SD大鼠背根神经节细胞新取材和培养方法

华中科技大学硕士学位论文SD大鼠背根神经节细胞新取材和培养方法姓名：艾玛AmmarAliAhmed申请学位级别：硕士专业：外科学（手外科）指导教师：康皓2011-05SD大鼠背根神经结细胞的新取材和培养方法华中科技大学同济医学院附属协和医院血液病研究所　硕士研究生：艾玛　 指导教师：　康皓 教授　中文摘要　【目的】：探讨有效摘取ＳＤ大鼠背根神经结（ＤＲＧ）的方法，为实验取材奠定基础。

　方法①取材　选择为三周ＳＤ大鼠，提取ＤＲＧ神经元。

用显微解剖获取足够数量的ＳＤ大鼠背根神经结，通过胰蛋白酶＋Ⅳ型胶原酶消化②分离：ＰＢＳ洗涤吸干液体，加入０．２％Ⅳ型胶原酶和０．２５％胰蛋白酶放入３７０Ｃ温箱边消化边用眼科剪剪碎背根神经结重复数次，直到消化液变得粘稠后就可以用滴管吹打，吹打后就可以完全的把背根组织彻底的消化掉，加入含有血清的ＮＢＬ培养基终止消化，用１０００转离心，③原代培养将细胞悬液加入底面铺有Ｌ一多聚赖氨酸的培养皿（ＰＰ　）中，放人３７℃、体积分数为０．０５的ＣＯ２培养箱中，让其自然贴壁。

定期在倒置相差显微镜下观察。

　【结果】背根神经结细胞原代贴壁生长较慢，一个月以后才能长满瓶底生长二天后２０倍光镜下背根神经结细胞。

细胞成簇生长，向四周放射状排列，细胞核圆形透亮，细胞形态多边型，向四周伸出较长的触须，其他类型细胞基本上已经死掉而悬浮在培养基里。

　【关键词】背根神经结；细胞培养　New method in extracting and culture of dorsal rootganglion neurons of SD ratDepartment of Hand surgery, Union Hospital, Tong Ji Medical School, HuazhongUniversity of Science and TechnologyPostgraduate: Ammar Ali Ahmed TaherSupervisor: Pro. KangHaoAbstract【Objectives】To explore the effective method applied in extracting of dorsal root ganglion (DRG) of SD rats, thus lay a foundation for obtainment of the experimental materials.【Methods】①Obtainment of materials: Select 3-week SD rats to extract DRG neuron. Sufficient dorsal root ganglions of SD rat may be obtained via microdissection and digested using trypsin + collagenase IV. ② Isolation:The liquid is washed and sucked up with PBS, add 0.2% collagenase IV and 0.25% trypsin, after that it is placed in 37℃incubator for digestion, during which the dorsal root ganglion should be sheared with ophthalmic scissor for several time until the digestive juice becomes thick, and then a pipette is used to blow, the dorsal root tissue may be digested totally after blowing, add NBL medium containing serum to terminate the digestion process, and centrifuged at 1000r. ③Culture of primary generation of cells: Add cellular suspension in Petri dish (PP) with L-poly-lysine laid down at its bottom, and placed in incubator (37℃, 0.05 volume fraction of CO2) to allow natural adherent growth. The inverted phase contrast microscope is used regularly to perform the observation.【Results】The adherent growth of the primary generation of dorsal root ganglion cells isso slow that a month is needed to cover the bottom of the flask. The dorsal root ganglion cells under 20 x optical microscope grow for two days. The cluster of cells grows in radial arrangement to the surrounding; the nucleus appears round and transparent and cellular morphology in shape of polygon while the longer tentacles expand around the cell; other types of cell have been basically dead and suspend in the medium.【Keywords】Dorsal root ganglion; Cell culture独创性声明本人郑重声明，本学位论文是本人在导师指导下进行的研究工作及取得的研究成果的总结。

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（请在以上方框内打“v”）学位论文作者签名：指导教师签名：日期：年月日日期：年月日IntroductionThe neuron of dorsal root ganglion locates in dorsal root ganglion and peripheral nerve system (PNS) of vertebrates; the dorsal root ganglion contains Schwann cells, fibroblasts and satellite cells around neuron, in addition to rich sensory neurons of peripheral nerve system. As the pseudounipolar neuron, DRG neuron is originated from neural crest and formed via migration of pluripotent precursor cell. The axon is rounded by Schwann cell to form the myelin sheath. The dorsal root ganglion cell is a primary sensory neuron that can conduct nervous impulses like touch sensation, pain sensation, warm sensation and so forth. As the link between in vitro and in vivo environment of organism and spinal cord, its functions include transmission and regulation of various sensory receptions, and conduction of various injured perceptions, thus it plays an important role in development and maintain of neuropathology. It can conduct nervous impulses such as touch, pain and warm sensations. DRG is important in nervous system as a critical relay station that connects peripheral and central nervous fibers. DRG neuron involves directly in physiopathology of some hereditary and acquired neuropathies. The technology of culture of nervous tissue and cells established by Harrison in 1907, who was the first to culture successfully the implantation plate of neural tube of frog embryo in the lymph clot and observed the activity of neuronal process and its growth cone, so that Cajal’s neuron doctrine is supported. In the last two decades, the nervous culture has become a modern research approach with its modification in methodology and combination with other subjects, and become one of the bridges and links among various subject strategies while there is growing recognition in this field, resulting in effective combination between morphology and functionality, as well as basis and clinic. Its application is expanded while the culture of nervous tissue is combined with advanced research approaches and instruments at present. The technology of neuron culture plays an important role in researches from the growth and differentiation of neuron and glial cell, neurotransmitter, neuropeptide and neurotrophic factor, nervous endocrine, nervous immunity, neural regeneration and neural transplantation, clinicaldisease to electrophysiological record in a single neuron cell, etc. [1]. In 1950s, the formation of peripheral nervous myelin sheath from the dorsal root ganglion of chicken embryo in organic culture was observed at first by Murray and Peterson. In 1956, Naki reported the culture of cells isolated from the dorsal root ganglion neuron of chicken embryo, and observed the growth of growth cone of nervous fiber. The nerve growth factor (NGF), discovered by Levi Montalcini utilizing the culture technique of dorsal root ganglion cells of chicken embryo in the late 1950s, promoted significantly the development of the nervous culture. DRG culture model is used widely in nerv ous research fields such as the guidance and regeneration of axon [2], the growth and plasticity of synapse [3], myelinization of central nervous system and peripheral nervous system [4], the role of neurotrophic factor and the distribution of its receptors [5], the aging mechanism of nervous cells [6], gene therapy[7], tissue engineering [8] and so on, and become the new research approach for hereditary and acquired neuropathies (for example, Friedreich ataxia [9], nervous pain syndrome [10], peripheral neuropathy causey by diabetes mellitus [11], Guillain-Barre syndrome (GBS) and the autoimmunity disease of nervous system), due to the interior structure of DRG and its intrinsic biological significance, as well as its similarity to the neurons in other sites. Currently, animal model is one of the major methods for peripheral nervous research, used to research the peripheral neuropathy and its treatment via the culture of nervous tissue. The treatment and recovery of peripheral nervous injury are difficulties in medical community for many years; the recovery of sensorimotor function of peripheral nerve is not so good once it is injured. In recent years, the incidence of peripheral nervous injury is increasing resulted from incidents like traffic accident and working injury along with the significant improvements in national economy and living standard in the population; thus the effective method to treat peripheral nervous injury should be worked out as soon as possible. The efficacy of peripheral nervous injury is not satisfied although significant progress has been realized clinically in its treatment. The reasons of that may include ① slow regeneration rate of the nerve (generally 1 mm daily);② different degrees of denaturation and necrosis of proximal neuron; ③ the dismal effector expresses denervated changes, i.e. atrophy, denaturation and even necrosis, especially the skeletal muscle. The considerations for treatment of peripheral nervous injury focus on three methods: ① promote the regeneration of peripheral nerve; ② reduce the atrophy of targeted muscle; and ③ prevent proximal neuron from denaturation and necrosis. A problem that has been not solved clinically is how to delay the atrophy of targeted muscle. It is impossible to repair directly the brachial plexus as the avulsed wound of its nerve root is occurred clinically; the nervous transposition can only be applied in such repair clinically these days, such as the using of phrenic nerve, accessory nerve, intercostal nerves and so on; there are defects, however, like long regeneration distance, atrophy that has been resulted before the muscle is innervated, denaturation (especially the intrinsic muscles of hand) leading to the lost of the opportunity of functional recovery. The quality in recovery of peripheral nervous injury is improved significantly since the development of microsurgery in 1950s, especially as the basic research is deepened in the last three decades. The repair of peripheral nervous injury has developed from single microsurgery to the current laser technology, cell transplantation technology, tissue engineering technology and the like, along with the further understanding of the effect of neurotrophic factor, laminin, interleukin, immunological reaction and the like on the regeneration of peripheral nerve, thus significantly enrich and promote the studies on the repair of peripheral nervous injury. However, the treatment of denervated innervation and lost of function of targeted muscle resulted from avulsed wound of nerve root (for example, the common avulsed wound of nerve root of brachial plexus) and/or spinal cord injury with injured motor neuron and so on, remains a difficulty in modern medical field.Materials and methods1 Materials1.1 Selection of experimental animals: 7 male/female healthy adult SD neonatal rats (3weeks) are selected as breeding rats (purchased from Institute of Experimental Animal of Chinese Academy of Medical Sciences).1.2 Reagents1.2.1 Reagents used in cultureNeurobasal medium (Sigma)NBL medium (Neurobasal medium added with 2.49g/l D-glucose, 0.073g/L L-glutamine, 10ml/L FBS fetal bovine serum (GIBCO) and 1:50 B27 additive (GIBCO)).Collagenase IV (Sigma)0.25% trypsin (Sigma)1.2.2 Reagents used in immunohistochemistry stainingNSE rabbit anti-mouse NSE antibody (primary antibody): (Boster)4% paraformaldehyde (Boster)CY3 goat anti-rabbit IgG (secondary antibody): (Boster)1.2.3 Nissl staining reagentsDAPI (colorant) Roche1.3 Main instruments and devices:35mm, 60mm and 100mm medium; 96-well culture plate;0.05 CO2 incubator (thermo.co. USA);Clean bench (Suzhou); MOTlC anatomic microscope;Invert microscope (Olympus, Japan);Digital camera (NIKON COOI PIX950, Japan).1.4 Experimental instrumentsOphthalmic scissor, micro forceps, micro scissor and Petri dishes2 Experimental methods2.1 Obtainment and separation of dorsal root ganglion cells7 SD neonatal rats (3 weeks) are not killed after anesthesia. Following soaked in 0.5%iodophor for 30 min, the lumbar spine of rat in prone position is obtained and placed in PBS dish under the sterilized condition; the spinal cord is exposed when spinous process is uncovered following the both sides of the spinous process are cut off with ophthalmic scissor (Figure 1 and 2); the spinal cavity is exposed after the spinal cord is lifted and cut off, and the nervous fiber connected to spinal cord is removed completely.(Figure 3 and 4) The site that dorsal root ganglion locates, (Figure 4) the dorsal root ganglion can be drawn out while the residue nervous fiber is pulled gently with fiber forceps along the direction of red arrow; (Figure 5) the expansion part is retained after trimmed with micro forceps to remove excessive nervous fiber and placed in prepared small flask. The liquid is washed and sucked up with PBS, add 0.2% collagenase IV and 0.25% trypsin, after that it is placed in 37℃incubator for digestion of 30 min during which the dorsal root ganglion should be sheared with ophthalmic scissor for several time until the digestive juice becomes thick, and then a pipette is used to blow, the dorsal root tissue may be digested totally after blowing, add NBL medium containing serum to terminate the digestion process, centrifuged for 5 min at 1000r, washed with PBS for 3 times and add medium to suspend the cells.2.2 Culture of primary generation of dorsal root ganglion cellsAdd cellular suspension in Petri dish (PP) laid down at the bottom with L-poly-lysine, and placed in incubator (37℃, 0.05 volume fraction of CO2) to allow natural adherent growth. The Olympus inverted phase contrast microscope is used regularly to perform the observation.2.3 Subculture of dorsal root ganglion cellsThe trypsin of 2.5 g/L mass concentration is used for digestion after the growing dorsal root ganglion cells occupied 70-80% area of the bottom of culture flask and the passage is done at the ratio of 1:2.2.4 Drawing of growth curveTake the first generation of cells in logarithmic phase and remove the medium, use phosphate buffer to wash twice and the trypsin of 2.5 g/L mass concentration to digest for 1-3 min at 37℃, add medium containing serum to terminate the digestion, use pipette to blow the cells off the wall, the cell suspension is prepared as a result. The suspension is counted with counting plate to adjust the cellular concentration to 108.The preparation i s inoculated in 96-well plate with 200 µL in each well; the plate is cultured in incubator (37℃, 0.05 volume fraction of CO2). The test is conducted at 1, 2, 3, 4, 5, 6, 7, 8, 9 d ays, respectively. The absorbance value of individual well is measured on ELISA reader with CCK8 method and the values are used to drawn the growth curve of dorsal root ganglion cells.2.5 The immunohistochemistry determination of dorsal root ganglion cellsThere is specific surface marker nestin on the nervous stem cell, thus NSE can be used to identify if the cultured cell is neuron. In cell-seeded coverslip cultured in vitro is fixed with 4% paraformaldehyde and stained with immunofluorescence cytochemistry technology. It is washed for three times with PBS (pH7.2-7.4) and then blocked with10% goat serum followed by washing with PBS again for three times; the primary antibody is NSE monoclonal rabbit anti-rat antibody that is incubated at 4℃ overnight and washed for three times; the secondary antibody is CY3 goat anti-rabbit antibody that is incubated at 37℃ for 60 min and washed thoroughly with PBS, and then strike dyeing with DAPI is done for 1 min. The express of NSE is observed and photographed under confocal microscope following the staining.ResultsThe growth features of dorsal root ganglion cellsThe primary generation of dorsal root ganglion cells grows so slow that a month is needed to cover the bottom of the flask. (Figure 6) The dorsal root ganglion cells under 20 x optical microscope grow for two days. (Figure 7) The dorsal root ganglion cells under 20 x optical microscope grow two days after the growth began. The cluster of cells grows in radial arrangement to the surrounding; the nucleus appears round and transparent and cellular morphology in shape of polygon while the longer tentacles expand around the cell; other types of cell have been basically dead and suspend in the medium. (Figure 8 and 9) The dorsal root ganglion cells under 40 x optical microscope after the medium was changed for the cells.The growth curve of dorsal root ganglion cellsThe cultured dorsal root ganglion cells grow slow and their curve appears basically a straight line, suggesting that no cell division has been occurred.The identification of phenotype of dorsal root ganglion cellsThe red NSE positive express is observed in cytoplasm and the blue one locating in the middle is observed in nucleus (see Figure 11).FiguresFigure 1. The schematic diagram of spinal anatomy, the spinal bone should be cut off along the red line.Figure 2. The schematic diagram of spinal anatomy, the spinal bone should be cut off along the red line.Figure 3. The location of dorsal root ganglion.Vertebral foramenLamina of vertebral archSpinous processSuperior articular processTransverse process Vertebral bodyInferior articular processCut line Cut lineUncinate processAnterior ramus of cervical nerveVertebral arteryCervical dorsal rami nervesCervical nerve root Cut line Cut line Superior articular processTransverse processSpinous processInferior articular processSuperior vertebral notchVertebral body Inferior vertebral notchDorsal root ganglionFigure 4. The location of dorsal root ganglion whic h can be drawn out along the direction of red arrow.Figure 5. The dorsal root ganglion is trimmed with fiber scissor. Dorsal root ganglion Draw out the dorsal root ganglion Vertebral body The site where the operation is conducted under microscopeFigure 6. The dorsal root ganglion cells under 20 x optical microscope grow for two days.Figure 7. The dorsal root ganglion cells under 20 x optical microscope grow two days after the growth began.Figure 8. The dorsal root ganglion cells under 40 x optical microscope grow two days after the growth began.Figure 9. The dorsal root ganglion cells under 40 x optical microscope grow two days after the growth began.Figure 10. The stained nucleus of dorsal root ganglion cell.DiscussionThe dorsal root ganglion is the sensory neuron of peripheral nervous system with concentrated cell bodies, high density and simple structure. The interior structure of DRG and its biological significance are similar to the neurons in other sites. The establishment of in vitro stable culture system for DRG facilitates the neurobiological researches including axon guidance, synaptogenesis, signal transduction of neuron, neurotrophic factor and so on. The survival, growth and purification of DRG cultured in vitro are discussed as follows:1. The factors affecting the survival and behavior of in vitro cultured DRG1.1 The survival sign of neuron cultured with isolated cell from nervous tissue: adherentgrowth is realized within 24 hours after the implantation and tens of microns of protrusion grows gradually [12].1.2 Obtainment of DRG for in vitro culture: The animals used for DRG culture at presentinclude chicken, rat and mouse while human dorsal root ganglion is seldom used due to medical ethics and limited resource. The age of the experimental animal affects directly the culture result of DRG. The cultured ganglion with different age can survive along with the development of culture conditions and methods. The nervous tissue from embryo and neonatal animal that is generally obtained survives easily under in v itro culture conditions [13], because the proliferation of neuron takes place in embryo phase and less division will occur in postnatal and in vitro neuron during the early growth of nervous system; on the other hand, the neuron of animal embryo has a low degree of morphological differentiation and chemodifferentiation, high in vitro survival capacity, whereas the neuron of the postnatal or adult animal has a high degree of differentiation, matured development of neurite, thus the in vitro survival rate of neuron is lower than that of embryo source. Similarly, although the neuron of dorsal root ganglion from adult or aged animal can survive when cultured in vitro [14] [15], the culture condition necessary in the culture is more strict and difficult. The research results of ourpre-experiment show that 3-week neonatal rat is advisable to be used in the experiment.For anatomic method, the spinal cord is often exposed by dissection from ventral side during the isolation of DRG. In the experiment, we have tried dorsal dissection following the path used for isolation of ganglion of adult rat, thus removal of spinal cord with DRG from vertebral canal is simple; survival of H during in vitro culture is easy with longer survival period; the procedure of obtainment is simple with more amount of DRG and shorter time used in obtainment.1.3 Extracellular matrix: After the neuron “migrates” outside of the organism, its survivaldepends on if it can adhere to a surface, therefore, the neuron can only survive on the culture plate coated with matrix; for isolated neuron, adhesion to growth matrix as soon as possible is one of the factors affecting the in vitro survival of neuron. The interaction between extracellular matrix and nervous cell will affect the neurons in their adhesion, survival, differentiation, phenotypic express and the migration and growth of axon [16].The common matrix comprises of PLL, LN, collagen (type I, III, IV and V) and fibronectin. While coated with PLL and LN matrix, the DRG cells disperse well; an allowable substrate provided by LN will bind with the receptors on the surface of the cell to stimulate the growth of axon [17], moreover, PLL and LN can upregulate the NSE express of neuron [18]. We use collagenase IV in the experiment for such substance has the activity faster than digestion of nervous tissue and less damage to neuron.1.4 Inoculum density: Proper inoculum density facilitates the survival of neuron and thegrowth of axon, and the interval of cell adhesion is important too; high inoculum density causes small spacing among adherent neurons and faster growth speed of neurite while the nervous cells are difficult to survival at low inoculum density. The reason might be the effect of signal transduction mechanism among the cells. The inoculum density of isolated and cultured cells is not less than 104/ml in general or rare survival will be resulted; the proper inoculum density is 105-106/ml.1.5 In vitro culture medium of neuron: The condition of in vitro culture medium of neuronshould be similar to in vivo living environment as much as possible, and the medium should contain the nutritive material s and inorganic salts necessary for the growth and survival of the cell, as well as provides appropriate PH value and osmotic pressure. The developmental trend of in vitro medium for neuron is changed from the medium containing natural compositions (such as serum, plasma and embryonic extract liquid, etc.) to the one without natural composition. The serum, the mostly used natural composition, contains various active substances for modulation of the growth and metabolism of the cell, such as hormone, growth factor, transporter, vitamin and microelements, etc. However, the uncertainty in the chemical compositions of serum is adverse to analyze the lab results, so that it is replaced gradually by serum-free defined medium and the amount of information used in researching the nervous system with in vitro culture method is greatly increased [19]. The defined medium for nervous tissue includes DMEM/F12, N1, N2 and N3. In our experiment, the serum medium Neurobasal combined with B27 additive supports the in vitro survival and growth of neuron, and effectively inhibits the proliferation of non-nervous cells. The isolation of individual cells within nervous tissue is realized based on the different adherent speed of such cells. The method is simple and effective, but both purity and amount of isolated stem cells are low.1.6 Others: In addition to the factors that affecting the survival of neuron, other importantfactors during the culture include the cleanness of cover glass, the control of digestion time and amount of trypsin, the mechanical impact to neuron in medium exchange and observation. Unclean cover glass will lead to difficult adhesion of neuron. The digestion time of trypsin should be adequate while longer digestion time will damage neuron and shorter time will prevent nervous tissue from isolation of single cell. The small neurite is very sensitive to the minor vortex flow in the culture plate, therefore, caution should be taken in each operation to reduce the damage to neuron plus proper adjustment ofosmotic pressure and pH, as well as control of every step, thus the distinct single cell with smooth surface and intact cytoplasm can be isolated.2. Grow cone (GC): In 1956, Naki reported firstly that the development of growth cone ofnervous fiber was observed after the single homogeneous DRG cell was cultured among the cells isolated from dorsal root ganglion neuron of chicken embryo. As the palm-shaped expansion structure at the end of neurite in neurogenesis phase and nervous regeneration phase, the growth cone determines the way of growth and expansion of guided neurite. As a high motile and active structure with irregular shape, it presents 1-3 or more filopodia or lamellipodia while repeated extension and retraction in the form of transformative filum terminale result in regular changes in number, size and shape. The growth cone is the transient structure that can be observed easily at 24h and 72h during culture until the appearance of connected postsynaptic elements. In our experiment, the adherent growth of primary generation of dorsal root ganglion cells is slow and a month is needed to cover the bottom of the flask. The dorsal root ganglion cells can be observed two days after growth under 2x optical microscope. The cluster of cells grows in radial arrangement to the surrounding; the nucleus appears round and transparent and cellular morphology in shape of polygon while the longer tentacles expand around the cell; other types of cell have been basically dead and suspend in the medium.3. Purification and culture of neuron: The obtainment of high purity of neuron culturesystem facilitates the research on biological characteristics and functions of specific cell.The DGR contains rich neurons, Schwann cells, fibroblasts and satellite cells, how to obtain rich neurons remains one of the difficulties in DRG isolation and culture. There is no rapid and effective method for purification of nervous cells; in order to get purer and more homogeneous nervous cells during the culture, the most direct method is to add mitotic inhibitor (such as 5-FU, Ara and C) in the medium to inhibit the growth of other cells like gliocyte and fibroblast. The toxic effect of mitotic inhibitor on mitoticcell is theoretically realized via block of DNA synthesis and inhibition of cell division, such drugs may cause the neuron dead in the anaphase. We explore the differential velocity adherent time in the pre-experiment in reference to the differential velocity adherent culture method by Meyer that is used in purification of neuron, the optimal time of differential velocity adhesion prior to inoculation for DRG cell suspension is 50 min, during this period the non-nervous cell with high adherent capacity a nd fast adherent speed is removed while the non-adherent neuron remained. In the study, the serum-free medium Neurobasal combined with B27 additive inhibits the division and proliferation of fibroblast and Schwann cell and promotes selectively the survival of neuron, thus the purification rate of DRG is 91% or so. The non-purified DRG culture system that survives for 3-4 weeks is longer significantly than that of purified DRG system. The Schwann cell can maintain the survival of neuron and promote the growth of neurite through excretion of extracellular matrix like collagen and neurotrophic factors like cell adhesion molecule [20] and NGF. The adherent rate of DRG neuron depends on different culture medium and time. The purpose of isolation and purification of neuron cell can be achieved only when culture medium more sensitive to non-nervous cell is selected to allow more non-nervous cells to adhere in advance. The culture medium that is more favorable for the growth and survival of neuron and growth of axon should be chosen during the culture after the purification of DRG neuron is done.4. Isolation and culture of nervous tissue: The cultures of nervous tissue includeessentially explant culture and isolation culture. In explant culture, the growth of neurite reflects the growth characteristics of the neuronal cluster, but not that of single neuron.In isolation culture, the more homogeneous neurons can be obtained easily: the overall single neuron may be observed if the inoculum density is low. The neuron during culture may differentiate to dendrite and axon with the morphology and function corresponding to the sites in vivo. In 1956, Naki reported firstly the culture of cells。