Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR CULTURING FLUID-FILLED SENSORY ORGANS
Field of the Invention
This invention relates to iro vi~r~o 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 its vit~~o 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 ire oilru 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 auditory sensory epithelium, the Organ of Corti, involves fine
microsurgical
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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 resultin;; 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.
Summar~of the Invention
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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 bony labyrinth, including the Organ of Corti, W
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 introducing a fluid-
filled
sensory organ into a culture chamber containing liquid culture medium, and
moving the
culture chamber (for example, rotatin" vibratin~~ 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 culture medium. Preferably the fluid-
filled sensory
organ is continuously, or almost continuously, in motion and suspended in the
liquid
culture medium, i.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 provides 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
3 5 sensory organ.
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
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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 arid 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 (.see, 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 evaluatin'J 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
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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 mew 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 i~~ oilno 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
I S filled sensory organs in accordance with the methods of the present
invention are
disclosed in U.S. Patent Serial No: 5,437,998; LJ.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 l, 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 or 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
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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
S 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 Oz to diffi.~se through wall portion 24 and into
the cell culture
1 S media within lumen 26. Correspondingly, COz 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
2S 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
lonbitudinal 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
3S 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 (RCC~~~'~'1), the High Aspect Ratio Vessel (HARVTM)
and the
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Cylindrical Cell Culture Vessel (CCC~'~'") which are manufactured by
Synthecon, Inc.
(8054 El Rio, Houston, Texas).
In the practice of the present invention, NeuralbasalT~"' media from Gibco BRL
(Gibco BRL media are produced by Life Technologies, Corporate Headquarters,
S 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 DN1E, BM E and M-199 with fetal calf serum or
horse
serum. All of the foregoing media are sold by Gibco -BRL. When using
NeuralbasalT"'
IO medium, N2 or B27 supplements play a more si<~nificant role when extended
periods of
culture (>96 hr) are attempted.
The methods of the present invention permit the continuous culture of fluid-
filled
senson~ organs over an extended time period without significant degeneration
of the
organ, tissues or cells. The methods of the present invention permit the
continuous
1 S culture of fluid-filled sensory organs for a period extending from a few
minutes to more
then 1 SO 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 1 SO hours.
The following examples merely illustrate the best mode now contemplated for
20 practicing the invention, but should not be construed to limit the
invention.
Example 1
Excision and Ifa 67/rv 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 S min
25 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 3S mm plastic culture dish (Nape Nunc International, 2000 North Aurora Road,
Naperville, IL 60563). Using surgical forceps, the bony inner ear labyrinth
was visualized
and separated from the temporal bone. The overlying connective tissue, stapes
bone,
30 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
3S to the HARVT~' or CCCV'~"' vessel which contains SO or SS ml of
Neuralbasal~'~"' Media
supplemented with either N2 or B27 media supplement (both sold by Gibco-BRL,
Catalogue number 17504-036), 10 U/ml of penicillin and .25 ~g/~I of fungizone.
The
B27 supplement is sold as a SOX concentrate which is used at a working
concentration of
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0.5X (e.g., 550 ~l of 50X B27 stock solution is added to 55 ml of
NeuralbasalT"' Media).
The N2 supplement stock solution is 100X and is used at a working
concentration of 1X
(e.g., 550 ~l of 100X N2 stock solution is added to 55 ml of NeuralbasalTM
Media). The
vessel is then placed in a tissue culture incubator at 37°C and in a
95% air/5% C02
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~'~"'/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 I >>rn 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°~o 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~'~M vessel containing 50 or 55 ml of NeuralbasalTM Media
supplemented with either N2 or B27 media supplement, 10 U/ml of penicillin and
.25 ~g/~l of fungizone. The vessel is then placed in a tissue culture
incubator at 37°C and
in a 95% 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''~ medium ( 1 x) sold by Gibco.
Table 1. Neuralbasal'~~1 medium composition
Com onent m~/liter~tM
Inorganic salts
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CaCI~ (anh drous)200 1,800
Fe (NO ) 9H~0 0.1 0.2
KCL 400 5,360
M~C1~ (anhydrous)77.3 812
NaCI 3,000 51,300
NaHCO 2 200 26,000
NaH~PO H~0 125 900
D-glucose 4,500 25,000
Phenol Red 8.1 23
HEPES x,600 10,000
Sodium Pyruvate 25 230
Amino Acids
L-alanine 2.0 20
L-ar rinine HCL 84 400
L-as ara rille 0.83 5
H~0
L-c steine 1.21 10
L- lutamate
GI cine 30 400
L-histidine HCL 42 200
H~0
L-isoleucine 105 800
L-lysine HCL 146 5
L-methionine 30 200
L- hen lalanine 66 400
L- roline 7.76 67
L-serine 42 400
L-threonine 95 800
L-tr to han 16 80
L-t rosine 72 400
L-valine 94 800
D-Ca antothenate 4 8
Choline chloride 4 28
Folic acid 4 8
i-Inositol 7.2 40
Niacinamide 4 30
Pyridoxal HCL 4 20
Riboflavin 0.4 10
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Thiamine HCL 4 10
Vitamin B 12 0:34 0.2
The following antibiotics may be added to Neuralbasalw'~ medium. Fungizone
reagent (amphotericin B, 0.25~tg/ml, and sodium desoxycholate, 0.25~g/ml)
which is sold
by Gibco-BRL, Catalog number 1704-036. Penicillin G (10 units/ml) which is
sold by
S Sigma, Catalog number P 3414. Neomycin sulfate ( 1 mM), sold by Sigma,
Catalog
number N 6386. NeuralbasalT~'~' 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)
Com orient 1JV Li uid m /L
INORGANIC SALTS:
CaCh (anh d.)
200.00
KCL 400.00
M~SO anh d. 98.00
NaCL 6800.00
NaHCO 2200.00
NaH~PO HBO 140.00
OTHER COI\1PONENTS:
D-Glucose 1000.00
Phenol Red 10.00
Sodium Succinate -
Succinic Acid -
AMINO ACIDS:
L-Alanine -
L-Are>inine HCL 126.00
L-As ara~;ine HBO - '
L-As artic Acid -
L-C stine -
L-Cystine 2HCL 31.00
L-Cystine 2Na -
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Com ~onent IX Li uid m /L
L-Glutamic acid -
L-Glutamine 292.00
L-Alan 1 L-Glutamine -
Glycine -
L-Histidine HCL H O 42.00
L-Isoleucine 52.00
L-Leucine 52.00
L-L sine HCL 73.00 '
L-Methionine 15.00
L-Phenylalanine 32.00
L-Proline -
L-Serine -
L-Threonine 48.00
L-T to han 10.00
L-T rosine
L-T rosine 2Na 2H~0 52.00
D-Valine 92.00
L-Valine -
VITAMINS:
D-Ca Pantothenate 1.00
Choline Bitartrate -
Choline Chloride 1.00
Folic Acid 1.00
I-lnositol 2.00
Niacinamide 1.00
Pyridoxal HCL 1.00
Riboflavin 0.10
Thiamine HCL I.00
Table 3 shows the composition of B>\1E Basal Medium ( 1 x) sold by Gibco.
TABLE 3
BME Basal Medium
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_. _
i
Com orient 1 N Li uid m /L
INORGANIC SALTS:
CaCh (anh d.) 200.00
KCL 400.00
M >CL~ (anh d. -
M SO (anh d.) 97.70
M SO 7H~0 -
NaCL 6800.00
NaHCO 2200.00
NaH~PO HBO 140.00
OTHER COMPONENTS:
D-Glucose I 000.00
Phenol Red 10.00
AMINO ACIDS:
L-Ar mine HCL 21.00
L-C stine -
L-C stine 2HCL 16.00
L-Glutamine -
L-Histidine 8.00
L-Histidine HCL HBO -
L-Isoleucine 26.00
L-Leucine 26.00
L-L sine HCL 36.47
L-Methionine 7.50
L-Phen lalanine 18.50
L-Threonine 24.00
L-T to han 4.00
L-T rosine -
L-T rosine 2Na 2H~0 26.00
L-Valine I 23.50
VITAM1NS:
Biotin 1.00
D-Ca Pantothenate 1.00
Choline Chloride 1.00
Folic Acid 1.00
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Com orient IX Li uid m /L
I-Inositol 2.00
Niacinamide 1.00
P ridoxal HCL 1.00
Riboflavin 0.10
Thiamine HCL 1.00
Table 4 shows the composition of Medium 199 ( lx) sold by Gibco.
Table 4
Medium 199
Com orient I?~ Li uid m /1.
INORGANIC SALTS:
CaCL~ anhyd.) 200.00
Fe NO 9H~0
KCL 400.00
KH~PO -
M >SO 98.00
M 1S0 (anh d. -
NaCL 6800.00
NaHCO
2200.00
NaH~PO 2H~0 -
Na~HPO 7H~0 -
NaH~I'O HBO 140.00
OTHER COMPONENTS:
Adenine Sul hate 10.00
Adenosine-5-tri hos hate 1.00
Adenosine-5- hos hate 0.20
Cholesterol 0.20
2-deos -D-ribose 0.50
Deoxyribose -
D-Glucose 1000.00
Glutathione reduced 0.05
Guanine HCL 0.30
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Com ~onent I?~ Li uid m /L
HEPES -
H oxanthine Na 0.40
Phenol Red -
Ribose 0.50
Sodium Acetate 50.00
Sodium Acetate 3H~0 ~ -
Th mine 0.30
Tween 80 ~~ 20.00
Uracil 0.30
Xanthine Na 0.34
AM1N0 ACIDS:
L-Alanine 25.00
L-Ar~linine HCL 70.00
L-As artic Acid 30.0
L-C stine HCL HBO 0.10
L-C stine 2HCL 26.00
L-Glutamic acid 75.00
L-Glutamine 100.00
Gl cine 50.00
L-Histidine HCL H O 22.00
L-Hydroxy roline 10.00
L-lsoleucine 40.00
L-Leucine 60.0
L-L sine HCL 70.00
L-Methionine 15.00
L-Phen lalanine 25.00
L-Proline 40.00
L-Serine 25.00
L-Threonine 30.00
L-T to han 10.00
L-T rosine 2Na 2H~0 58.00
L-Valine 25.00
VITAI\11>\'S:
Ascorbic Acid ~ 0.05
CA 02364024 2001-08-31
WO 00/54583 PCT/US00/05736
-l S-
I
Com ~onent I
1 ~ Li uid m /L
a-Toco herol Phos hate sodium0.01
salt
Biotin 0.01
Calciferol 0.10
D-Ca Pantothenate 0.01
Choline Chloride O.SO
Folic Acid 0.01
I-Inositol O.OS
Menadione j 0.01
Niacin 0.025
Niacinamide 0.025
Para-aminobenzoic Acid ~ O.OS
P ridoxal HCL 0.025
P ridoxine HCL 0.025
Riboflavin 0.01
Thiamine HCL 0.01
Vitamin A (acetate 0.14
N2 medium includes insulin, selenium, putrescine, transferrin and progesterone
and is described in Bottenstein, J.E. and Sato, G.H., Proc. Nat'1 Acad. Sci.
(U. S. A ) 7C( 1 ): S 14-S 17 ( 1979), which publication is incorporated
herein by reference.
S 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 epithelia 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
I S and photographed under epifluorescence microscopy.
Cross-sectional data indicated that the normal cytoarchitecture of the inner
ear
sensory epithelia 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
CA 02364024 2001-08-31
WO 00/54583 PCT/US00/05736
-16-
function. These spaces occur between hair-cells and supporting-cells and are
maintained
after prolon;Jed 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-l4mice 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 (RCCVT"~) that contained
Neuralbasal~'~~' medium with N2 or B27, 1-<,lutamine, penicillin and
amphotericin B. The
RCCVT"" was attached to a rotary motor inside an incubator at 37°C and
having an
atmosphere that included 5% CO~. 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-VI, 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 (3-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 /3-
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.
