哺乳动物内耳毛细胞的体外培养（英文）
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摘要
由于内耳的血脑屏障作用,药物渗入到内耳比较困难。新生小鼠内耳毛细胞的体外培养体系的建立,为体外进行支持细胞转分化机制的研究和进行体外药物损伤毛细胞实验等提供实验技术的前提。为了避免毛细胞体外培养过程中污染杂菌,解剖内耳耳蜗的整个过程十分重要。处死小鼠后,将其浸泡在75%酒精中1～3分钟,防止鼠毛污染培养基。打开内耳耳蜗之前,选用添加了青霉素的磷酸盐缓冲液(1×PBS);培养过程中使用的是仅仅添加青霉素的培养基来减少对毛细胞的损伤。在基底膜培养的第一步,选用DMEM(包含5%马血清体积比和5%胎牛血清)作为组织粘附培养的培养基,保证足够的营养,同时更好地维持整个基底膜培养状态下的形态。在之后的培养中,选用DMEM(添加了10%胎牛血清、1%N2和1%B27)作为长期的培养基。使用含有表皮生长因子的N2和B27的培养基进行基底膜以及椭圆囊之后的培养,有助于维持毛细胞的体外生长时间。选用鼠尾胶包被盖玻片后培养,可以增加基底膜和椭圆囊的粘附作用,保证毛细胞静纤毛的向上生长。该文章展现了哺乳动物毛细胞的体外培养的具体方法,能够较好地维持耳蜗基底膜在体外培养的形态,并增加毛细胞体外培养的存活时间。
Auditory function in vertebrates depends on the transduction of sound vibrations into electrical signals by inner ear hair cells.In general,hearing loss resulting from hair cell damage is irreversible because the human ear has been considered to be incapable of regenerating or repairing these sensory elements following severe injury.Therefore,regeneration and protection of inner ear hair cells have become an exciting,rapidly evolving field of research during the last decade.However,mammalian auditory hair cells are few in number,experimentally inaccessible,and barely proliferate postnatally in vitro.Various in vitro primary culture systems of inner ear hair cells have been established by different groups,although many challenges remain unresolved.Here,we briefly explain the structure of the inner ear,summarize the published methods of in vitro hair cell cultures,and propose a feasible protocol for culturing these cells,which gave satisfactory results in our study.A better understanding of in vitro hair cell cultures will substantially facilitate research involving auditory functions,drug development,and the isolation of critical molecules involved in hair cell biology.
Auditory function in vertebrates depends on the transduction of sound vibrations into electrical signals by inner ear hair cells.In general,hearing loss resulting from hair cell damage is irreversible because the human ear has been considered to be incapable of regenerating or repairing these sensory elements following severe injury.Therefore,regeneration and protection of inner ear hair cells have become an exciting,rapidly evolving field of research during the last decade.However,mammalian auditory hair cells are few in number,experimentally inaccessible,and barely proliferate postnatally in vitro.Various in vitro primary culture systems of inner ear hair cells have been established by different groups,although many challenges remain unresolved.Here,we briefly explain the structure of the inner ear,summarize the published methods of in vitro hair cell cultures,and propose a feasible protocol for culturing these cells,which gave satisfactory results in our study.A better understanding of in vitro hair cell cultures will substantially facilitate research involving auditory functions,drug development,and the isolation of critical molecules involved in hair cell biology.
引文
Abdouh A,Despres G,Romand R,1993.Hair cell overproduction in the developing mammalian cochlea in culture.Neuroreport,5(1):33-36.https://doi.org/10.1097/00001756-199310000-00008
Anniko M,van de Water TR,1978.Organ culture of the postnatal mouse crista ampullaris part I.Arch Oto-RhinoLaryngol,220(1-2):129-132.https://doi.org/10.1007/BF00456306
Betlejewski S,2008.Science and life-the history of marquis Alfonso Corti.Otolaryngol Pol,62(3):344-347.https://doi.org/10.1016/S0030-6657(08)70268-3
Bird JE,Daudet N,Warchol ME,et al.,2010.Supporting cells eliminate dying sensory hair cells to maintain epithelial integrity in the avian inner ear.J Neurosci,30(37):12545-12556.https://doi.org/10.1523/JNEUROSCI.3042-10.2010
Brandon CS,Voelkel-Johnson C,May LA,et al.,2012.Dissection of adult mouse utricle and adenovirus-mediated supporting-cell infection.J Vis Exp,(61):e3734.https://doi.org/10.3791/3734
Bucks SA,Cox BC,Vlosich BA,et al.,2017.Supporting cells remove and replace sensory receptor hair cells in a balance organ of adult mice.eLife,6:e18128.https://doi.org/10.7554/eLife.18128
Chang DT,Chai R,DiMarco R,et al.,2015.Protein-engineered hydrogel encapsulation for 3-D culture of murine cochlea.Otol Neurotol,36(3):531-538.https://doi.org/10.1097/MAO.0000000000000518
Corns LF,Johnson SL,Kros CJ,et al.,2014.Calcium entry into stereocilia drives adaptation of the mechanoelectrical transducer current of mammalian cochlear hair cells.Proc Natl Acad Sci USA,111(41):14918-14923.https://doi.org/10.1073/pnas.1409920111
Ding DL,Stracher A,Salvi RJ,2002.Leupeptin protects cochlear and vestibular hair cells from gentamicin ototoxicity.Hear Res,164(1-2):115-126.https://doi.org/10.1016/S0378-5955(01)00417-8
Ding DL,He JC,Allman BL,et al.,2011.Cisplatin ototoxicity in rat cochlear organotypic cultures.Hear Res,282(1-2):196-203.https://doi.org/10.1016/j.heares.2011.08.002
Ding DL,Qi WD,Yu DZ,et al.,2013.Addition of exogenous NAD+prevents mefloquine-induced neuroaxonal and hair cell degeneration through reduction of caspase-3-mediated apoptosis in cochlear organotypic cultures.PLo S ONE,8(11):e79817.https://doi.org/10.1371/journal.pone.0079817
Dror AA,Avraham KB,2009.Hearing loss:mechanisms revealed by genetics and cell biology.Annu Rev Genet,43:411-437.https://doi.org/10.1146/annurev-genet-102108-134135
Eatock RA,2000.Adaptation in hair cells.Annu Rev Neurosci,23:285-314.https://doi.org/10.1146/annurev.neuro.23.1.285
Friedman TB,Griffith AJ,2003.Human nonsyndromic sensorineural deafness.Annu Rev Genomics Hum Genet,4:341-402.https://doi.org/10.1146/annurev.genom.4.070802.110347
Furness DN,Richardson GP,Russell IJ,1989.Stereociliary bundle morphology in organotypic cultures of the mouse cochlea.Hear Res,38(1-2):95-109.https://doi.org/10.1016/0378-5955(89)90131-7
Gaboyard S,Chabbert C,Travo C,et al.,2005.Threedimensional culture of newborn rat utricle using an extracellular matrix promotes formation of a cyst.Neuroscience,133(1):253-265.https://doi.org/10.1016/j.neuroscience.2005.02.011
Géléoc GS,Holt JR,2014.Sound strategies for hearing restoration.Science,344(6184):1241062.https://doi.org/10.1126/science.1241062
Gubb D,García-Bellido A,1982.A genetic analysis of the determination of cuticular polarity during development in Drosophila melanogaster.J Embryol Exp Morphol,68(1):37-57.
Hawkins JE Jr,Johnsson LG,Aran JM,1969.Comparative tests of gentamicin ototoxicity.J Infect Dis,119(4-5):417-426.https://doi.org/10.1093/infdis/119.4-5.417
Hudspeth AJ,1989a.How the ear’s works work.Nature,341(6241):397-404.https://doi.org/10.1038/341397a0
Hudspeth AJ,1989b.Mechanoelectrical transduction by hair cells of the bullfrog’s sacculus.Prog Brain Res,80:129-135.https://doi.org/10.1016/S0079-6123(08)62206-2
Hudspeth AJ,1997.How hearing happens.Neuron,19(5):947-950.https://doi.org/10.1016/S0896-6273(00)80385-2
Kalinec GM,Webster P,Lim DJ,et al.,2003.A cochlear cell line as an in vitro system for drug ototoxicity screening.Audiol Neurootol,8(4):177-189.https://doi.org/10.1159/000071059
Koehler KR,Hashino E,2014.3D mouse embryonic stem cell culture for generating inner ear organoids.Nat Protoc,9(6):1229-1244.https://doi.org/10.1038/nprot.2014.100
Landegger LD,Dilwali S,Stankovic KM,2017.Neonatal murine cochlear explant technique as an in vitro screening tool in hearing research.J Vis Exp,(124):e55704.https://doi.org/10.3791/55704
Lelli A,Asai Y,Forge A,et al.,2009.Tonotopic gradient in the developmental acquisition of sensory transduction in outer hair cells of the mouse cochlea.J Neurophysiol,101(6):2961-2973.https://doi.org/10.1152/jn.00136.2009
Li-Korotky HS,2012.Age-related hearing loss:quality of care for quality of life.Gerontologist,52(2):265-271.https://doi.org/10.1093/geront/gnr159
Lin JC,Zhang XD,Wu FF,et al.,2015.Hair cell damage recruited Lgr5-expressing cells are hair cell progenitors in neonatal mouse utricle.Front Cell Neurosci,9:113.https://doi.org/10.3389/fncel.2015.00113
Malgrange B,Thiry M,van de Water TR,et al.,2002.Epithelial supporting cells can differentiate into outer hair cells and Deiters’cells in the cultured organ of Corti.Cell Mol Life Sci,59(10):1744-1757.https://doi.org/10.1007/PL00012502
Matz G,Rybak L,Roland PS,et al.,2004.Ototoxicity of ototopical antibiotic drops in humans.Otolaryngol-Head Neck Surg,130(S3):S79-S82.https://doi.org/10.1016/j.otohns.2003.12.007
May LA,Kramarenko II,Brandon CS,et al.,2013.Inner ear supporting cells protect hair cells by secreting HSP70.JClin Invest,123(8):3577-3587.https://doi.org/10.1172/JCI68480
May-Simera H,2016.Evaluation of planar-cell-polarity phenotypes in ciliopathy mouse mutant cochlea.J Vis Exp,(108):e53559.https://doi.org/10.3791/53559
Meiteles LZ,Raphael Y,1994.Scar formation in the vestibular sensory epithelium after aminoglycoside toxicity.Hear Res,79(1-2):26-38.https://doi.org/10.1016/0378-5955(94)90124-4
Meyer-Bisch C,2005.Measuring noise.Med Sci(Paris),21(5):546-550.https://doi.org/10.1051/medsci/2005215546
Oesterle EC,Rubel EW,1993.Postnatal production of supporting cells in the chick cochlea.Hear Res,66(2):213-224.https://doi.org/10.1016/0378-5955(93)90141-M
Oesterle EC,Tsue TT,Reh TA,et al.,1993.Hair-cell regeneration in organ cultures of the postnatal chicken inner ear.Hear Res,70(1):85-108.https://doi.org/10.1016/0378-5955(93)90054-5
Ou HC,Lin V,Rubel EW,2013.“In-bone”utricle culturesa simplified,atraumatic technique for in situ cultures of the adult mouse(Mus musculus)utricle.Otol Neurotol,34(2):353-359.https://doi.org/10.1097/MAO.0b013e31827ca330
Parker M,Brugeaud A,Edge AS,2010.Primary culture and plasmid electroporation of the murine organ of Corti.JVis Exp,(36):e1685.https://doi.org/10.3791/1685
Qian D,Jones C,Rzadzinska A,et al.,2007.Wnt5a functions in planar cell polarity regulation in mice.Dev Biol,306(1):121-133.https://doi.org/10.1016/j.ydbio.2007.03.011
Quint E,Hackney CM,Furness DN,1996.The effect of neomycin on organotypic cultures of the adult guinea-pig utricle.Ann N Y Acad Sci,781(1):683-685.https://doi.org/10.1111/j.1749-6632.1996.tb15759.x
Rastel D,Abdouh A,Dahl D,et al.,1993.An original organotypic culture method to study the organ of Corti of the newborn rat in vitro.J Neurosci Methods,47(1-2):123-131.https://doi.org/10.1016/0165-0270(93)90028-P
Roberson DW,Rubel EW,1994.Cell division in the gerbil cochlea after acoustic trauma.Am J Otol,15(1):28-34.
Romand R,Chardin S,1999.Effects of growth factors on the hair cells after ototoxic treatment of the neonatal mammalian cochlea in vitro.Brain Res,825(1-2):46-58.https://doi.org/10.1016/S0006-8993(99)01211-1
Ruben RJ,1967.Development of the inner ear of the mouse:a radioautographic study of terminal mitoses.Acta Otolaryngol,Suppl 220:1-44.
Shang JL,Cafaro J,Nehmer R,et al.,2010.Supporting cell division is not required for regeneration of auditory hair cells after ototoxic injury in vitro.J Assoc Res Otolaryngol,11(2):203-222.https://doi.org/10.1007/s10162-009-0206-7
Smith ME,Groves AK,Coffin AB,2016.Editorial:sensory hair cell death and regeneration.Front Cell Neurosci,10:208.https://doi.org/10.3389/fncel.2016.00208
Sobkowicz HM,Bereman B,Rose JE,1975.Organotypic development of the organ of Corti in culture.J Neurocytol,4(5):543-572.https://doi.org/10.1007/BF01351537
Spencer NJ,Cotanche DA,Klapperich CM,2008.Peptide-and collagen-based hydrogel substrates for in vitro culture of chick cochleae.Biomaterials,29(8):1028-1042.https://doi.org/10.1016/j.biomaterials.2007.11.006
Taura A,Nakashima N,Ohnishi H,et al.,2016.Regenerative therapy for vestibular disorders using human induced pluripotent stem cells(iPSCs):neural differentiation of human iPSC-derived neural stem cells after in vitro transplantation into mouse vestibular epithelia.Acta Otolaryngol,136(10):999-1005.https://doi.org/10.1080/00016489.2016.1183169
von Békésy G,1956.Current status of theories of hearing.Science,123(3201):779-783.https://doi.org/10.1126/science.123.3201.779
Werner M,van de Water TR,Andersson T,et al.,2012.Morphological and morphometric characteristics of vestibular hair cells and support cells in long term cultures of rat utricle explants.Hear Res,283(1-3):107-116.https://doi.org/10.1016/j.heares.2011.11.003
Werner M,van de Water TR,Hammarsten P,et al.,2015.Morphological and morphometric characterization of direct transdifferentiation of support cells into hair cells in ototoxin-exposed neonatal utricular explants.Hear Res,321:1-11.https://doi.org/10.1016/j.heares.2014.12.011
White PM,Doetzlhofer A,Lee YS,et al.,2006.Mammalian cochlear supporting cells can divide and trans-differentiate into hair cells.Nature,441(7096):984-987.https://doi.org/10.1038/nature04849
Yamahara K,Yamamoto N,Nakagawa T,et al.,2015.Insulinlike growth factor 1:a novel treatment for the protection or regeneration of cochlear hair cells.Hear Res,330:2-9.https://doi.org/10.1016/j.heares.2015.04.009
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Zhao HB,2001.Long-term natural culture of cochlear sensory epithelia of guinea pigs.Neurosci Lett,315(1-2):73-76.https://doi.org/10.1016/S0304-3940(01)02357-6
Anniko M,van de Water TR,1978.Organ culture of the postnatal mouse crista ampullaris part I.Arch Oto-RhinoLaryngol,220(1-2):129-132.https://doi.org/10.1007/BF00456306
Betlejewski S,2008.Science and life-the history of marquis Alfonso Corti.Otolaryngol Pol,62(3):344-347.https://doi.org/10.1016/S0030-6657(08)70268-3
Bird JE,Daudet N,Warchol ME,et al.,2010.Supporting cells eliminate dying sensory hair cells to maintain epithelial integrity in the avian inner ear.J Neurosci,30(37):12545-12556.https://doi.org/10.1523/JNEUROSCI.3042-10.2010
Brandon CS,Voelkel-Johnson C,May LA,et al.,2012.Dissection of adult mouse utricle and adenovirus-mediated supporting-cell infection.J Vis Exp,(61):e3734.https://doi.org/10.3791/3734
Bucks SA,Cox BC,Vlosich BA,et al.,2017.Supporting cells remove and replace sensory receptor hair cells in a balance organ of adult mice.eLife,6:e18128.https://doi.org/10.7554/eLife.18128
Chang DT,Chai R,DiMarco R,et al.,2015.Protein-engineered hydrogel encapsulation for 3-D culture of murine cochlea.Otol Neurotol,36(3):531-538.https://doi.org/10.1097/MAO.0000000000000518
Corns LF,Johnson SL,Kros CJ,et al.,2014.Calcium entry into stereocilia drives adaptation of the mechanoelectrical transducer current of mammalian cochlear hair cells.Proc Natl Acad Sci USA,111(41):14918-14923.https://doi.org/10.1073/pnas.1409920111
Ding DL,Stracher A,Salvi RJ,2002.Leupeptin protects cochlear and vestibular hair cells from gentamicin ototoxicity.Hear Res,164(1-2):115-126.https://doi.org/10.1016/S0378-5955(01)00417-8
Ding DL,He JC,Allman BL,et al.,2011.Cisplatin ototoxicity in rat cochlear organotypic cultures.Hear Res,282(1-2):196-203.https://doi.org/10.1016/j.heares.2011.08.002
Ding DL,Qi WD,Yu DZ,et al.,2013.Addition of exogenous NAD+prevents mefloquine-induced neuroaxonal and hair cell degeneration through reduction of caspase-3-mediated apoptosis in cochlear organotypic cultures.PLo S ONE,8(11):e79817.https://doi.org/10.1371/journal.pone.0079817
Dror AA,Avraham KB,2009.Hearing loss:mechanisms revealed by genetics and cell biology.Annu Rev Genet,43:411-437.https://doi.org/10.1146/annurev-genet-102108-134135
Eatock RA,2000.Adaptation in hair cells.Annu Rev Neurosci,23:285-314.https://doi.org/10.1146/annurev.neuro.23.1.285
Friedman TB,Griffith AJ,2003.Human nonsyndromic sensorineural deafness.Annu Rev Genomics Hum Genet,4:341-402.https://doi.org/10.1146/annurev.genom.4.070802.110347
Furness DN,Richardson GP,Russell IJ,1989.Stereociliary bundle morphology in organotypic cultures of the mouse cochlea.Hear Res,38(1-2):95-109.https://doi.org/10.1016/0378-5955(89)90131-7
Gaboyard S,Chabbert C,Travo C,et al.,2005.Threedimensional culture of newborn rat utricle using an extracellular matrix promotes formation of a cyst.Neuroscience,133(1):253-265.https://doi.org/10.1016/j.neuroscience.2005.02.011
Géléoc GS,Holt JR,2014.Sound strategies for hearing restoration.Science,344(6184):1241062.https://doi.org/10.1126/science.1241062
Gubb D,García-Bellido A,1982.A genetic analysis of the determination of cuticular polarity during development in Drosophila melanogaster.J Embryol Exp Morphol,68(1):37-57.
Hawkins JE Jr,Johnsson LG,Aran JM,1969.Comparative tests of gentamicin ototoxicity.J Infect Dis,119(4-5):417-426.https://doi.org/10.1093/infdis/119.4-5.417
Hudspeth AJ,1989a.How the ear’s works work.Nature,341(6241):397-404.https://doi.org/10.1038/341397a0
Hudspeth AJ,1989b.Mechanoelectrical transduction by hair cells of the bullfrog’s sacculus.Prog Brain Res,80:129-135.https://doi.org/10.1016/S0079-6123(08)62206-2
Hudspeth AJ,1997.How hearing happens.Neuron,19(5):947-950.https://doi.org/10.1016/S0896-6273(00)80385-2
Kalinec GM,Webster P,Lim DJ,et al.,2003.A cochlear cell line as an in vitro system for drug ototoxicity screening.Audiol Neurootol,8(4):177-189.https://doi.org/10.1159/000071059
Koehler KR,Hashino E,2014.3D mouse embryonic stem cell culture for generating inner ear organoids.Nat Protoc,9(6):1229-1244.https://doi.org/10.1038/nprot.2014.100
Landegger LD,Dilwali S,Stankovic KM,2017.Neonatal murine cochlear explant technique as an in vitro screening tool in hearing research.J Vis Exp,(124):e55704.https://doi.org/10.3791/55704
Lelli A,Asai Y,Forge A,et al.,2009.Tonotopic gradient in the developmental acquisition of sensory transduction in outer hair cells of the mouse cochlea.J Neurophysiol,101(6):2961-2973.https://doi.org/10.1152/jn.00136.2009
Li-Korotky HS,2012.Age-related hearing loss:quality of care for quality of life.Gerontologist,52(2):265-271.https://doi.org/10.1093/geront/gnr159
Lin JC,Zhang XD,Wu FF,et al.,2015.Hair cell damage recruited Lgr5-expressing cells are hair cell progenitors in neonatal mouse utricle.Front Cell Neurosci,9:113.https://doi.org/10.3389/fncel.2015.00113
Malgrange B,Thiry M,van de Water TR,et al.,2002.Epithelial supporting cells can differentiate into outer hair cells and Deiters’cells in the cultured organ of Corti.Cell Mol Life Sci,59(10):1744-1757.https://doi.org/10.1007/PL00012502
Matz G,Rybak L,Roland PS,et al.,2004.Ototoxicity of ototopical antibiotic drops in humans.Otolaryngol-Head Neck Surg,130(S3):S79-S82.https://doi.org/10.1016/j.otohns.2003.12.007
May LA,Kramarenko II,Brandon CS,et al.,2013.Inner ear supporting cells protect hair cells by secreting HSP70.JClin Invest,123(8):3577-3587.https://doi.org/10.1172/JCI68480
May-Simera H,2016.Evaluation of planar-cell-polarity phenotypes in ciliopathy mouse mutant cochlea.J Vis Exp,(108):e53559.https://doi.org/10.3791/53559
Meiteles LZ,Raphael Y,1994.Scar formation in the vestibular sensory epithelium after aminoglycoside toxicity.Hear Res,79(1-2):26-38.https://doi.org/10.1016/0378-5955(94)90124-4
Meyer-Bisch C,2005.Measuring noise.Med Sci(Paris),21(5):546-550.https://doi.org/10.1051/medsci/2005215546
Oesterle EC,Rubel EW,1993.Postnatal production of supporting cells in the chick cochlea.Hear Res,66(2):213-224.https://doi.org/10.1016/0378-5955(93)90141-M
Oesterle EC,Tsue TT,Reh TA,et al.,1993.Hair-cell regeneration in organ cultures of the postnatal chicken inner ear.Hear Res,70(1):85-108.https://doi.org/10.1016/0378-5955(93)90054-5
Ou HC,Lin V,Rubel EW,2013.“In-bone”utricle culturesa simplified,atraumatic technique for in situ cultures of the adult mouse(Mus musculus)utricle.Otol Neurotol,34(2):353-359.https://doi.org/10.1097/MAO.0b013e31827ca330
Parker M,Brugeaud A,Edge AS,2010.Primary culture and plasmid electroporation of the murine organ of Corti.JVis Exp,(36):e1685.https://doi.org/10.3791/1685
Qian D,Jones C,Rzadzinska A,et al.,2007.Wnt5a functions in planar cell polarity regulation in mice.Dev Biol,306(1):121-133.https://doi.org/10.1016/j.ydbio.2007.03.011
Quint E,Hackney CM,Furness DN,1996.The effect of neomycin on organotypic cultures of the adult guinea-pig utricle.Ann N Y Acad Sci,781(1):683-685.https://doi.org/10.1111/j.1749-6632.1996.tb15759.x
Rastel D,Abdouh A,Dahl D,et al.,1993.An original organotypic culture method to study the organ of Corti of the newborn rat in vitro.J Neurosci Methods,47(1-2):123-131.https://doi.org/10.1016/0165-0270(93)90028-P
Roberson DW,Rubel EW,1994.Cell division in the gerbil cochlea after acoustic trauma.Am J Otol,15(1):28-34.
Romand R,Chardin S,1999.Effects of growth factors on the hair cells after ototoxic treatment of the neonatal mammalian cochlea in vitro.Brain Res,825(1-2):46-58.https://doi.org/10.1016/S0006-8993(99)01211-1
Ruben RJ,1967.Development of the inner ear of the mouse:a radioautographic study of terminal mitoses.Acta Otolaryngol,Suppl 220:1-44.
Shang JL,Cafaro J,Nehmer R,et al.,2010.Supporting cell division is not required for regeneration of auditory hair cells after ototoxic injury in vitro.J Assoc Res Otolaryngol,11(2):203-222.https://doi.org/10.1007/s10162-009-0206-7
Smith ME,Groves AK,Coffin AB,2016.Editorial:sensory hair cell death and regeneration.Front Cell Neurosci,10:208.https://doi.org/10.3389/fncel.2016.00208
Sobkowicz HM,Bereman B,Rose JE,1975.Organotypic development of the organ of Corti in culture.J Neurocytol,4(5):543-572.https://doi.org/10.1007/BF01351537
Spencer NJ,Cotanche DA,Klapperich CM,2008.Peptide-and collagen-based hydrogel substrates for in vitro culture of chick cochleae.Biomaterials,29(8):1028-1042.https://doi.org/10.1016/j.biomaterials.2007.11.006
Taura A,Nakashima N,Ohnishi H,et al.,2016.Regenerative therapy for vestibular disorders using human induced pluripotent stem cells(iPSCs):neural differentiation of human iPSC-derived neural stem cells after in vitro transplantation into mouse vestibular epithelia.Acta Otolaryngol,136(10):999-1005.https://doi.org/10.1080/00016489.2016.1183169
von Békésy G,1956.Current status of theories of hearing.Science,123(3201):779-783.https://doi.org/10.1126/science.123.3201.779
Werner M,van de Water TR,Andersson T,et al.,2012.Morphological and morphometric characteristics of vestibular hair cells and support cells in long term cultures of rat utricle explants.Hear Res,283(1-3):107-116.https://doi.org/10.1016/j.heares.2011.11.003
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