WO2017032224A1 - 一种嗅鞘细胞的制备方法 - Google Patents

一种嗅鞘细胞的制备方法 Download PDF

Info

Publication number
WO2017032224A1
WO2017032224A1 PCT/CN2016/094529 CN2016094529W WO2017032224A1 WO 2017032224 A1 WO2017032224 A1 WO 2017032224A1 CN 2016094529 W CN2016094529 W CN 2016094529W WO 2017032224 A1 WO2017032224 A1 WO 2017032224A1
Authority
WO
WIPO (PCT)
Prior art keywords
olfactory
cells
cell
olfactory ensheathing
ensheathing
Prior art date
Application number
PCT/CN2016/094529
Other languages
English (en)
French (fr)
Inventor
黄红云
高文勇
肖娟
Original Assignee
黄红云
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 黄红云 filed Critical 黄红云
Priority to EP16838484.0A priority Critical patent/EP3339427B1/en
Priority to JP2018528369A priority patent/JP6812435B2/ja
Priority to US15/754,171 priority patent/US10829734B2/en
Publication of WO2017032224A1 publication Critical patent/WO2017032224A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0622—Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/062—Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/02—Atmosphere, e.g. low oxygen conditions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

  • the invention belongs to the technical field of cell culture, and relates to a method for preparing olfactory ensheathing cells, which comprises the separation, passage, cryopreservation, differentiation of olfactory ensheathing cells and preparation of required reagents.
  • Olfactory ensheathing cells originate from the olfactory basement membrane and are distributed in the olfactory bulb and olfactory mucosa.
  • the primitive olfactory neurons are accompanied by a large number of basal cells that migrate from the olfactory mucosa to the axon to the cerebral vesicle.
  • the olfactory ensheathing cells direct the olfactory axons to reach the cerebral vesicles. These cells form the early olfactory bulb, and then the olfactory bulb evanates and migrates.
  • a thin layer is formed on the surface, and then they penetrate the colloidal membrane to guide the formation of the olfactory nerve layer and the small spherical layer, which becomes a new gelatinous membrane covering the surface of the olfactory bulb.
  • the olfactory ensheathing cells can still migrate through the adult stage. This gelatinous membrane barrier between the peripheral and central nerves.
  • Within the olfactory bulb it is the only glial cell that contacts and coats the olfactory axons. In the entire central nervous system pathway, it coats the olfactory axons to prevent contact with other central nervous system cells.
  • OEC secretes neurotrophic factors, axon growth stimulating substances, promotes axonal regeneration and helps myelination. It is a special glial cell similar in function to Schwann cells and oligodendrocytes, with neurotrophic effects. , protection, regulation or stimulation, promote myelination and axon regeneration, inhibit gliosis and hyperplasia and other nerve repair effects.
  • These characteristics of olfactory ensheathing cells provide a good internal environment for the repair and functional reconstruction of damaged or degenerated nerves. The properties of olfactory ensheathing cells make it the best choice for nerve repair.
  • Ethical factors The olfactory bulb tissue derived from aborted fetuses is susceptible to ethical and moral constraints.
  • the cell purity is not high: (1) cytarabine and chemical reagents are broad-spectrum cytostatics, inhibiting the growth of fibroblasts, but also inhibit the olfactory ensheathing cells, and the amount is difficult to grasp. (2) Physical methods also remove a large number of olfactory ensheathing cells while removing fibroblasts. (3) Using serum-containing medium, fibroblasts tend to grow faster.
  • the object of the present invention is to provide a method for preparing a large number of active olfactory ensheathing cells quickly and easily, thereby providing a sufficient source of olfactory ensheathing cells for clinical nerve repair treatment.
  • a method for preparing olfactory ensheathing cells comprising the steps of:
  • (1) culture the olfactory ensheathing cell single cell derived from the middle nasal olfactory mucosa is cultured in an olfactory sheath cell culture medium at 37 ° C under 5% CO 2 to make it adhere to the wall;
  • the cells obtained by filtering the culture supernatant obtained in the step (1) were mixed with the olfactory ensheathing cell culture medium, and cultured at 37 ° C under 5% CO 2 to grow adherently.
  • the olfactory sheath cells are human olfactory mucosal olfactory ensheathing cells.
  • the olfactory ensheathing cell culture medium is DMEM/DF12 (Gibco) as a basal medium, and a final concentration of 20-60 ng/ml of EGF, 20-80 ng/ml of FGF, and 1-2 ml of N2 (100 ⁇ ) is added. ), 2-3 ml of B27 (50 ⁇ ), 0.1 ⁇ g/ml of T3 (Sigma).
  • the human olfactory mucosal olfactory ensheathing cell culture medium is DMEM/DF 12 (Gibco), 20 ng/ml EGF (Pepro Tech), 20 ng/ml FGF (Pepro Tech), 1% N2 (Gibco), 2% B27. (Gibco), T3 (Sigma) neurotrophic factor.
  • the method for preparing the olfactory ensheathing cell single cell comprises the following steps:
  • the middle nasal olfactory mucosa tissue block is digested with an enzyme to obtain a single cell.
  • the preparation method of the olfactory ensheathing cell single cell comprises the following steps:
  • the middle nasal olfactory mucosa tissue was digested with collagenase I and neutral protease to obtain single cells.
  • the neutral protease is tissue dispase II;
  • the preparation method of the olfactory ensheathing cell single cell comprises the following steps:
  • the collagenase I is used at a concentration of 0.1% and the neutral protease is used at a concentration of 0.2%.
  • the middle turbinate olfactory mucosa tissue block is washed before use to remove surface residual bloodstain; further preferably, the surface residual blood is removed by using penicillin to physiological saline at a ratio of 1:1; and further preferably, in the culture
  • the washed olfactory mucosa was cut into a 1 mm 3 tissue block with sterile scissors in a dish.
  • the seeding density of the culture in the step (1) is 1 ⁇ 10 4 cells/ml.
  • the cells are adherently grown to 90%.
  • the seeding density of the culture in the step (2) is 1 ⁇ 10 4 cells/ml.
  • the cells are adherently grown to 90%.
  • the preparation method further comprises the step of passage or cryopreservation of the olfactory sheath cells;
  • the collected passage cell culture supernatant is filtered and mixed with the olfactory mucosal olfactory ensheathing cell culture medium at a ratio of 1:3, and configured as a passage medium;
  • the preparation method further comprises the step of cryopreserving the olfactory ensheathing cells; preferably, the cryopreservation is cryopreservation; preferably, the step of cryopreserving the olfactory ensheathing cells comprises: at step When the cells in the (2) are adherently grown to 90%, the culture supernatant is filtered to obtain a frozen storage solution of the olfactory sheath cells.
  • the composition of the olfactory ensheathing cell cryopreservation solution is autologous serum, culture supernatant, DMEM/F12 and DMSO.
  • the composition of the olfactory ensheathing cell cryopreservation solution is autologous serum, culture supernatant, DMEM/F12 and DMSO in a volume ratio of 5:2:2:1.
  • the preparation method further comprises a differentiation culture step of the olfactory ensheathing cells; preferably, the step of culturing the olfactory ensheathing cells comprises adding a glial differentiation culture medium to the olfactory ensheathing cells and differentiating and culturing. More preferably, the composition of the glial differentiation culture medium is an olfactory ensheathing cell medium and autologous serum at a volume ratio of 87:13.
  • the preparation method is:
  • Step 1 The olfactory ensheathing cell culture medium prepared by using neurotrophic factor.
  • Step 2 Tissue extraction: Under local anesthesia conditions, a proper amount of middle nasal olfactory mucosa tissue was taken.
  • Step 3 Tissue block pretreatment: cleaning the surface residual blood trace of the taken tissue block, and processing the tissue block to make it a 1 mm 3 tissue block.
  • Step 4 Combining digestion with two enzymes: collecting the shredded tissue blocks, adding 2 volumes of collagenase I and neutral protease, and shaking and digesting in a 37 ° C water bath.
  • Step 5 Primary culture: The individual cells obtained by the collection and digestion are placed in a centrifuge tube and mixed with the olfactory ensheathing cell culture medium in a cell culture flask, and placed in a 37 ° C, 5% CO 2 incubator for cultivation.
  • Step 6 Subculture: When the cells are adherently grown to about 90%, the collected passage cell culture supernatant is mixed with the olfactory ensheathing cell culture medium at a ratio of 1:3, and the subculture medium is disposed at a cost.
  • Step 7 Cryopreservation: When the cells are adherent to about 90%, the supernatant of the cryopreserved cells is filtered for the special cryopreservation of olfactory ensheathing cells (autologous serum, cell culture supernatant, DMEM/F12, DMSO); cryopreservation according to routine procedures.
  • olfactory ensheathing cells autologous serum, cell culture supernatant, DMEM/F12, DMSO
  • the preparation method of the invention relates to tissue extraction, double enzyme combined digestion, subculture, ultra-low temperature freezing Storage, differentiation culture, preparation of required reagents.
  • the invention has the advantages that the method for extracting and culturing the olfactory ensheathing cells can rapidly obtain a large number of olfactory ensheathing cells, and can maintain the proliferative ability of the olfactory ensheathing cells for a long time, and the cells obtained after passage to the 11th generation still have olfactory ensheathing cells.
  • Figure 1 shows that the adherent cells were observed to have a fusiform shape, a bipolar elongated protrusion, a tripolar elongated protrusion, and a polygon under ordinary inverted micromirrors.
  • A is green fluorescence, S100 immunocytochemical staining
  • B red fluorescence, p75 ( L-NGFR) immunocytochemical staining
  • C blue fluorescence, Hoechst labeled nuclei
  • D is a full spectrum fluorescence map.
  • Figure 1 Microscopic examination of olfactory ensheathing cells, magnification: 10 ⁇ / 0.25.
  • Figure 2 Immunohistochemical staining of olfactory ensheathing cells by confocal microscopy, magnification: 20 ⁇ / 0.5.
  • the olfactory ensheathing cell culture medium was prepared using DMEM/DF12 (Gibco) as the basic medium, and the culture factors added included EGF with a final concentration of 20-60 ng/ml, FGF of 20-80 ng/ml, and N2 of 1-2 ml (100).
  • EGF EGF with a final concentration of 20-60 ng/ml
  • FGF FGF of 20-80 ng/ml
  • N2 1-2 ml (100).
  • ⁇ ) 2-3 ml of B27 (50 ⁇ ) and 0.1 ⁇ g/ml of T3 (Sigma) were sterilized by filtration in a clean bench, and stored at 4 ° C until use.
  • tissue pieces transported back to the laboratory were washed with physiological saline containing 100 U/ml penicillin to remove residual blood on the surface of the mucosal tissue block, and cut into tissue pieces having an area of 1 mm 3 with a sterile ophthalmic scissors in a glass culture dish.
  • Collect the chopped tissue block add 2 volumes of collagenase I and neutral protease, shake and digest in a 37 °C water bath for 15 min, then repeatedly blow with a thick elbow pipette, naturally settle for 1 min, transfer the supernatant to the centrifuge tube and The digestion was terminated, and the tissue was digested into a single cell suspension by repeating this three times.
  • the cell suspension after the termination of digestion was collected and centrifuged at 1200 r/4 min, and cleared with DMEM/F12. Wash and centrifuge at 1200r/4min for 3 times.
  • the olfactory ensheathing cell culture medium was added to make a single cell suspension, and seeded at a density of 1 ⁇ 10 4 cells/ml in a Poly-L-lysine-treated oblique plastic flask, at 37 ° C, 5% CO 2 .
  • the culture was carried out in a constant temperature and humidity incubator.
  • the supernatant of the culture medium was collected and mixed with the olfactory ensheathing cell medium at a ratio of 1:3 through a 0.22 ⁇ m filter, and the subculture medium was configured at a cost.
  • the cells were collected and the appropriate amount of passage medium was added to prepare the cells.
  • the suspension was stained with trypan blue, and the viable cells were counted under a microscope, and then passaged, and the density of the passaged cells was 1 ⁇ 10 4 /cm 3 .

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Neurology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Neurosurgery (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Acoustics & Sound (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种嗅鞘细胞的制备方法，所述方法包括细胞培养基的配置、组织取材和预处理、酶消化、细胞培养、冻存和分化培养的步骤，制备出的嗅鞘细胞能长时间维持增殖能力，在传代至第11代仍然具有嗅鞘细胞的活性。

Description

一种嗅鞘细胞的制备方法 技术领域
本发明属于细胞培养技术领域，涉及一种嗅鞘细胞的制备方法，该制备方法包括嗅鞘细胞的分离、传代、冻存、分化以及所需试剂的配制。
背景技术
嗅鞘细胞(olfactory ensheathing cells，OECs)起源于嗅基底膜，分布在嗅球和嗅粘膜。原始嗅觉神经元伴随大量基板细胞从嗅粘膜传出轴突向端脑泡方向迁移，嗅鞘细胞引导嗅神经轴突到达端脑泡，这些细胞形成早期的嗅球，接着嗅球发生外翻，迁移细胞覆在其表面形成一薄层，接着它们穿透胶质界膜引导形成嗅神经层和小球层，成为覆盖在嗅球表面新的胶质界膜，在成年阶段嗅鞘细胞依然能迁徙穿过周围神经和中枢神经之间这种胶质界膜屏障。在嗅球内，它是唯一接触和包被嗅神经轴突的胶质细胞。在整个中枢神经系统通路中，它包被嗅神经轴突，以防止与其他中枢神经系统细胞接触。OEC可分泌神经营养因子、轴突生长刺激物质，能促进轴突再生和帮助髓鞘形成，是在功能上与施旺细胞和少突胶质细胞相似的一种特殊胶质细胞，具有神经营养、保护、调控或刺激，促髓鞘化和轴突再生，抑制胶质增生瘢痕形成等神经修复作用。嗅鞘细胞这些特性为损伤或退变神经的修复及功能重建提供了很好内环境。嗅鞘细胞的特性使得其成为神经修复的最佳选择。已发表的关于嗅鞘细胞移植的文章也强有力的证明了嗅鞘细胞是神经修复的最佳材料。然而，现有嗅鞘细胞的制备方法：用流产3—5个月的胎儿，取脑内嗅球组织，用含一定量的动物血清直接培养，然后使用一定量的阿糖胞苷、物理方法或者使用化学试剂抑制减少成纤维细胞，达到纯化嗅鞘细胞的目的。采用高浓度的动物血清和DMSO混合成嗅鞘细胞冻存保护液。存在着诸多不足之处：
1、伦理因素：采用流产胎儿来源的嗅球组织，易受到伦理道德的约束。
2、细胞纯度不高：⑴阿糖胞苷和化学试剂是广谱细胞抑制剂，在抑制成纤维细胞生长的同时也会对嗅鞘细胞产生抑制，而且用量较难把握。⑵物理方法在去除成纤维细胞的同时也会丢失大量的嗅鞘细胞。⑶采用含血清培养基，成纤维细胞容易更快生长。
3、无法持续传代培养：(1)使用阿糖胞苷和化学试剂是广谱细胞抑制剂，会影响嗅鞘细胞的传代次数。(2)使用血清做为基础培养基，嗅鞘细胞容易不定向分化，成纤维细胞生长迅速，且多次传代很难保持生物学特性。
4、会有潜在风险：来源于异体的血清、组织标本，容易感染病毒和细菌、容易发生排异、过敏等未知危险。
发明内容
为了解决现有技术中存在的上述缺陷，本发明的目的是提供一种快速、简便获得大量活性嗅鞘细胞的制备方法，从而为临床神经修复治疗提供充足的嗅鞘细胞来源。
用于实现上述目的的技术如下：
一种嗅鞘细胞的制备方法，该制备方法包括以下步骤：
(1)培养：将来源于中鼻甲嗅粘膜的嗅鞘细胞单细胞置于37℃，5％CO2的条件下在嗅鞘细胞培养基中培养，使之贴壁生长；
(2)将步骤(1)得到的培养上清液过滤得到的细胞再与嗅鞘细胞培养基混合，置于37℃，5％CO2的条件下培养，使之贴壁生长。
优选地，所述嗅鞘细胞为人嗅粘膜嗅鞘细胞。优选地，所述嗅鞘细胞培养基为DMEM/DF12(Gibco)做基础培养基，添加最终浓度为20-60ng/ml的EGF、20-80ng/ml的FGF、1-2ml的N2(100×)、2-3ml的B27(50×)、0.1μg/ml的T3(Sigma)。
优选地，所述人嗅粘膜嗅鞘细胞培养基为DMEM/DF 12(Gibco)、20ng/ml EGF(Pepro Tech)、20ng/ml FGF(Pepro Tech)、1％N2(Gibco)、2％B27(Gibco)、T3(Sigma)神经营养因子。
优选地，所述嗅鞘细胞单细胞的制备方法包括以下步骤：
用酶将中鼻甲嗅粘膜组织块消化得到单细胞。
更优选地，所述嗅鞘细胞单细胞的制备方法包括以下步骤：
用胶原蛋白酶Ⅰ和中性蛋白酶将中鼻甲嗅粘膜组织块消化得到单细胞。
优选地，所述中性蛋白酶为组织分散酶Ⅱ；
更优选地，所述嗅鞘细胞单细胞的制备方法包括以下步骤：
用胶原蛋白酶Ⅰ和中性蛋白酶将1mm3的中鼻甲嗅粘膜组织块在37℃水浴中震荡消化得到单细胞，其中所述胶原蛋白酶和中性蛋白酶与中鼻甲嗅粘膜组织块的体积比为2:1。
优选地，所述胶原蛋白酶Ⅰ使用浓度为百分之0.1和中性蛋白酶使用浓度为百分之0.2。
进一步优选地，所述中鼻甲嗅粘膜组织块消化前经过清洗以去除表面残余血迹；更进一步优选地，使用青霉素与生理盐水配比1∶1，去除表面残余血迹；再进一步优选地，在培养皿中用无菌剪刀将洗浄的嗅粘膜剪成面积为1mm3组织块。
优选地，所述步骤(1)中的培养的接种密度为1×104个细胞/ml。优选地，所述细胞贴壁生长至90％。
优选地，所述步骤(2)中的培养的接种密度为1×104个细胞/ml。优选地，所述细胞贴壁生长至90％。
优选地，所述制备方法还包括将所述嗅鞘细胞传代或冻存的步骤；
更优选地，所述传代中,收集传代细胞培养上清液过滤与嗅粘膜嗅鞘细胞培养基比1∶3进行混合，配置成传代的培养基；
优选地，所述制备方法还包括冻存所述嗅鞘细胞的步骤；优选地，所述冻存为超低温冻存；优选地，所述超低温冻存所述嗅鞘细胞的步骤包括：在步骤(2)中的细胞贴壁生长至90％时，将培养上清液过滤，制成嗅鞘细胞冻存液冷冻保存。更优选地，所述嗅鞘细胞冻存液的组成为自体血清、培养上清液、DMEM/F12和DMSO。进一步优选地，所述嗅鞘细胞冻存液的组成为体积比为5∶2∶2∶1的自体血清、培养上清液、DMEM/F12和DMSO。
优选地，所述制备方法还包括所述嗅鞘细胞的分化培养步骤；优选地，所述嗅鞘细胞的分化培养步骤包括向所述嗅鞘细胞添加胶质细胞分化培养液并分化培养。更优选地，所述胶质细胞分化培养液的组成为体积比为87∶13的嗅鞘细胞培养基和自体血清。
在本发明的具体实施方案中，所述制备方法为：
步骤1、用神经营养因子配制出的嗅鞘细胞培养基。
步骤2、组织取材：局部麻醉条件下，夹取适量中鼻甲嗅粘膜组织。
步骤3、组织块预处理：清洗所取组织块的表面残余血迹，处理组织块使其成为1mm3组织块。
步骤4、双酶组合消化：收集剪碎的组织块，加入2倍体积的胶原蛋白酶Ⅰ和中性蛋白酶，37℃水浴锅震荡消化。
步骤5、原代培养：收集消化得到的单个细胞置于离心管中与嗅鞘细胞培养基充分混合种植于细胞培养瓶中，并置于37℃，5％CO2培养箱中培养。
步骤6、传代培养：细胞贴壁生长至90％左右时，收集传代细胞培养上清液过滤与嗅鞘细胞培养基比1∶3进行混合，配置成本次传代的培养基。
步骤7、超低温冻存：细胞贴壁生长至90％左右时，收集冻存细胞的上清液过滤用于配置嗅鞘细胞专用冻存液(自体血清、细胞培养上清液、DMEM/F12、DMSO)；按照常规程序冷冻保存。
步骤8、分化培养：添加配制的胶质细胞分化培养液(嗅鞘细胞培养基∶自体血清＝87∶13)应用常规方法分化培养。
本发明的制备方法涉及组织取材、双酶组合消化、传代培养、超低温冻 存、分化培养、所需要试剂的配制。本发明优点是提供的嗅鞘细胞的提取和培养方法能快速获得大量嗅鞘细胞，并且能长时间维持嗅鞘细胞的增殖能力，在传代至第11代后获得的细胞仍然具有嗅鞘细胞的活性，如附图说明所示：图1显示，在普通倒置微镜下观察到贴壁的细胞呈梭形、双极细长突起、三极细长突起、多边形。放大倍数：10×/0.25；在图2显示的嗅鞘细胞共聚焦显微镜免疫组化化学染色鉴定结果中，(A)是绿色荧光，S100免疫细胞化学染色；(B)是红色荧光，p75(L-NGFR)免疫细胞化学染色；(C)是蓝色荧光，Hoechst标记细胞核；(D)是全光谱荧光图。放大倍数：20×/0.5。所培养的细胞高表达嗅鞘细胞阳性标记物S100、P75(L-NGFR)。
附图的简要说明
图1：嗅鞘细胞的镜检结果，放大倍数：10×/0.25。
图2：嗅鞘细胞共聚焦显微镜免疫组化化学染色鉴定，放大倍数：20×/0.5。
实施发明的最佳方式
1、嗅鞘细胞培养基的配制
用DMEM/DF12(Gibco)做基础培养基配制嗅鞘细胞培养基，其中添加的培养因子包括最终浓度为20-60ng/ml的EGF、20-80ng/ml的FGF、1-2ml的N2(100×)、2-3ml的B27(50×)、0.1μg/ml的T3(Sigma)于超净台内过滤除菌，4℃下储存备用。
2、组织取材
获取组织块前两天，清理鼻腔并确定无感染等；获取前进行鼻内局部麻醉，将浸有含1.2％-1.5％丁卡因的生理盐水的纱棉片用枪状镊塞入鼻腔中，局部麻醉2次，每次5-10min；使用筛窦咬钳取中鼻甲靠外上1/3处的嗅粘膜组织，将取得的组织块放入玻璃皿；再将玻璃皿放入冰盒中，立即带回培养室。
3、组织预处理
将运送回实验室的组织块用含100U/ml青霉素的生理盐水清洗粘膜组织块的表面残余血迹，在玻璃培养皿中用无菌眼科剪刀剪成面积为1mm3的组织块。
4、双酶组合消化
收集剪碎的组织块，添加2倍体积的胶原蛋白酶Ⅰ和中性蛋白酶，37℃水浴锅内震荡消化15min后用粗弯头吸管反复吹打，自然沉降1min，转移上清液到离心管中并终止消化,如此重复3次可以将组织消化为单细胞悬液,将消化终止后的细胞悬液收集到一起并以1200r/4min离心,用DMEM/F12清 洗并以1200r/4min离心，共3次。
5、原代培养
加入嗅鞘细胞培养基将其制成单细胞悬液，以1x104个细胞/ml的密度接种于经Poly-L-lysine处理过的斜口塑料培养瓶中，于37℃，5％CO2恒温恒湿培养箱中进行培养。
6、传代培养
当细胞融合生长至90％左右，收集培养基上清并经0.22μm滤器过滤与嗅鞘细胞培养基比1∶3进行混合，配置成本次传代培养基，收集细胞加入适量传代培养基制成细胞悬液，经台盼蓝染色后，在显微镜下对活细胞计数，再进行传代，传代细胞的密度为1×104个/cm3。
7、超低温冻存
细胞融合生长至90％左右，收集培养基上清经0.22μm滤器过滤，配置嗅鞘细胞专用冻存液(自体血清∶细胞培养上清液∶DMEM/F12：DMSO体积比＝5∶2∶2∶1)，常规收集用于冻存的细胞，添加冻存保护液，调节细胞的最终密度为5×106个/ml～5×107个/ml；置于4℃下60min，-20℃下70min，-80℃下超低温冷冻过夜后，置于液氮罐中。
8、分化培养
配制胶质细胞分化培养液(嗅鞘细胞培养基∶自体血清体积比＝87∶13)，4℃下保存备用；添加配制的胶质细胞分化培养液应用常规方法分化培养。
以上是结合实施例对本发明做出的具体说明，但本发明并不限于上述实施例，在本领域普通技术人员所具备的知识范围内，还可以在不脱离本发明宗旨的前提下做出各种变化，或直接或间接将本发明运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。

Claims (9)

  1. 一种嗅鞘细胞的制备方法，该制备方法包括以下步骤：
    (1)培养：将来源于中鼻甲嗅粘膜的嗅鞘细胞单细胞置于37℃，5％CO2的条件下在嗅鞘细胞培养基中培养，使之贴壁生长；
    (2)将步骤(1)得到的培养上清液过滤得到的细胞再与嗅鞘细胞培养基混合，置于37℃，5％CO2的条件下培养，使之贴壁生长。
  2. 根据权利要求1所述的方法，其中，所述嗅鞘细胞为人嗅粘膜嗅鞘细胞。
  3. 根据权利要求1或2所述的方法，其中，所述嗅鞘细胞培养基为DMEM/DF12(Gibco)做基础培养基，添加最终浓度为20-60ng/ml的EGF、20-80ng/ml的FGF、1-2ml的N2(100×)、2-3ml的B27(50×)、0.1μg/ml的T3(Sigma)；
    优选地，所述人嗅粘膜嗅鞘细胞培养基为DMEM/DF 12(Gibco)、20ng/ml EGF(Pepro Tech)、20ng/ml FGF(Pepro Tech)、1％N2(Gibco)、2％B27(Gibco)、T3(Sigma)神经营养因子。
  4. 根据权利要求1-3中任一项所述的方法，其中，所述嗅鞘细胞单细胞的制备方法包括以下步骤：
    用酶将中鼻甲嗅粘膜组织块消化得到单细胞；
    优选地，所述嗅鞘细胞单细胞的制备方法包括以下步骤：
    用胶原蛋白酶Ⅰ和中性蛋白酶将中鼻甲嗅粘膜组织块消化得到单细胞；
    优选地，所述中性蛋白酶为组织分散酶Ⅱ；
    更优选地，所述嗅鞘细胞单细胞的制备方法包括以下步骤：
    用胶原蛋白酶Ⅰ和中性蛋白酶将1mm3的中鼻甲嗅粘膜组织块在37℃水浴中震荡消化得到单细胞，其中所述胶原蛋白酶Ⅰ和中性蛋白酶与中鼻甲嗅粘膜组织块的体积比为2:1；
    优选地，所述胶原蛋白酶Ⅰ使用浓度为百分之0.1和中性蛋白酶使用浓度为百分之0.2。
  5. 根据权利要求1-4中任一项所述的方法，其中，所述中鼻甲嗅粘膜组织块消化前经过清洗以去除表面残余血迹；优选地，使用青霉素与生理盐水配比1∶1，去除表面残余血迹；更优选地，在培养皿中用无菌剪刀将洗净的嗅粘膜剪成面积为1mm3组织块。
  6. 根据权利要求1-5中任一项所述的方法，其中，所述步骤(1)中的培养的接种密度为1×104个细胞/ml；优选地，所述细胞贴壁生长至90％；
    优选地，所述步骤(2)中的培养的接种密度为1×104个细胞/ml；更优选 地，所述细胞贴壁生长至90％。
  7. 根据权利要求1-5中任一项所述的方法，其中，所述制备方法还包括将所述嗅鞘细胞传代或冻存的步骤；
    优选地，所述传代中,收集传代细胞培养上清液过滤与嗅粘膜嗅鞘细胞培养基比1∶3进行混合，配置成传代的培养基；
    优选地，所述制备方法还包括冻存所述嗅鞘细胞的步骤；优选地，所述冻存为超低温冻存；优选地，所述超低温冻存所述嗅鞘细胞的步骤包括：在步骤(2)中的细胞贴壁生长至90％时，将培养上清液过滤，制成嗅鞘细胞冻存液冷冻保存；更优选地，所述嗅鞘细胞冻存液的组成为自体血清、培养上清液、DMEM/F12和DMSO；进一步优选地，所述嗅鞘细胞冻存液的组成为体积比为5∶2∶2∶1的自体血清、培养上清液、DMEM/F12和DMSO。
  8. 根据权利要求1-7中任一项所述的方法，其中，所述制备方法还包括所述嗅鞘细胞的分化培养步骤；优选地，所述嗅鞘细胞的分化培养步骤包括向所述嗅鞘细胞添加胶质细胞分化培养液并分化培养；更优选地，所述胶质细胞分化培养液的组成为体积比为87∶13的嗅鞘细胞培养基和自体血清。
  9. 根据权利要求1-8中任一项所述的方法，其中，在所述制备方法包括以下步骤：
    步骤1、用神经营养因子配制出的嗅鞘细胞培养基；
    步骤2、组织取材：局部麻醉条件下，夹取适量中鼻甲嗅粘膜组织；
    步骤3、组织块预处理：清洗所取组织块的表面残余血迹，处理组织块使其成为1mm3组织块；
    步骤4、双酶组合消化：收集剪碎的组织块，加入2倍体积的胶原蛋白酶Ⅰ和中性蛋白酶，37度水浴锅震荡消化；
    步骤5、原代培养：收集消化得到的单个细胞置于离心管中与嗅鞘细胞培养基充分混合种植于细胞培养瓶中，并置于37℃，5％CO2培养箱中培养；
    步骤6、传代培养：细胞贴壁生长至90％左右时，收集传代细胞培养上清液过滤与嗅鞘细胞培养基比1∶3进行混合，配置成本次传代的培养基；
    步骤7、超低温冻存：细胞贴壁生长至90％左右时，收集冻存细胞的上清液过滤用于配置嗅鞘细胞专用冻存液(自体血清、细胞培养上清液、DMEM/F12、DMSO)；按照常规程序冷冻保存；
    步骤8、分化培养：添加配制的胶质细胞分化培养液(嗅鞘细胞培养基∶自体血清＝87∶13)应用常规方法分化培养。
PCT/CN2016/094529 2015-08-21 2016-08-11 一种嗅鞘细胞的制备方法 WO2017032224A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP16838484.0A EP3339427B1 (en) 2015-08-21 2016-08-11 Preparation method for olfactory ensheathing cells
JP2018528369A JP6812435B2 (ja) 2015-08-21 2016-08-11 嗅神経鞘細胞の調製方法
US15/754,171 US10829734B2 (en) 2015-08-21 2016-08-11 Preparation method for olfactory ensheathing cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN2015105160552 2015-08-21
CN201510516055.2A CN105062956B (zh) 2015-08-21 2015-08-21 人嗅粘膜嗅鞘细胞分离、传代、冻存、分化技术

Publications (1)

Publication Number Publication Date
WO2017032224A1 true WO2017032224A1 (zh) 2017-03-02

Family

ID=54492476

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/094529 WO2017032224A1 (zh) 2015-08-21 2016-08-11 一种嗅鞘细胞的制备方法

Country Status (5)

Country Link
US (1) US10829734B2 (zh)
EP (1) EP3339427B1 (zh)
JP (1) JP6812435B2 (zh)
CN (1) CN105062956B (zh)
WO (1) WO2017032224A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108441475A (zh) * 2018-03-21 2018-08-24 山东省齐鲁干细胞工程有限公司 一种培养中鼻甲来源嗅鞘细胞的方法

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105062956B (zh) 2015-08-21 2018-02-09 北京市虹天济神经科学研究院 人嗅粘膜嗅鞘细胞分离、传代、冻存、分化技术
CN105349491B (zh) * 2015-12-15 2019-09-24 北京市虹天济神经科学研究院 来源于嗅粘膜神经元细胞分离制备技术
EP4060025A4 (en) * 2019-11-15 2023-12-06 Beijing Hongtianji Neuroscience Academy PRODUCTION PROCESS FOR ODOR PRECURSOR CELLS
CN115261324B (zh) * 2022-07-13 2024-05-17 华中农业大学 鱼类嗅觉神经元的培养方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001030982A1 (en) * 1999-10-27 2001-05-03 Griffith University Olfactory ensheathing cells isolated from the lamina propria
WO2007069927A2 (en) * 2005-12-14 2007-06-21 Akademia Medyczna Im. Piastow Slaskich Methods of the obtaining of olfactory ensheathing cells and their application
CN105062956A (zh) * 2015-08-21 2015-11-18 黄红云 人嗅粘膜嗅鞘细胞分离、传代、冻存、分化技术

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0218316D0 (en) 2002-08-07 2002-09-11 Medical Res Council Improvements relating to therapy
AR046076A1 (es) * 2003-07-18 2005-11-23 Otsuka Pharma Co Ltd Procedimiento para obtener una poblacion homogenea de celulas precursoras de oligodendrocitos y poblacion obtenida
WO2005046598A2 (en) 2003-11-07 2005-05-26 University Of Florida Research Foundation, Inc. Culturing and differentiating neural precursor cells
JP4844042B2 (ja) 2005-08-18 2011-12-21 ソニー株式会社 平面型表示装置
JP2007222045A (ja) * 2006-02-22 2007-09-06 Yamaguchi Univ 中枢神経障害性疾患の回復誘発細胞
CN101591641A (zh) * 2008-05-30 2009-12-02 北京市虹天济神经科学研究院 嗅鞘细胞培养上清液诱导嗅干细胞分化方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001030982A1 (en) * 1999-10-27 2001-05-03 Griffith University Olfactory ensheathing cells isolated from the lamina propria
WO2007069927A2 (en) * 2005-12-14 2007-06-21 Akademia Medyczna Im. Piastow Slaskich Methods of the obtaining of olfactory ensheathing cells and their application
CN105062956A (zh) * 2015-08-21 2015-11-18 黄红云 人嗅粘膜嗅鞘细胞分离、传代、冻存、分化技术

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
DING, DONG ET AL.: "The Results of Olfactory Ensheathing Cells from Different Purification Methods", PROGRESS IN MODERN BIOMEDICINE, vol. 14, no. 19, 31 July 2014 (2014-07-31), XP009508945 *
GUO, XIN ET AL.: "Comparison of Growth of Human Olfactory Epithelium Ensheathing Cells in Different Culture Conditions", ZHEJIANG CLINICAL MEDICAL JOURNAL, vol. 16, no. 9, 30 September 2014 (2014-09-30), pages 1366, XP009508947 *
HIGGINSON, J.R. ET AL.: "The Culture of Olfactory Ensheathing Cells (OECs) - a Distinct Glial Cell Type", EXPERIMENTAL NEUROLOGY, 15 September 2010 (2010-09-15), XP055366553 *
JIANG, XIAORONG ET AL.: "Isolation, Culture and Purification of Olfactory Ensheathing Cells from Human Fetal Olfactory Mucosa", PROCEEDINGS OF THE 5 TH ANNUAL CONFERENCE OF INTERNATIONAL ASSOCIATION OF NEURORESTORATOLOGY, THE 9 TH ANNUAL CONFERENCE OF GLOBAL COLLEGE OF NEUROPROTECTION & NEUROREGENERATION AND THE 4 TH INTERNATIONAL SPINAL CORD INJURY TREATMENTS AND TRIALS SYMP, 31 December 2012 (2012-12-31), XP009508954 *
NASH, H.H. ET AL.: "New Method of Purification for Establishing Primary Cultures of Ensheathing Cells from the Adult Olfactory Bulb", GLIA, vol. 34, no. 2, 15 April 2001 (2001-04-15), XP002434762 *
See also references of EP3339427A4 *
SHENG, WEIBIN ET AL.: "Isolation, Culture and Identification of Olfactory Ensheathing Cells from Adult Rat Olfactory Mucosa", JOURNAL OF CLINICAL REHABILITATIVE TISSUE ENGINEERING RESEARCH, vol. 12, no. 16, 15 April 2008 (2008-04-15), XP009508961 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108441475A (zh) * 2018-03-21 2018-08-24 山东省齐鲁干细胞工程有限公司 一种培养中鼻甲来源嗅鞘细胞的方法
CN108441475B (zh) * 2018-03-21 2020-09-29 山东省齐鲁干细胞工程有限公司 一种培养中鼻甲来源嗅鞘细胞的方法

Also Published As

Publication number Publication date
US10829734B2 (en) 2020-11-10
US20180245040A1 (en) 2018-08-30
EP3339427A1 (en) 2018-06-27
JP2018526025A (ja) 2018-09-13
EP3339427A4 (en) 2019-01-16
EP3339427B1 (en) 2024-04-17
CN105062956B (zh) 2018-02-09
CN105062956A (zh) 2015-11-18
JP6812435B2 (ja) 2021-01-13

Similar Documents

Publication Publication Date Title
LU500561B1 (en) In vitro construction method and use of liver organoids
WO2017032224A1 (zh) 一种嗅鞘细胞的制备方法
CN110551684B (zh) 一种人脐带间充质干细胞制备方法
US6875605B1 (en) Modular cell culture bioreactor and associated methods
CN104450611B (zh) 一种人羊膜间充质干细胞原代分离及培养方法
CN106754674A (zh) 从人胎盘羊膜制备羊膜间充质干细胞的方法及其应用
TW201827592A (zh) 從臍帶的羊膜分離間質幹細胞的方法、從臍帶的羊膜分離的間質幹細胞群以及用於從臍帶的羊膜分離間質幹細胞的細胞培養基
WO2008060037A1 (en) Method for the simultaneous primary-isolation and expansion of endothelial stem/progenitor cell and mesenchymal stem cell derived from mammal including human umbilical cord
JP2010538681A (ja) 人または動物胚から間葉系幹細胞を抽出及びその分泌物を抽出する方法
CN105950550A (zh) 一种间充质干细胞无血清培养基、细胞分离培养方法
CN104263699A (zh) 细胞移植用临床治疗级真皮多能干细胞规模化制备培养方法
CN106085952A (zh) 一种胎盘绒毛板间充质干细胞及其提取方法
CN106754664B (zh) 一种诱导骨骼肌肌源性干细胞成脂分化的培养基及其应用和成脂分化方法
RU2433172C2 (ru) Способ получения гомогенной популяции стволовых клеток и ее применение
CN106520676A (zh) 从人胎盘羊膜制备人羊膜上皮细胞的方法及其应用
CN110564681B (zh) 一种乳牙牙髓干细胞的分离培养和神经定向分化方法
CN114807034A (zh) 一种人多能干细胞来源的Müller细胞的制备方法
CN106661545B (zh) 制备诱导多能干细胞的方法及由其制备的诱导多能干细胞
CN104031881B (zh) 一种人类嗅黏膜间充质干细胞的分离培养方法
US10542743B2 (en) Isolation, expansion and characterization of wharton's jelly mesenchymal stem cells
CN112280733B (zh) 一种羊膜干细胞的提取方法
CN110484491B (zh) 羊膜和羊水来源的内皮祖细胞获取方法及其纯化培养方法
US20220098553A1 (en) Method for mesenchymal stem cell isolation and osteoblast differentiation
CN106367380A (zh) 可在体外制备肝芽的细胞共培养方法和肝芽
CN109666642A (zh) 一种树鼩大脑皮层少突胶质前体细胞体外分离纯化的方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16838484

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2018528369

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 15754171

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2016838484

Country of ref document: EP