作者：戴惟葭， Shelley CulpStewwart, Anna Cheng, John Flanagan, C Ross Ethier
作者单位：100053 中国北京，首都医科大学宣武医院眼科；加拿大多伦多大学眼科与视觉科学系； 加拿大多伦多大学机械与工业工程系
【摘要】目的：探索人眼视神经乳头星形胶质细胞体外培养的方法，为进一步研究星形胶质细胞在青光眼性视神经病变中的作用打下基础。
方法：取材新鲜人眼视神经乳头组织和筛板组织，进行星形胶质细胞和筛板细胞的体外培养与传代试验。
结果：组织块培养4～8wk后，原代细胞开始生长，星形胶质细胞在形态学和生长特性上与筛板细胞明显不同，β1型星形胶质细胞可以在无血清的培养液中生长良好，通过无血清培养液选择性培养可以在第二代传代过程中分离出纯化的星形胶质细胞，并可在第三至第四代收获大量细胞以备后续的研究。
结论：精细准确的组织解剖分离对于获得纯化细胞至关重要，采用无血清培养液选择分离获得星形胶质细胞方法经济简单，细胞纯度高，便于进一步储存和研究。
【关键词】 细胞培养；青光眼，开角型；视神经
Human optic nerve head astrocytes culture in vitro: I. the primary culture and passage
WeiJia Dai, Shelley CulpStewwart, Anna Cheng, John Flanagan, C Ross Ethier
Department of Ophthalmology, Xuanwu Hospital, Capital Medical University, Beijing 100053, China Department of Ophthalmology & Vision Science, University of Toronto, Canada Institute of Biomaterials and Biomedical Engineering, Department of Mechanical and Industrial Engineering, University of Toronto, Canada
Abstract
AIM: To culture astrocytes from human donor eyes in order to understand the function of astrocytes in remodelling events in the glaucomatous optic nerve head (ONH).
METHODS: Primary cultures were prepared by explantation of human ONH tissue in order to get astrocytes. Laminar criborsa (LC) cells were prepared concurrently for comparison. Astrocyte cultures could be separated from LC cells by selecting medium. Similar procedures were used for LC.
RESULTS: Primary cells grew from human optic nerve head explants 48 weeks after explantation. Astrocytes had different morphologies and growth characteristics from LC cells. Type 1B astrocyte cells could grow in medium without FBS. Purified cultures were obtained by second passage and could be harvested by third to fifth passage, which were prepared to use for further study, including being characterized by positive glial fibrillary acidic protein (GFAP) and neural cell adhesion molecule (NCAM) staining.
CONCLUSION: Precise dissection of fragment is the most important step to get clear explants for primary culture. Economic and rapid method could be useful to select cells by different mediums, which will help us to get more purified cells for further study.
KEYWORDS: cell culture; glaucoma, open angle; optic nerve head
INTRODUCTION
Glaucomatous optic neuropathy is characterized by a progressive loss of retinal ganglion cells associated with visual functional deficits, usually in response to elevated intraocular pressure. As the ganglion cells die in glaucoma, there is a progressive thinning of the nerve fibre layer (NFL). The mechanism of this pathology, however, is not totally clear. So far substantial research has shown that damage to the optic nerve axons occurs at the level of the lamina cribrosa in the optic nerve head [1,2]. Increasingly, glial cells in the CNS have been cited as participants in the pathologic course of neuronal damage after mechanical, ischemic and various other insults. The mechanisms by which glial cells are involved in CNS neuronal damage may vary [3,4], but many of the mechanisms are surprisingly similar to those proposed in glaucoma. Glial cells, especially, play an important role in supporting axons and forming the interface between connective tissue surfaces and surrounding blood vessels [35]. Different kinds of astrocytes are located in pre and postlaminar region, as well as in lamina cribrosa. In glaucoma, there is extensive remodeling of the OHN extracellular matrix (ECM), with increased expression of ECM and proteolytic enzymes, cell adhesion molecules, and neurotoxic mediators, by reactive ONH astrocytes [3]. In order to understand all the functions that astrocytes perform in glaucoma development, we attempted to culture the human optic nerve astrocytes in vitro.
MATERIALS AND METHODS
Donors Sixtyfive human eyes without history of eye disease (age 14101) were obtained within 2448 hours of death from Toronto Eye Bank. Eyes were transported to the laboratory in a humidified atmosphere container.
Procedures of Dissection Fresh eyes were rinsed and carefully dissected in sterilized PBS (supplemented with penicillin/streptomycin) under dissecting microscope. The whole anterior segment and posterior part of vitreous, retina pigment and remnant nonneural tissues were removed. The resulting cylinder of tissue included the optic nerve head, lamina cribrosa and post laminar myelinated nerve, as well as small amounts of sclera and choroid (Figure 1A). Under a dissecting microscope, the post laminar myelinated nerve was identified and excised using fine scissors. The central vessels were carefully teased out of the remaining piece of tissue (Figure 1EH). The ONH tissue was cut into several smaller pieces and put into a flask with a drop of DMEM (see explants culture).
Explants Culture Tissue culture flasks(25cm2)were conditioned with 1mL prewarmed growth medium Dulbeccos modified Eagles medium (DMEM, Sigma Company)/F12 (SigmaAldrich, Canada), supplemented with 100mL/L FBS, LGlutamine(2mmol/L),Gentamycin(10mg/L),and Fungizone(250μg/L).
Figure 1Procedure of dissection A: tissue with sclera (Sc), chloroid (C) and retina (R); B:tissue with dura mater (DM); C: tissue with pia mater; D: side view of picture A (LC: laminar cribrosa; MN: myelinated nerve); E: yellow lines indicate the cuts that will be made in the next steps; F: yellow line indicates the cut of optic nerve head; G: separation part of optic nerve head from lamina tissue; H: vessels in the middle of cutting explant (20×)（略）
Each explant piece was placed into the culture flask, and kept standing in a humidified incubator (37℃ 50mL/L CO2 , Thermo Forma) for 1520 minutes allowing the explants to adhere to the flask. After 5 days in the incubator an additional 4mL of media was carefully added to the flask. After another 10 days, the media on the cells was changed biweekly. Three to 8 weeks later, cells were observed growing out from the explants.
Passage When the primary cells from explants were confluent, they were passaged and split in 1∶2. For astrocytes, cells from ONH would be selected and cultured in serumfree astrocyte medium (AGMFBS) (ABM supplemented with growth factors, cytokines, antibiotics and supplements; Cambrex).Once the population looked homogeneous, serum was included in the media at all future media changes. For LC cells, the cells were cultured in lowglucose DMEM (SigmaAldrich) supplemented with 100mL/L FBS, LGlutamine (2mmol/L) and Gentamycin (10mg/L).
RESULTS
The Best Dissecting Method for Cell Culture We were searching a best way to dissecting tissue and got the purified culture explants as best as we can. Follow the procedure we did, a clear anatomic tissue with nicely identified layers could be observed very well (Figure 2) which could help us to get the best explants for our further culture.
Primary Cell Culture from Human Optic Nerve Head Initial outgrowth from the explants appeared within 38 weeks of culture and reached confluence in 412 weeks. Cells grew out from the explant and contained a mixed population of cells with two major cell types: astrocyte cells (AST) and laminar cribrosa cells (LC). The former was starshaped with long processes (Figure 3A) while the latter a large flat polygonal appearance with oval clear nuclei (Figure 3B). Fibroblastlike cells could be easily identified due to their rapid growth rates and multilayering characteristics.
At the first passage, astrocytes cells could be selected by changing the medium to FBS free medium, in which LC cells could not survive. After this selection process, the cultures appeared to be essentially pure astrocytes. We also found that AST cells could be passaged to 7th passage and grew very well while LC cells could not survive nicely after 4th passage.
DISCUSSION
Glial cells are structurally and functionally linked to neuronal tissues, including the optic nerve and retina. The transition of quiescent mature astrocytes to the activated phenotype represents one of the earliest and most dominant responses of the central nervous system to injury. Pathologically, the activated cells may be characterized by increased size and number, by altered cellular properties, such as upregulation of glial fibrillary acidic protein (GFAP), and by additional cellular changes that may cause or relieve neuronal impairment [4,6,7]. Such astrocytic changes have been found in the glaucomatous optic nerve and retina as well as in experimental glaucoma models [8,9].
Figure 2Details in dissection fragment a: retina on the face of optic nerve head; b: optic nerve head; c: lamina tissue including prelamina and postlamina; d: myelinated optic nerve fibers (20×)（略）
Figure 3 A:2nd passage of AST cells after FBS free selection. Morphological characters (arrows) showed typical starshaped with long extension processes; B: 1st passage of 2nd day growth LC cells with flat body of polygonal short processes (200×)（略）
In the optic nerve, glial cells include astrocytes, oligodendrocytes and microglia. Astrocytes are the major glial cells type in the nonmyelinated optic nerve head in most mammalian species. In the human optic nerve head, two subpopulations of astocytes are distinguished. Type 1A astroctyes are interspersed in the glial columns and at the edge of cribriform plates. They express GFAP but not neural cell adhesion molecule (NCAM). Type 1B astrocytes express both GFAP and NCAM. They are the major glial cell population in this region and are the primary cells involved in synthesis of the optic nerve head ECM during development and throughout life [10]. At the vitreal surface of the optic disc, which is lined with Type 1B astrocytes, is the source of the cells used for this study (see materials and methods). In our results, Type 1B astrocytes showed a starshaped morphology, and positive characterization will be shown in our next paper where were GFAP and NCAM positive. The results are consistent with those of Hernandez [11]. The method we used is economical and easy to perform because the astrocytes grow well in AGM without FBS while the LC cells cannot survive in this medium. The precise dissection of explant from ONH is quite important to get the purified cells.
The function of Type 1B astrocytes is to form the glial columns, surround blood vessels in the prelaminar region, form the cribriform plates in the lamina cribrosa and separate the sclera from the optic nerve in the insertion area. Under normal intraocular pressure, the astrocytes remain quiescent. In glaucoma, the lamina cribrosa undergoes significant deformation in response to changes in intraocular pressure (IOP), which generates biomechanical stress on astrocytes and other cell types of LC [12,13], leading to the reactive astrocyte phenotype, optic nerve head remodelling and loss of retinal ganglion cell axons. Histopathologic studies in human donor eyes also demonstrate that astrocytes undergo an activation process in glaucoma1 [14].
Cultures of adult human ONH astrocytes as a useful in vitro model have been reported in several studies. Several studies will be done using the cultured cells to simulate the physical stresses caused by elevated IOP. Such in vitro experiments will help us to understand the mechanisms involved in the remodelling of ECM in glaucoma and provide us a model with which to research medications for optic nerve protection in the future [1517].
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