inner and outer hair cells are noted; the large internal sulcus cells with basal nuclei and large apical eosinophilic cytoplasm are seen, The amorphous

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tectorial membrane is seen on the apical surfaces of these developing internal sulcus cells. A developing stria vascularis with its ectodermal and mesodermal components is apparent. F i g u r e 17 s h o w s p e r i l y m p h a t i c spaces, a utriculosaccular area and two semicircular canals. The entry of the endolymphatic duct into the utriculosaccular area is noted in the upper right hand corner of this section. Figure 18 is a further serial section which shows the same elements as in Figure 17, with the addition of the be-

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ginning of the endolymphatic duct. Figures 28 and 29 are higher magnzcation microphotographs, illustrating the relationship of the endolymphatic duct with the utriculosaccular space. Figure 19 is a continuation of the serial sections showing two ampullae with their associated cristae and the endolymphatic duct. Figure 30 is a higher magnification from Figure 19 showing the detail of the crista ancl its cupula and the macula. Figure 31

shows in greater detail the crista and its cupula as seen in Figures 19 and 30. Pigmented cells are noted under the crista. Figure 20 is a further serial section showing the same two ampullae and the enlarging endolymphatic duct. Figures 32 and 33 are medium power photomicrographs of serial sections showing the ampulla with its crista, cupula and its semicircular canal. Figure 21 is a further serial sectioil

I N N E R E A R I N O R G A N CCILTURE

showing one crista and two semicircular canals. The endolymphatic duct has increased in relative size. Figure 34 is a higher power of a further serial section showing the relationship of the endolymphatic duct to the semicircular canal and the formation of an endolymphatic sac. Figure 22 is a further serial section showing two semicircular canals and

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the endolymphatic sac. Figure 35 is a higher magnification of the endolymphatic sac in an area which may represent the pars rugosa. Figure 23 is :I further serial section illustrating the same structures seen in Figure 22. Figures 24 and 25 are the last two representative serial sections showing the most peripheral portion of the semicircular canal and the endolymphatic sac.

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Fig. 26. A high power microphotograph of a n~aculaeseen in Figures 15 and 16. The perilymphatic space and a tuft of sensory hairs projecting into the lumen of the organ culture are identified. ( X 500). Fig. 27. A high power microphotograph of a cochlear duct area seen in Figure 16. The spiral limbus and stria vascularis are labeled. An asterisk is over the luminal surface of a developing organ of Corti and multiple arrowheads indicate a forming tectorial membrane. ( X 340). Figs. 28 and 29. Two serial medium power microphotographs illustrating the communication of the endolymphatic duct with the utriculosaccular area. ( X 180).

The significant structures found in specimen 1312A, are summarized in Table I. DISCUSSION

The organ culture system has been demonstrated to have a high degree of r e p r ~ d u c i b i l i t y Observations .~~ from the time lapse studies and the sequential drawings of living culture suggest that there may be two different mechanisms for the formation of the semicircular canals and the cochlear duct. The semicircular canals appear to be formed initially by production of primary folds that are then extended and thinned. This is followed by a condensation of

tissue away from the center of the rudimentary canal. After the arc of the canal has formed, an ampullary swelling occurs and the ampullae develop. The cochlear duct seems to be formed by an extension of the ventral portion of the otocyst. This is followed by a flexure of the developing cochlear duct. The duct continues to extend as it begins to coil to assume its cochlear formation. As this forms, the medial wall of the otocyst is probably responsible for the sensory epithelium of the utricle, saccule and cochlea. The lateral wall probably forms the stria vascularis. The apical end of the cochlear duct appears

lNNER EAR IN ORGAN CULTURE

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Fig. 30. A medium power microphotograpl~of a maculae (arrow) and a cristae (Fig. 18). The cupula of the cristae is indicated by an asterisk. ( X 200). Fig. 31. A high power microphotograph of the cristae of Figure 30. A tuft of sensory hairs projecting into a cupula (asterisk) is noted. A pigmented cell in tlle perilymphatic space below the cristae is indicated. ( X 640).

to be formed first and the remainder of the cochlear duct is formed later. This would agree with previous studies of terminal mitosis.24 TABLE I SUMMARY O F DEVELOPMENT

Organ Culture Specimen #I31 2A1 Age at Day of Explantation - Twelfth Gestation Day No. of days in uitro - 10 Cartilaginous Present otic capsule Present Perilymphatic spaces Semicircular Three present with ampullae canals Cristae Three present with associated cupulae Maculae Two present in utriculosaccular area Stato-Acoustico Present ganglion Endolymphatic Present duct Endolympliatic Present sac Organ of Corti Present - Good inner sulcus cells with forming tectorial membrane Stria vascularis Present and forming General Excellent -Well defined areas of sensory structures morphology with a definite relationship to overall morphology

The ability to grow the inner ear in organ culture now allows an investigator to directly manipulate the development of the ear, either surgically and/or biochemically. There are at least two areas of research in which the organ culture technique has been found in other systems to be critical in understanding biological and pathological problems. The first of these is in the field of experimental embryology and elucidation of the mechanisms by which orderly development of the eye takes place.25 There is almost no knowledge at this time concerning the physical and biochemical mechanisms necessary for the normal development of the mammalian inner ear. It is expected that with the use of organ culture techniques many of these mechanisms can be defined. The second area in which organ culture and/or tissve culture has played a substantive role is in the understanding, prevention and cure of human disease.2G There are numerous examples, among which are viral diseases, such as polio and rubella; degenerative diseases, such as Niemann-Pick, Gaucher, Tay-Sachs and multiple sclerosis; and other abnormalities of metabolism, such as the mucopolysaccharidoses and maple syrup

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Figs. 32 and 33. Two serial section, medium po\r7er microphotographs that depict the area of the semicircular canal. Asterisks denote the cupula of the cristae in the ampulla of the semicircular canals. The arc of the semicircular canal is indicated by arrows. ( X 180). Fig. 34. A medium power microphotograph of the endolymphatic duct as it enlarges to form an endolymphatic sac. ( X 180). Fig. 35. A medium power microphotograph of an area of the endolymphatic sac that is similar to the pars rugosa in oioo. ( X 180).

disease; and in the work of cancer, e.g., Burkitts' lymphoma. This very incomplete list illustrates many of the ways in which organ culture can be used to attack the fundamental problems of disease.

The same broad general principles can be applied to the studies of inner ear deafness. There are several areas of investigation in which the organ culture technique has immediate application. The organ culture system can be

I N N E R EAR IN O R G A N CULTURE

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used in the examination of genetic inner completely chemically defined media ear degeneration and it could be used allows for even more ways of maniputo study the effects of viruses in the lating the environment. Additionally, developing inner ear. Recent work by other work has been instituted in the DeolZ7again points out the significance ear in order to begin to grow, selecof thyroid abnormality in inner ear de- tively, various parts of the organ culgeneration. The organ culture system ture. This has been done successfully is an ideal tool with which to investi- with other organs and there appears to gate these problems, both experimen- be no reason why this cannot be done tally and clinically. Another area which in the ear. Whether or not we would can easily be studied with this tech- have one uniform type of tissue from nique is the effect of ototoxics in the the inner ear is an open question at this inner ear. The organ culture system point. Much effort will be spent in this allows for quantification of dosage and area because of its obvious significance for a sophisticated biochemical analysis to the further study of inner ear disease. of the effects of treatment. The list of investigations which can This discussion has only pertained to be done with the organ culture system the organ culture technique as it was can be greatly extended. The system applied in this study. More recently, has been adequately developed and the technique has been further refined should be used to attack the problems so that organ cultures can now be of inner ear deafness. If the same exgrown under completely chemically de- perience found in other disease sysfined media. The techniques used for reporting the organ cultures in this tems is reproducible, even in part, in paper depended upon the use of fetal work with the inner ear, there should calf serum for portions of the media be substantive contributions to the unwhich were undefined. The ability to derstanding, prevention and cure of grow the organ of the inner ear in a inner ear deafness. DEPT. OF OTORHINOLOGY, ALDERTEINSTEINCOLLEGEOF MEDICINE,1300 MORRISPARK -

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AvE., BRONX, N. Y. 10461.

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ACKNOWLEDGMENTS -The authors wish to thank Ms. Cathy Shea for her technical assistance, Mr. Milton Kurtz for the microphotography, and Drs. Murray Bornstein, Kenneth Shulman and Robert Rosinde for the generous loan of equipment and technical assistance. REFERENCES

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( e d ) : Cells and Tissues in Culture, London h New York, Academic Press, 1965, vol. 2, p 521

8. Friedmann I: The chick embryo otocyst in tissue culture: A model ear. J Laryngol Oto182:185-201, 1968 9. Friedmann I: Innervation of the developing fowl embryo otocyst. Acta Otolaryngol 67:224, 1969 10 Friedmann I, Bird ES: The effect of ototo~icantibiotics on isolated chick embryo otocyst, J path Bact 81:81, 1961

11. Friedmann I, Bird ES: Virus particles in tissue culture of a healthy chick embryo OtocYst. J Ultrastruct Res 5:428, 1961 12. ~ ~ I, ~ i ES: i ~ ~l~~~~~~ d ~ micro-d scopic studies of the isolated fowl embryo otocyst in tissue culture. J Ultrastruct Res 20: 356-365, 1967 13. Lawrence M, Merchant D!:

Tissue cul-

~

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ture technique for the study of the isolated otic vesicle. Ann Otol Rhinol Laryngol 62: 770. 1953 14. Orr MF: Development of acoustic ganglia in tissue cultures of embryonic chick otocysts. Exp Cell Res 40:68, 1965 15. Orr MF: Histogenesis of sensory epithelium in reaggregates of dissociated embryonic chick otocysts. Dev Biol 17:39, 1968 16. Su ahara Y: Morphological studies on the memtranous labyrinth cultured in vitro. J Otolaryngol Jap 67:81, 1964

21. Van De Water TR, Ruben RJ: Quantification of the "in vitro" development of the mouse embrvo inner ear. Ann Otol Rhino1 Laryngol (sbppl No. 4 ) 82: 18-21, 1973 22. Van De Water TR, Ruben RJ: Or an culture of the mammalian inner ear: A toof to study inner ear deafness. Laryngoscope, 1974 (in press) 23. Neuman RE, Tytell AA: Serumless medium for cultivation of cells of normal and malignant origin. Proc Soc Exp Biol Med 104:252-256, 1960

17. Maximow A: Tissue cultures of young mammalian embryos. Entry to Embryology 6:80, 1925

24. Ruben RJ: Development of the inner ear of the mouse: A radioautographic study of terminal mitosis. Acta Otolaryngol (Suppl) (Stockh) 220, 1967

18. Sobkowicz HM, Bereman B, Rose JE: Development of the organ of Corti of the newborn mouse in culture. J Acoust Soc Am 55:459, 1974

25. Coulombre AJ: The eye, in DeHaan RL, Ursprung H ( eds ) : Organogenesis. New York, Holt Rinehart & Winston, 1965, pp 219254

19. Van De Water TR, Ruben RJ: Organ culture of the mammalian inner ear. Acta Otolaryngol 71 :303-312, 1971

26. Nitowsky HM: Use of cell culture techniques in diagnosis and studies of inherited diseases. Seminars in Hematology 9, No. 4: 403-429, 1974

20. Van De Water TR, Heywood P, Ruben RJ: Development of sensory structures in organ cultures of the twelfth and thirteenth gestation day mouse embryo inner ears. Ann Otol Rhinol Laryngol (Suppl No. 4 ) 82:l-17, 1973

27. Deol MS: An experimental approach to the understanding and treatment of hereditary syndromes with congenital deafness and hypothyroidism. J Med Genet 10:235-242, 1973