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• • A—HUMAN NECESSITIES
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/60—Transcription factors

Definitions

• This invention relates to /// vitro cell and tissue culture methods applicable to fluid- filled sensory organs, such as the inner ear.
• 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
• 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,
• the present invention provides methods tor culturing fluid-filled sensory organs /// vit/ o
• 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
• the culture chamber is completely filled with liquid cultui e medium
• 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
• 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
• 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
• the methods of the present invention permit the continuous culture of fluid-filled sensory organs over an extended time pe ⁇ od 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
• 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.
• 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.
• 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.
• 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.
• 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).
• antioxidants e.g., catalase, superoxide dismutase and fatty acids such as linoleic and linolenic acid.
• 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.
• 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
• the fluid-filled sensory organ is continuously moved within the culture chamber so that the fluid
• FIGURE 1 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
• 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
• Wall portion 24 may be constructed at least partially of a gas permeable material, such as silicone rubber.
• a gas permeable material such as silicone rubber.
• 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
• 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
• gas permeable material in the construction of at least part of wall portion 24 permits 0 2 to diffuse through wall portion 24 and into the cell culture media within lumen 26 Correspondingly, C0 2 can diffuse out of lumen 26
• 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.
• an air filter is preferably also employed to protect the air pump valves from dirt.
• 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.
• 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.
• RCCVTM Rotary Cylindrical Culture Vessel
• HAVTM High Aspect Ratio Vessel
• CCCV 'I M Cylindrical Cell Culture Vessel
• NeuralbasalTM 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.
• 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
• 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.
• 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 (NeuralbasalTM 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
• an inner ear excised and prepared in the foregoing manner is transferred to the HARVTM or CCCVTM vessel which contains 50 or 55 ml of NeuralbasalTM Media supplemented with either N2 or B27 media supplement (both sold by Gibco-BRL,
• B27 supplement is sold as a 50X concentrate which is used at a working concentration of 0 5X (e.g., 550 ⁇ l of 50X B27 stock solution is added to 55 ml of NeuralbasalTM Media)
• the N2 supplement stock solution is 100X and is used at a working concentration of IX (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% CO 2 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
• 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
• an eye excised and prepared in the foregoing manner is transferred to the HARVTM or CCCVTM vessel containing 50 or 55 ml of NeuralbasalTM Media supplemented with either N2 or B27 media supplement, l O 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%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 NeuralbasalTM medium composition
• NeuralbasalTM medium Fungizone reagent (amphote ⁇ cin B, 0 25 ⁇ g/ml, and sodium desoxycholate, 0 25 ⁇ g/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 NeuralbasalTM medium may also be supplemented with L-Glutamine (2mM)
• Table 2 shows the composition of Minimum Essential Media (MEM)(lx) sold by Gibco
• Table 3 shows the composition of BME Basal Medium (lx) sold by Gibco.
• Table 4 shows the composition of Medium 199 ( lx) sold by Gibco
• N2 medium includes insulin, selenium, putrescine, transfer ⁇ n 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
• 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
• 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
• 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
• 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
• RCCVTM was attached to a rotary motor inside an incubator at 37°C and having an atmosphere that included 5% C0 2
• 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

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• 2000
  • 2000-03-03 EP EP00914825A patent/EP1161142A4/de not_active Withdrawn
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