Fig. 1 A complex set of “audio devices”
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Division of Medical Genetics and Genomics, the Children’s Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China
Institute of Genetics, Zhejiang University and Department of Genetics, Zhejiang University School of Medicine, Hangzhou 310058, China
Division of Medical Genetics and Genomics, the Children’s Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China
Institute of Genetics, Zhejiang University and Department of Genetics, Zhejiang University School of Medicine, Hangzhou 310058, China
1.Division of Medical Genetics and Genomics, the Children’s Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China
2.Institute of Genetics, Zhejiang University and Department of Genetics, Zhejiang University School of Medicine, Hangzhou 310058, China
纸质出版日期: 2019-02 ,
收稿日期: 2017-12-20 ,
录用日期: 2018-03-07
引用本文
Lu-wen Zhang, Xiao-hui Cang, Ye Chen, 等. 哺乳动物内耳毛细胞的体外培养[J]. 浙江大学学报(英文版)(B辑:生物医学和生物技术), 2019, 20(2):170-179.
Lu-wen Zhang, Xiao-hui Cang, Ye Chen, et al. In vitro culture of mammalian inner ear hair cells[J]. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology), 2019, 20(2):170-179.
Lu-wen Zhang, Xiao-hui Cang, Ye Chen, 等. 哺乳动物内耳毛细胞的体外培养[J]. 浙江大学学报(英文版)(B辑:生物医学和生物技术), 2019, 20(2):170-179. DOI: 10.1631/jzus.B1700613.
Lu-wen Zhang, Xiao-hui Cang, Ye Chen, et al. In vitro culture of mammalian inner ear hair cells[J]. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology), 2019, 20(2):170-179. DOI: 10.1631/jzus.B1700613.
Division of Medical Genetics and Genomics, the Children’s Hospital, Zhejiang University School of Medicine, Hangzhou 310058, China
Institute of Genetics, Zhejiang University and Department of Genetics, Zhejiang University School of Medicine, Hangzhou 310058, China
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.
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.
Hearing loss is one of the major health concerns in the world, and seriously affects the lives of millions of people. It may be caused by a number of factors, including genetic defects, aging, noise trauma, and excessive use of ototoxic drugs (Abdouh et al.,
Sounds received by the ear are conveyed through a series of in-built systems consisting of the outer ear, ear canal, eardrum, middle ear ossicles, inner ear, cochlea, and nerves running into the brain (Fig.
Fig. 1 A complex set of “audio devices”
(a) Schematic diagram of sound transmission: 1, ossicles; 2, semicircular canal; 3, crista ampullaris; 4, external ear; 5, tympanic membrane; 6, oval window; 7, round window; 8, cochlea; 9, auditory vestibular nerve; 10, vestibular nerve. (b–d) One of the most notable features of hair cells in the mammalian inner ear is the hair bundle. These hair bundles comprise numerous modified microvilli, called stereocilia, stacked in rows of increasing height. (b) Mouse cochlear hair cells at middle turn, one row of inner hair cells and three rows of outer hair cells exhibit typical “W” arrangement. (c, d) Hair cells in the mouse utricle (c) and crista ampullaris (d). The zoomed-in images show the stereocilia of the type I (lower left in c, d) and type II (lower right in c, d) hair cells. All stereocilia were stained with phalloidin (b–d)
There is a single row of inner hair cells and three rows of outer hair cells in the spiral organ (organ of Corti) of humans. The cochlea of a newborn human infant contains approximately 16 000 hair cells (Géléoc and Holt,
Fig. 2 Structure of cochlear duct
(a) Schematic diagram of cochlear duct: 1, oval window; 2, cochlear apex; 3, tympanic membrane; 4, round window; 5, representative position of sound resonance. (b) The inner ear labyrinth of a P7 C57BL/6 mouse. The white arrow indicates the basal membrane, the red arrow indicates the saccule, the green arrow indicates the cochlear axis, the yellow arrow indicates the utricle, and the blue arrows indicate the three cristae ampullaris
The membranous labyrinth is a continuous system of ducts filled with endolymph, and it lies within the bony labyrinth, surrounded by the perilymph. Because of the complex architecture, hair cells in the inner ear are difficult to access. Corti used surface preparations to observe the mammalian organ of ear in vitro for the first time in 1851 (Betlejewski,
Study | Specimen | System/culture medium | Supplement | Culture/experimental time |
---|---|---|---|---|
Yamashita and Vosteen ( 参考文献 1975 | Guinea pig Corti | Rose chamber method | Horse serum, fetal calf serum, human ascitis fluid, Gey’s balanced salt solution | 20 d |
Sobkowicz et al. ( 参考文献 1975 | Mouse cochlea | Collagen slice; MEM | Horse serum, HEPES, modified Simms salt solution, L-glutamine, D-glucose | 27 d |
Anniko and van de Water ( 参考文献 1978 | Mouse crista ampullaris | Neaman/Tytell medium | 1% L-glutamine, 1% Na-pyruvate, 10% fetal calf serum | 7 d |
Oesterle and Rubel ( 参考文献 1993 | Chicken cochlea | Floating culture; BME | Earle’s balanced salt solution, D-glucose, FBS | 7 d |
Rastel et al. ( 参考文献 1993 | Rat Corti | Collagen gel drop floating; MEM | 40% horse serum, L-glutamine, glucose, HEPES, HBSS | 12 d |
Romand and Chardin ( 参考文献 1999 | Cochlea | DMEM/F12 | D-glucose, L-glutamine, sodium selenium, apotransferrin, albumin bovine | 10 d |
Spencer et al. ( 参考文献 2008 | Chicken cochlea | Artificial perilymph media | N1, N2 | 5 d |
Lelli et al. ( 参考文献 2009 | Mouse cochlea | MEM | 10 mmol/L HEPES | 8 d |
Parker et al. ( 参考文献 2010 | Mouse cochlea | DMEM | 5% FBS, 5% horse serum | 7–10 d |
Ding et al. ( 参考文献 2011 | Mouse cochlea | Collagen gel drop; BEM | BSA, serum-free supplement, 20% glucose, penicillin G, 200 mmol/L glutamine, 1×BME | 48 h |
May et al. ( 参考文献 2013 | Mouse utricle | Floating culture; DMEM/F12 | 5% FBS | 24 h |
Ou et al. ( 参考文献 2013 | Mouse utricle | In bone culture; DMEM | 1% FBS | 3 d |
Lin et al. ( 参考文献 2015 | Mouse utricle | Floating culture; DMEM/F12 | 1% N2, 2% B27, b-FGF, IGF-1, EGF, heparin sulfate | 10 d |
Werner et al. ( 参考文献 2015 | Rat utricle | DMEM | 1% N1, 10% FBS, glucose | 28 d |
Taura et al. ( 参考文献 2016 | Mouse utricle | Matrigel-coated glass coverslips; DMEM | 10% FBS | 10 d |
Landegger et al. ( 参考文献 2017 | Cochlea | DMEM | 1% FBS, 1% N2, and 1% ampicillin | 7 d |
MEM: minimal essential medium; BME: basal minimal Eagle’s medium; DMEM: Dulbecco’s modified Eagle’s medium; F12: Ham’s F12 medium; HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; FBS: fetal bovine serum; HBSS: Hank’s balanced salt solution; N1: N1 supplement; N2: N2 supplement; BSA: albumin from bovine serum; B27: B27 supplement; b-FGF: basic fibroblast growth factor; IGF-1: insulin-like growth factor 1; EGF: epidermal growth factor
The first paper on in vitro culturing was published more than 40 years ago (Betlejewski,
Gel embedding methods have been developed for inner ear hair cell cultures. Rastal et al. (
C57BL/6 mice were purchased from SLAC laboratory Animal Co. (Shanghai, China). The mice were housed in temperature-and humidity-controlled rooms with 12-h light/dark cycles and with free access to food and water. All protocols were approved by the Animal Care and Use Committee of Zhejiang University, Hangzhou, China, and were in compliance with the “Guidelines for the Care and Use of Laboratory Animals” published by the National Academy Press (NIH Publication No. 85-23, Revised in 1996; https://www.nabr.org/animal-welfare-2/animal-welfare-in-practice/guide-for-the-care-and-use-of-laboratory-animals).
DMEM (GBICO, cat: SH30243.01); fetal bovine serum (GBICO, cat: 10099141); heat-inactivated horse serum (GBICO, cat: 26050070); N2 supplement (Thermo Fisher, cat: 17502048); B27 (GBICO, cat: 12587-010); Hank’s balanced salt solution (HBSS, Life Technologies, cat: 14025076); phosphate-buffered saline (PBS, Sangon, cat: E607016-0500); rat tail collagen, Type 1 (BD Biosciences, cat: 4236); BME (Sigma-Aldrich, cat: B9638); penicillin (Sigma-Aldrich, cat: A6140); phalloidin (Enzo, cat: ALX-350-268-MC01); 4,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, cat: D9542); 4% paraformaldehyde (PFA, Beijing Leagene Biotechnology, cat: DF0135-500ml); Triton X-100 (Sangon, cat: 9002-93-1).
Surgical forceps (VETUS, #5 Tweezers 3C); surgical scissors (ROBOZ, cat: RS-6702); 4-well plates (BD Falcon, cat: 353654); microscope slide coverslips (Thermo, cat: D9542); dissecting microscope (Lecia, EZ4); sterilizer (VORTEX-5); biosafety cabinet (BCM-1300); cell incubator (Heal Force).
2.4.1 In vitro culture of mouse cochlear hair cells
(1) Postnatal mice (3 to 5 d after birth) are anaesthetized using isoflurane-saturated gas (Fig.
Fig. 3 Diagrams of dissecting steps
(a) Postnatal 5-d-old C57BL/6 mice were sacrificed for inner ear dissection. (b) Bisect the mouse head and remove the brain tissue. (c) The yellow triangles show the locations of inner ear. (d–f) Carefully separate the cochlea from the extraneous bones. The red triangle shows the cochlea. (g, h) Remove the stria vascularis, spiral bone plate, Reissner’s membrane, and tectorial membrane. The blue triangle shows the Reissner’s membrane; the white triangle shows the basilar membrane. (i) Representative structure of the basilar membrane. (j) The hair cells were labeled with phalloidin. (a–h) Bar=1 mm (each grid)
2.4.2 In vitro culture of mouse utricle hair cells
(1) Mice were sacrificed, and the temporal bone was obtained as mentioned above (Figs.
The organotypic cultures were fixed in 4% PFA (4 °C/overnight) and permeabilized with 0.01% Triton X-100 (5 min/room temperature). Then, the hair cells were stained with phalloidin (1:200, in PBS) for 20 min at room temperature. The slides were washed three times in PBS, and then the nuclei were labeled by DAPI (10 μmol/L stock solution; 1:1000 in PBS) for 10 min at room temperature. The slides were washed three more times in PBS, covered with a glass coverslip over fluorescent mounting media, and left to dry in the dark before the edges were sealed with nail hardener. All staining processes were manipulated in a light avoidance condition.
Viable culture preparations from the mammalian inner ear are still problematic. Currently, there is no satisfactory way to culture inner ear tissues for a very long time period. Utricle, one of the sensory epitheliums in the inner ear membranous labyrinth, is easier to obtain for culturing hair cells in vitro. As shown in Fig.
Fig. 4 In vitro culturing of mouse utricle hair cells
(a) Mouse utricles were isolated and cultured in vitro for 1 d. (b) After 5 d’s culture, most of the cells lost their hair bundle (the white triangles indicate the hair cells and the yellow triangle shows the supporting cells). The stereocilia were stained with phalloidin (green). The nuclei were labelled by DAPI (blue)
For cochlear cell culture, mice of postnatal 3–5-d old were used. Compared with adult mice, the cochleae of newborn mice can be easily dissected to obtain the culture specimen since the temporal bone is not fully calcified (Fig.
Fig. 5 Isolation of the inner ear from mouse
(a) Representative image of inner ear from P7 C57BL/6 mouse. (b) The inner ear of adult mouse was embedded within the bony labyrinth. The red arrow indicates the cochlea, the black arrow indicates the oval window, and the blue arrow indicates the round window
Fig. 6 In vitro culturing of mouse cochlea hair cells
The cochlea was isolated from P5 C57BL/6 mouse for both immunohistochemical staining and in vitro culturing. (a) Epithelial cells were observed emerging from the basilar membrane. (b) The fresh isolated cochlea cells were stained with phalloidin. The nuclei were stained with DAPI. (c) The cochlea maintained a good shape and cell viability after culturing for 5 d. (d) Outer hair cells gradually disappeared with the increase of culturing time. The red arrows show the inner hair cells, the white arrows show the outer hair cells, and the white triangles show the loss of hair cells
In the last decade, rapid progress has been made in the area of in vitro culture of mammalian inner ear hair cells, and various culturing systems have been published to date. The development of in vitro hair cell cultures not only improves our understanding of the molecular control of progenitor cell fates within the developing cochlea, but also facilitates the study of drug delivery and gene therapy targets. However, gradually increased apoptosis of hair cells continues to be an issue. In general, hair cells cultured in vitro survive for only 1–2 weeks. Recently, 3D cell culture systems have gained increasing importance in tissue engineering and drug testing because of their advantages in providing physiologically relevant information. Using these 3D cell culture strategies, researchers have succeeded in maintaining primary cultures with greater stability and longer lifespans than those in regular 2D cultures. Further research should be conducted to enable prolonged survival of hair cells and eliminate the influence of antibiotics, thereby significantly contributing to the understanding of hair cell biology.
Compliance with ethics guidelines
Lu-wen ZHANG, Xiao-hui CANG, Ye CHEN, and Min-xin GUAN declare that they have no conflict of interest.
All applicable institutional and/or national guidelines for the care and use of animals were followed.
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