EP1161142A1

EP1161142A1 - Methods for culturing fluid-filled sensory organs

Info

Publication number: EP1161142A1
Application number: EP00914825A
Authority: EP
Inventors: Jonathan Kil; Hubert Lowenheim; Anish H. Sudra
Original assignee: Individual
Current assignee: Otogene AG; Otogene USA Inc
Priority date: 1999-03-05
Filing date: 2000-03-03
Publication date: 2001-12-12
Also published as: EP1161142A4; AU3616400A; CA2364024A1; WO2000054583A1

Abstract

The present invention provides methods for culturing fluid-filled sensory organs in vitro. In particular, the present invention provides methods for culturing the eyeball, including the retina, and the inner ear, including the Organ of Corti, in vitro. The methods of the present invention involve little or no microsurgical dissection of the entire fluid-filled sensory organ, thereby preserving the structural and functional integrity of the sensory epithelium. The methods of the present invention include the steps of (a) introducing a fluid-filled sensory organ (such as an inner ear or an eye ball) into a culture chamber containing liquid culture medium and (b) moving the culture chamber so that the fluid-filled sensory organ moves within the culture chamber. Preferably the culture chamber is cylindrical or annular in shape and is rotated about its longitudinal axis.

Description

METHODS FOR CULTURING FLUID-FILLED SENSORY ORGANS

Field of the Invention This invention relates to /// vitro cell and tissue culture methods applicable to fluid- filled sensory organs, such as the inner ear. Background of the Invention

In vitro culture of cells and tissues provides a relatively convenient way of measuring and manipulating biochemical, physiological, and developmental processes. Removal of tissue from its normal cytoarchitecture, i.e., removal from the anatomical structures that normally surround and support the excised tissue, may, however, affect the biochemical, physiological and developmental responses of the excised tissue, thereby making it difficult to interpret data obtained from in vitro studies Moreover, surgical excision of the tissue of interest, which may be small and delicate, can result in tissue damage One possible solution to this problem is to remove and culture the tissue of interest together with as much of the surrounding cytoarchitecture as possible. Due to the debilitating effects of loss of sight or hearing, there is particular interest in studying the tissues and cell types that mediate the perception of light and sound The retina is the specialized tissue within the eye ball that includes cell types (rods and cones) that respond to light The Organ of Corti, located within the bony labyrinth, contains the specialized hair cells that respond to sound The ability to culture the intact eyeball or intact bony labyrinth //; vitro would provide investigators with the ability to study and manipulate the normal biochemistry, physiology and development of the cells and tissues responsible for light and sound perception, and to thereby develop therapies for treating blindness and deafness

Traditional organotypic culture of fluid-filled sensory organs, such as the mammalian auditor ' sensory epithelium, the Organ of Corti, involves fine microsurgical dissection and adherence of the epithelium to either a glass slide or coverslip The slide or coverslip provides the necessary support to maintain the Organ of Corti's delicate cytoarchitecture and is first coated with a biological adhesive to make the epithelium adhere in its proper orientation For example, inner ear sensory hair-cells lie superior to their underlying, non-sensory supporting-cells and can be maintained in culture using this method.

Maintenance of proper cytoarchitecture can also be achieved by suspending the organ in either a collagen, fibrin or thrombin clot However, this method inhibits future genetic manipulation and/or the delivery of growth factors to the appropriate target cells, since collagen, fibrin and thrombin all impair the delivery of large proteinaceous molecules including growth factors, plasmids and viruses Floating the microdissected, sensory epithelium reduces the need for such methods, but after prolonged culture periods, the organ collapses or folds upon itself resulting in the degeneration or loss of the sensory cells. In addition, all of the foregoing methods are inherently prone to variability since they rely upon fine microsurgical techniques that often require extensive training Additionally, all of the foregoing techniques were developed using tissues from neonatal animals that are much easier to surgically manipulate However, the results of such studies are difficult to interpret and apply to the more mature adult system Culturing the adult sensory system using the foregoing methods does not result in the maintenance of normal cytoarchitecture Even with extensive training, the shear force imparted during microsurgical manipulation on tissues that are comprised of only two cell layers, makes them problematic to grow and manipulate For these reasons, the in vitro study of sensory cell regeneration is very difficult Preferably, a method of culturing a fluid-filled sensory organ or structure, will involve little or no microsurgical dissection of the entire fluid-filled sensory organ or structure (i.e., inner ear or eye) Such a technique would be of added benefit, in that the sensory epithelium of the fluid-filled structure (i.e., organ of Corti or retina) could be maintained in its native, local environment Unfortunately, utilizing prior art culture techniques, when the whole inner ear is surgically dissected and floated in culture media, the sensory epithelium within the inner ear quickly degenerates with an almost complete loss of both sensory cells and non-sensory cells

Consequently, there is a need for a method of culturing fluid-filled sensory organs, such as the bony labyrinth (including the Organ of Corti) and eyeball, in vitro, that involves little or no microsurgical dissection of the entire fluid-filled sensory organ, and that preserves the structural and functional integrity of the sensory epithelium

Summary of the Invention -J-

The present invention provides methods tor culturing fluid-filled sensory organs /// vit/ o In particular, the present invention provides methods for culturing the eyeball, including the retina, and the bony labyrinth, including the Organ of Corti, in vit/o The methods of the present invention involve little or no microsurgical dissection of the entire fluid-filled sensory organ, thereby preserving the structural and functional integrity of the sensory epithelium

The methods of the present invention include the steps of introducing a fluid-filled sensory organ into a culture chamber containing liquid culture medium, and moving the culture chamber (for example rotating vibrating or rocking the culture chamber) so that the fluid-filled sensory organ moves within the culture chamber Preferably the culture chamber is completely filled with liquid cultui e medium Preferably the fluid-filled sensory organ is continuously, or almost continuously, in motion and suspended in the liquid cultuie medium, / e , preferably the fluid-filled sensory organ rarely or never contacts the walls of the culture chamber during culture, thereby minimizing physical damage to the fluid-filled sensory organ Also, the fluid-filled sensory organ is preferably moved in a manner that minimizes the turbulence and shear forces that are experienced by the fluid- filled sensory organ during culture within the culture chamber For example, the culture chamber may be cylindrical or annular in shape, or disc-shaped, and may be rotated horizontally about its longitudinal axis during culture of a fluid-filled sensory organ The culture chamber can include an inlet to permit the inflow of oxygen, such as inflow across a membrane that is permeable to oxygen but impermeable to microorganisms The culture chamber also can include at least one sampling port through which samples of the liquid medium, or of the fluid-filled sensory organ tissue can be removed, and other substances can be introduced into the liquid medium In another aspect, the present invention pi ovides assays for biologically active molecules, the assays including the steps of (a) introducing a fluid-filled sensory organ selected from the group consisting of an inner ear and an eye ball into a culture chamber containing liquid culture medium, (b) contacting the fluid-filled sensory organ with a biologically active substance to be assayed, the fluid-filled sensory organ being contacted with the substance before, during or after being introduced into the culture chamber, (c) culturing the fluid-filled sensory organ, said culturing step including moving the culture chamber so that the fluid-filled sensory organ moves within the culture chamber, and (d) measuring a response in the fluid-filled sensory organ induced by the substance Preferably the culture chamber is continuously rotated during culture of the fluid-filled sensory organ

The methods of the present invention permit the continuous culture of fluid-filled sensory organs over an extended time peπod without significant degeneration of the organ, tissues or cells The methods of the present invention permit the continuous culture of fluid-filled sensory organs for a period extending from a few minutes to more then 150 hours

Brief Description of the Drawings The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same becomes better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIGURE 1 shows a representative embodiment of an apparatus, including a rotatable culture vessel, useful in the practice of the present invention for culturing fluid- filled sensory organs.

Detailed Description of the Preferred Embodiment The methods of the present invention permit the in vitro culture of intact, fluid- filled sensory organs, including the eyeball and the bony labyrinth (which includes the Organ of Corti) The anatomy of the inner ear is well known to those of ordinary skill in the art ( ee, e.g., Gray's Anatomy, Revised American Edition ( 1977), pages 859-867, incorporated herein by reference). Culturing the intact sensory organ minimizes the trauma and damage to the cells and tissues involved in sensory perception, such as the inner ear sensory epithelium including sensory hair cells and their associated supporting cells, because the fluid-filled sensory organ is not dissected to expose and/or remove the sensory tissue The methods of the present invention permit investigation of the normal development, biochemistry and physiology of cells involved in sensory perception. The methods of the present invention also permit targeted destruction of specific cell types, such as the sensory hair cells of the inner ear, in order to investigate the regeneration of specific cell types. More specifically, the methods of the present invention permit screening, assaying and otherwise evaluating biologically active molecules, such as molecules that are capable of inducing, suppressing, or otherwise altering, the growth, development, physiology or biochemistry of one or more cell types of the cultured, fluid-filled sensory organ. Examples of molecules that can be screened, assayed or otherwise evaluated using the methods of the present invention include, but are not limited to: proteins; peptides; growth factors; steroids, mitogens (including insulin); differentiation-inducing factors (including triiodo- 1 -thyronine, retinyl acetate and folate); inhibitors of cell death; and protective molecules (that protect the cultured, fluid-filled sensory organ from the biochemical stress of tissue culture), such as antioxidants (e.g., catalase, superoxide dismutase and fatty acids such as linoleic and linolenic acid). Additionally, the methods of the present invention can be used to introduce genes, proteins, peptides and other macromolecules into some or all of the cell types of a fluid-filled sensory organ. Further, the methods of the present invention can be used to test and evaluate methods of delivering genes, proteins and other macromolecules into some or all of the cell types of a fluid-filled sensory organ In particular, the methods of the present invention can be used to culture an inner ear and selectively lesion inner ear sensory hair cells and test the ability of the associated, non-sensory support cells to form new sensory hair cells The methods of the present invention include the steps of introducing an intact, fluid-filled sensory' organ into a culture chamber containing liquid medium adapted to permit the m vitro culture of the fluid-filled sensory organ, and moving the culture chamber so that the liquid medium and the fluid-filled sensory organ move within the culture chamber, preferably in a manner that minimizes the turbulence and shear forces that are experienced by the fluid-filled sensory organ Preferably, the fluid-filled sensory organ is continuously moved within the culture chamber so that the fluid-filled sensory organ is continuously suspended within the liquid medium Preferably, the culture chamber is rotated about its longitudinal axis

Presently preferred embodiments of an apparatus that is useful for culturing fluid- filled sensory organs in accordance with the methods of the present invention are disclosed in U S Patent Serial No 5,437,998, U S Patent Serial No 5,702,941 and U S Patent Serial No 5, 763,279, each of which patents is incorporated herein by reference

A representative embodiment of an apparatus useful in the practice of the present invention for culturing fluid-filled sensory organs is shown in FIGURE 1 Apparatus 10 includes a motor disposed within motor housing 12 mounted upon a base plate 14 A culture vessel 16 is mounted on motor housing 12 and includes a rear end cap 18 and a front end cap 20 which define the ends of a cylindrical culture chamber 22 that includes wall portion 24 (which is transparent in the embodiment shown in FIGURE 1 ) and lumen 26 A core 28, including an oxygen-permeable membrane 30, extends between rear end cap 18 and front end cap 20 Front end cap 20 is secured to core 28 by bolt 32 Front end cap 20 is penetrated by ports 34 An air filter 36 is mounted on motor housing 12 and is in gaseous connection with core 28 and oxygen-permeable membrane 30 Thus, in the embodiment shown in FIGURE 1 , culture vessel 16 has a substantially horizontal, longitudinal axis In operation, air is filtered through air filter 36 and enters lumen 26 through oxygenator membrane 30 Lumen 26 contains liquid culture medium within which is suspended a fluid-filled sensory organ to be cultured Liquid culture medium can be introduced into lumen 26 through one or more ports 34 Similarly, substances to be screened, assayed oi otherwise evaluated for their effect on the biochemistry or physiology of the cultured, fluid-filled sensory organs can be introduced into lumen 26 through one or more ports 34 Individual ports 34 can be adapted in a variety of configurations for introduction or removal of substances (such as introduction of new media or removal of old media) into lumen 26, for example by fitting a luer lock syringe port During culture of a fluid-filled sensory organ, culture vessel 16 is continuously, or substantially continuously, rotated about its longitudinal axis by the action of a motor (not shown) within motor housing 12, so that the fluid-filled sensory organ within is kept in continuous, or substantially continuous, motion. The speed of rotation can be adjusted so that the cultured, fluid-filled sensory organ is constantly in motion, but rotation should not be fast enough to cause significant turbulence in the aqueous medium within lumen 26

Wall portion 24 may be constructed at least partially of a gas permeable material, such as silicone rubber. For example, half of wall portion 24 can be made from gas permeable material and the remaining portion can be made of nonpermeable material Gas permeable materials commonly available are opaque Thus, using nonpermeable material for at least part of wall portion 24 may provide an advantage in allowing visual inspection of the cultured, fluid-filled sensory organ

If so desired, the use of gas permeable material in the construction of at least part of wall portion 24 permits 0₂ to diffuse through wall portion 24 and into the cell culture media within lumen 26 Correspondingly, C0₂ can diffuse out of lumen 26 Thus, the use of gas permeable material in the construction of at least part of wall portion 24 may overcome the need for air injection into lumen 26 Air injection into the culture medium within lumen 26 may be utilized, however, if additional oxygen is required to culture a fluid-filled sensory organ. When an air pump is utilized to inject air into the culture medium, an air filter is preferably also employed to protect the air pump valves from dirt.

An alternative embodiment of an apparatus useful in the practice of the present invention is an annular vessel with walls that may be constructed at least partially of a gas permeable material. Annular is defined herein to include annular, toroidal and other substantially symmetrical ring-like shaped tubular vessels The annular vessel has closed ends and a substantially horizontal longitudinal central axis.

In another embodiment, an apparatus useful in the practice of the present invention comprises a tubular vessel constructed at least partially of a gas permeable material. The vessel has closed ends and a substantially horizontal longitudinal central axis around which it rotates The vessel furthermore has two slidably interconnected members wherein a first member fits slidably into a second member, forming a liquid tight seal therebetween and providing a variable volume tubular vessel The bioreactor has means for rotating the tubular vessel about its substantially horizontal longitudinal central axis. One or more vessel access ports are provided for transferring materials into and out of the vessel The embodiment of the culture apparatus with slidably interconnected members may be adjusted to provide the exact size bioreactor needed.

Three representative, commercially available apparatuses useful in the practice of the present invention for culturing fluid-filled sensory organs are known as the Rotary Cylindrical Culture Vessel (RCCV™), the High Aspect Ratio Vessel (HARV™) and the Cylindrical Cell Culture Vessel (CCCV^{'I M}) which are manufactured by Synthecon, Inc (8054 El Rio, Houston, Texas).

In the practice of the present invention, Neuralbasal™ media from Gibco BRL (Gibco BRL media are produced by Life Technologies, Corporate Headquarters, Gaithersburg, MD), which requires the addition of B27 or N2 media supplement, is the presently preferred culture medium. Other culture media can be successfully used, however, to culture fluid-filled sensory organs in the practice of the present invention. Other suitable media include DME, BME and M-199 with fetal calf serum or horse serum. All of the foregoing media are sold by Gibco -BRL. When using Neuralbasal™ medium, N2 or B27 supplements play a more significant role when extended periods of culture (>96 hr) are attempted

The methods of the present invention permit the continuous culture of fluid-filled sensory organs over an extended time period without significant degeneration of the organ, tissues or cells The methods of the present invention permit the continuous culture of fluid-filled sensory organs for a period extending from a few minutes to more then 150 hours For example, various embodiments of the methods of the present invention permit the continuous culture of fluid-filled sensory organs for periods in excess of two hours, 12 hours, 24 hours, 96 hours and 150 hours.

The following examples merely illustrate the best mode now contemplated for practicing the invention, but should not be construed to limit the invention.

Example 1 Excision and In Vitro Culture of Mouse Inner Ear The inner ear of a mouse was excised in the following manner. Postnatal day 7-14 Swiss Webster mice were decapitated and their skulls immersed in 70% ethanol for 5 min to disinfect. Under sterile conditions, the skull was cut into halves along the mid-sagittal axis and placed into 3 ml of culture media (Neuralbasal™ Media at pH 7.4; Gibco) in a 35 mm plastic culture dish (Nalge Nunc International, 2000 North Aurora Road, Naperville, 1L 60563) Using surgical forceps, the bony inner ear labyrinth was visualized and separated from the temporal bone The overlying connective tissue, stapes bone, facial nerve and stapedial artery were removed Using a fine forcep, a small hole about 2 mm in diameter was made through the apical turn of the lateral cochlear wall This surgically created conduit, along with the patent oval and round windows of the cochlea, permit ready diffusion of the culture media into the fluid-filled inner ear.

Typically, an inner ear excised and prepared in the foregoing manner is transferred to the HARV™ or CCCV™ vessel which contains 50 or 55 ml of Neuralbasal™ Media supplemented with either N2 or B27 media supplement (both sold by Gibco-BRL,

Catalogue number 17504-036), l O U/ml of penicillin and .25 μg/μl of fungizone The

B27 supplement is sold as a 50X concentrate which is used at a working concentration of 0 5X (e.g., 550 μl of 50X B27 stock solution is added to 55 ml of Neuralbasal™ Media) The N2 supplement stock solution is 100X and is used at a working concentration of IX (e.g., 550 μl of 100X N2 stock solution is added to 55 ml of Neuralbasal™ Media) The vessel is then placed in a tissue culture incubator at 37°C and in a 95% air/5% CO₂ environment The vessel is then rotated at 39 rpm for periods of 24-168 hr 50% media changes are made every 48 hr As few as 2 and as many as 12 inner ears have been successfully cultured in one vessel

To lesion the inner ear sensory hair-cells, the inner ear is placed in Neuralbasal ^{1 M}/N2 or B27 media that contain 1 mM neomycin sulfate (Sigma, P O Box 14508, St Louis, MO 63178) for 24-48 hr After this culture period, the media is completely replaced with media devoid of neomycin

Example 2 Excision and In Vitro Culture of Mouse Eye A mouse eye was excised as follows For culturing the eye, postnatal day 7-14 Swiss Webster mice were decapitated and the eyes removed from the orbit with a blunt forcep The eye was rinsed in 70% ethanol for 2 min The eye was transferred to a 35 mm culture dish, where the optic nerve was trimmed and any connective tissue attached to the sclera of the eye was removed under sterile conditions in 3 ml of culture media (same as in Example 1) The cornea of the eye was penetrated with a fine surgical blade and the lens was removed with surgical forceps Removal of the lens allows the retina to be in full communication with the culture media

Typically, an eye excised and prepared in the foregoing manner is transferred to the HARV™ or CCCV™ vessel containing 50 or 55 ml of Neuralbasal™ Media supplemented with either N2 or B27 media supplement, l O U/ml of penicillin and 25 μg/μl of fungizone The vessel is then placed in a tissue culture incubator at 37°C and in a 95%o air/5% CO environment The vessel is then rotated at 39 rpm for periods of 24-168 hr 50% media changes are made every 48 hr.

Example 3 Culture Media All concentrations set forth herein are working concentrations, i.e., the concentrations of the components in the medium in which the fluid-filled sensory organ is incubated

Table 1 shows the composition of Neuralbasal ^I medium ( l x) sold by Gibco Table 1 Neuralbasal™ medium composition

The following antibiotics may be added to Neuralbasal™ medium Fungizone reagent (amphoteπcin B, 0 25μg/ml, and sodium desoxycholate, 0 25μg/ml) which is sold by Gibco-BRL, Catalog number 17504-036 Penicillin G (10 units/ml) which is sold by Sigma, Catalog number P 3414 Neomycin sulfate (1 mM), sold by Sigma, Catalog number N 6386 Neuralbasal™ medium may also be supplemented with L-Glutamine (2mM)

Table 2 shows the composition of Minimum Essential Media (MEM)(lx) sold by Gibco

Table 2 Minimum Essential Media (MEM)

Table 3 shows the composition of BME Basal Medium (lx) sold by Gibco.

TABLE 3 BME Basal Medium

Table 4 shows the composition of Medium 199 ( lx) sold by Gibco

Table 4

Medium 199

N2 medium includes insulin, selenium, putrescine, transferπn and progesterone and is described in Bottenstein, J E and Sato, G H , Proc Nat'l Acad Sci (U S A ) 76(1): 514-517 ( 1979) which publication is incorporated herein by reference

Example 4 Assay for Sensory Epithelium Vitality During Long Term Culture

In the practice of the present invention, the microgravitational environment provided by the rotation of the culture vessel allows the sensory epithelium of the inner ear to be maintained for prolonged periods of culture (>168 hr ) without significant degradation or loss of the sensory hair-cells or non-sensory supporting-cells Data demonstrating the continued vitality of the sensory hair cells during prolonged culture were obtained by labeling the sensory epithe a with a probe against F-actin (phalloidin- FITC) that labels the surfaces of sensory and non-sensory cells, and with a hair-cell specific antibody against calbindin, a calcium binding protein Both labels were detected and photographed under epifluorescence microscopy

Cross-sectional data indicated that the normal cytoarchitecture of the inner ear sensory epitheha are maintained For example, the Organ of Corti has several fluid-filled spaces called the tunnel of Corti and spaces of Nuel that are necessary for normal auditory function These spaces occur bet een hair-cells and supporting-cells and are maintained after prolonged periods of culture In normal gravitational environments, (i.e., when the inner ear is floated without rotating the culture vessel) the sensory epithelia begin to degenerate Without rotation, within 24 hr the hair-cells are either completely missing or appear to be undergoing various endstages of cell death After 48 hr., the supporting- cells are completely missing, or are present but with the total loss of the tunnel of Corti and spaces of Nuel Rotating the vessel prevents this degradation and maintains normal cytoarchitecture

Example 5 Lipofection of Cells Within Inner Ears Cultured In Accordance with the Present

Invention

Inner ears from P7-14mice were removed The oval and round window membranes were cleared A 1 mm hole was made at the apex of the cochlea Inner ears were placed into a Rotary' Cylindrical Culture Vessel (RCCV™) that contained Neuralbasal ™ medium with N2 or B27. 1 -glutamιne, penicillin and amphotericin B The

RCCV™ was attached to a rotary motor inside an incubator at 37°C and having an atmosphere that included 5% C0₂ Cultured inner ears were rotated in a continuous orbit perpendicular to the ground at 40 rpm After 2, 4, and 6 days, inner ears were aldehyde fixed F-actin labeling with phalloidin-FITC and immunolabeling with antibodies against hair cell specific proteins (calbindin, myosin-Vl, myosin-VIIa) showed the rather normal appearance of hair cells and supporting cells in the organ of Corti Sections showed preservation of the tunnel of Corti

In separate cultures, cultured inner ears were exposed to 1 mM neomycin sulfate for 24 hours This treatment killed all the inner and outer hair-cells (>99%) throughout the entire length of the cochlea (confirmed by microscopic examination of inner ear sections) Lesioned inner ears were then lipofected with a cationic lipid and a plasmid encoding β-galactosidase (InVitrogen) for 6-24 hours and allowed to recover for 24-72 hours Tissues were aldehyde-fixed and processed using X-gal histochemistry X-gal labeling appeared in supporting cells in the organ of Corti and in several extrasensory regions Controls that were not lipofected with a plasmid encoding β-galactosidase did not show X-gal labeling

While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows

1 A method for culturing a fluid-filled sensory organ, the method comprising the steps of

(a) introducing a fluid-filled sensory organ selected from the group consisting of an inner ear and an eye ball into a culture chamber containing liquid culture medium, and

(b) moving the culture chamber so that the fluid-filled sensory organ moves within the culture chamber

2 The method of Claim 1 wherein the fluid-filled sensory organ is an inner ear

3 The method of Claim 1 wherein the fluid-filled sensory organ is a human inner ear

4 The method of Claim 1 wherein the fluid-filled sensory organ is an eye ball

5 The method of Claim 1 wherein the fluid-filled sensory organ is a human eye ball

6 The method of Claim 1 wherein the liquid medium comprises Neuralbasal ^I medium

7 The method of Claim 1 wherein the liquid medium comprises Minimal Essential Medium

8 The method of Claim 1 wherein the liquid medium comprises BME Basal Medium

9 The method of Claim I wherein the liquid medium comprises Medium 199

10 The method of Claim 1 wherein the culture chamber is rotated

1 I The method of Claim 1 wherein the culture chamber is vibrated

12 The method of Claim 1 wherein the culture chamber is rocked

13 The method of Claim 1 wherein the fluid-filled sensory organ is cultured for at least 2 hours 14 The method of Claim 1 wherein the fluid-filled sensory organ is cultured for at least 12 hours

15 The method of Claim 1 wherein the fluid-filled sensory organ is cultured for at least 24 hours

16 The method of Claim 1 wherein the fluid-filled sensory organ is cultured for at least 96 hours

17 The method of Claim 1 wherein the fluid-filled sensory organ is cultured for at least 150 hours

18 The method of Claim 1 wherein the culture vessel is in the form of a cylinder or annulus

19 The method of Claim 1 5 wherein the culture vessel is rotated

20 A method for culturing a fluid-filled sensory organ, the method comprising the steps of

(a) introducing a fluid-filled sensory organ selected from the group consisting of an inner ear and an eye ball into a culture chamber containing liquid culture medium; and

(b) rotating the culture chamber so that the fluid-filled sensory organ continuously moves within the culture chamber

21 The method of Claim 17 wherein the culture chamber is in the form of a cylinder or annulus and is rotated about its longitudinal axis