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Mar 2018
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Neurostore: A Novel Cryopreserving Medium for Primary Neurons
Neurostore: 一种新型原代神经元冷冻保存介质   

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Abstract

Primary neuronal culture from rodents is a key tool in neurobiology. However, the preparation of primary cultures requires precise planning, starting from animal mating. Furthermore, each preparation generates a high amount of cells that eventually go wasted. The possibility to cryopreserve primary neural cells represents a resource for in vitro studies and significantly reduces the sacrifice of animals. Here we describe that Neurostore buffer supports the cryopreservation of primary neurons.

Keywords: Neurons (神经元), Cryopreservation (冷冻保存), Long-term storage (长期保存), Reproducibility (再生性), Specimen sharing (样品共享)

Background

Primary rodent cultures are a primary tool in any neurobiology laboratory. Primary cultures last 2-3 weeks. Therefore cells are normally prepared when necessary and employed in experiments immediately upon isolation. Cryopreservation is a routine procedure to allow long-term storage of mammalian cells. A protocol to achieve cryopreservation of neuronal cells would spare animals sacrifice and avoid the waste of cells. Furthermore, cell storage might permit the creation of cultures archive, support the reproducibility of experiments, and improve specimen sharing among different laboratories. Several studies have proposed a protocol to achieve the cryopreservation of neurons (Ichikawa et al., 2007; Paynter, 2008; Ma et al., 2010). Here we describe Neurostore, a novel buffer that promotes the long-term storage of primary mouse neurons and assures high viability upon thawing.

Materials and Reagents

  1. P1000 tips (VWR, catalog number: GREI740280_60)
  2. 15 ml Falcon tubes (EuroClone, catalog number: ET5050B )
  3. Petri dishes 100 mm (EuroClone, catalog number: ET2100)
  4. Cryovials (Corning, catalog number: 430659)
  5. E 15.5 mouse embryos (Charles River, C57BL/6 Mouse, Strain Code: 027)
  6. Fetal Bovine Serum EU Approved (FBS) (Euro Clone, catalog number: ECS0180L), store at -20 °C
  7. Trypsin 2.5% in HBSS (Euro Clone, catalog number: ECB3051D), store at -20 °C
  8. Gentamicin (Euro Clone, catalog number: ECM0011B0), store at 4 °C
  9. HBBS w/o Calcium & Magnesium (Euro Clone, catalog number: ECB4007L), store at 4 °C
  10. Magnesium sulfate (MgSO4) (Sigma-Aldrich, catalog number: M7506), store at RT
  11. HEPES (Sigma-Aldrich, catalog number: H3375), store at RT
  12. L-Glutamine 100x (200 mM) (Euro Clone, catalog number: ECB3000D-20), store at -20 °C
  13. NeurobasalTM Medium (Thermo Fisher Scientific, catalog number: 21103049), store at 4 °C
  14. Poly-D-lysine hydrobromide (Sigma-Aldrich, catalog number: P6407), store at -20 °C
  15. B-27TM Supplement (50x), serum-free (Thermo Fisher Scientific, catalog number: 17504044)
  16. Liquid nitrogen storage system (Midsci, catalog number: 38K-CS200)
  17. Ultrapure water system Milli-Q® Direct (Merck Millipore, catalog number: C85358)
  18. Dissection medium (see Recipes)
  19. Disruption medium (see Recipes)
  20. Neutralization medium (see Recipes)
  21. Neuronal complete medium (see Recipes)
  22. Neurostore medium (see Recipes)
  23. Poly-D-Lysine coating solution (see Recipes)

Equipment

  1. 10 ml pipettes (EuroClone, catalog number: EPS10N)
  2. Cell cooler (Thermo Fisher Scientific, catalog number: 5100-0001)
  3. Water bath (Thermo Fisher Scientific, catalog number: TSGP10)
  4. Carl ZeissTM StemiTM DV4 Series Stereomicroscope with LED Illumination (Zeiss, catalog number: 0000001036143)
  5. SL 8 Small Benchtop Centrifuge (Thermo Fisher Scientific, catalog number: 75007220)
  6. CO2 incubator (Thermo Fisher Scientific, catalog number: 51030992 )
  7. Automatic CO2 Delivery Unit (Vet-Tech.co.uk, catalog number: AN044)
  8. CO2 Chamber (Vet-Tech.co.uk, catalog number: AN045BR)
  9. Dissecting scissors, straight 112 mm (VWR, catalog number: 233-0011)
  10. Dissecting scissors, straight 150 mm (VWR, catalog number: 233-0013)
  11. Dissecting Forceps, Superfine Tip, Straight (VWR, catalog number: 82027-402)
  12. Forceps Straight, blunt 105 mm (VWR, catalog number: 232-2111)
  13. Bürker Counting chamber, 0.1 mm (VWR, catalog number: HECH40423001)

Procedure

  1. Neuronal cells preparation and freezing procedure
    1. Euthanize the pregnant mice with Carbon dioxide (CO2) overdose, remove uterus with dissection scissors and free individual fetuses from the embryonic sack. Place embryos into 10 ml sterile Dissection medium (see Recipe 1) in a 100 mm Petri dish and keep it on ice.
      Note: Clean the abdomen with ethanol 70% before cutting the skin with scissors to avoid embryo contamination.
    2. Decapitate mouse embryos with dissection scissors, remove skin and skull and place the brain in a sterile 100 mm petri dish containing 10 ml cold Dissection medium.
      Note: Remove skin and skull with sharp superfine tip dissecting forceps.
    3. Under a stereomicroscope discard the olfactory bulb, cerebellum, and midbrain from the mouse brain using the superfine tip forceps. Then remove meninges and blood vessels.
      Note: Be careful during the meninges removal procedure. Since the hippocampus is surrounded by meninges, you could inadvertently remove and discard it.
    4. Dissect out the hippocampus and the cortex from each hemisphere with the superfine tip forceps.
      Note: Hippocampus can be easily identified as a “banana” shaped tissue. You can also find images and the procedure to dissect hippocampus in Salazar et al., 2017.
    5. Collect the specimens in 12 ml cold Dissection medium in a 15 ml Falcon tube placed on ice.
      Note: Store the isolated specimens on ice during the whole procedure. Move the dissected tissues from the Petri dish to the 15 ml Falcon tube using the superfine forceps immediately after tissue isolation.
    6. At the end of the dissection procedure let the tissues settle at the bottom of the Falcon tube by gravity. Wash tissues three times each with 10 ml fresh Dissection medium previously warmed at 37 °C in a water bath.
      Notes:
      1. Add the dissection medium from the top of the Falcon and let the tissues settle at the bottom of the Falcon tube by gravity
      2. Remove as much medium as possible between each wash but keep the tissues covered by the medium during each step. Remove the medium gently using a 10 ml pipette. Avoid vacuum. 
    7. Discard the medium and then add 10 ml disruption medium (see Recipe 2) to the tissue specimen in a 15 ml Falcon tube. Warm at 37 °C for 15 min and mix by inverting the Falcon every 2 min.
    8. Let tissues settle at the bottom of the Falcon tube by gravity. Then discard the disruption medium and wash tissues with 10 ml Neutralization medium (see Recipe 3).
    9. Let tissues settle at the bottom of the Falcon tube by gravity. Then wash tissues three times each with 10 ml fresh Dissection medium previously warmed at 37 °C.
    10. Let tissues settle at the bottom of the Falcon tube by gravity. Discard the medium and then add to the tissue 5 ml Neuronal complete medium (see Recipe 4) previously warmed at 37 °C.
    11. Mechanically disrupt the tissue with a 10 ml pipette and resuspend the tissue 15-20 times or until almost tissue clumps are dissolved. 
    12. Spin the cells at 600 x g for 10 min at room temperature in a 15 ml Falcon bench centrifuge. 
    13. Discard all the medium with a 10 ml pipette and add 1 ml Neuronal complete medium. Resuspend cells 10-20 times with a P1000 pipette.
      Note: Avoid cell clumps formation! If some tissue debris remains, let it settle at the bottom of the Falcon by gravity. Then collect the medium that contains all dissociated cells and transfer it to a new 15 ml Falcon tube. Discard the old 15 ml tube with the tissue debris.
    14. Add 4 ml Neuronal complete medium.
    15. Count neuronal cells. Average yield is about 8 x 106 cell/cortices, 2.5 x 105 cells/hippocampi obtained from E15.5 mice embryos. For cell counting dilute a small amount of cell suspension (20 μl) 1:4 in Neuronal complete medium and count cells in a Bürker Counting chamber.
      Note: Diluting cells facilitate cell counting. Include the round shaped and bright cells and exclude irregular shaped and dark cells in the counting. Upon counting, remember to apply the dilution factor.
    16. Warm Neurostore medium (see Recipe 5) and cell cooler at 37 °C in a water bath.
    17. Upon counting, spin cells at 600 x g for 10 min.
    18. Discard all the Neuronal complete medium with a 10 ml pipette.
    19. Gently resuspend the cell pellet in 1 ml of warm Neurostore medium with a P1000 pipette (about ten times). Once all cell clumps are removed, add warm Neurostore medium to reach the desired cellular concentration.
      Note: Suggested concentration is 5 x 106 cells/ml Neurostore medium. 
    20. Store 1 ml of Neurostore medium containing neurons in each cryovial.
      Note: Aliquot cells with a P1000 tip, 5 x 106 cells/cryovial.
    21. Move cell cooler to -80 °C immediately. For long-term storage (above one month and up to one year) store cells in a liquid nitrogen tank.

  2. Thawing procedure
    1. Warm bath, Dissection medium, and neuronal complete culture medium at 37 °C.
    2. Put 9 ml warm Dissection medium in a 15 ml Falcon tube.
    3. Move cryovials to the 37 °C bath. Usually, complete thawing takes 2-3 min.
    4. Add 1 ml Neurostore containing cells to the 15 ml Falcon tube with warm Dissection medium using a P1000 pipette.
    5. Pellet cells at 600 x g for 10 min.
    6. Discard the medium.
    7. Gently resuspend cells in 1 ml warm Neuronal complete culture medium with a P1000 pipette (about ten times).
      Note: Resuspend until cell clumps disappear.
    8. Add 4 ml warm neuronal complete culture medium.
    9. Count the cells (as reported in Procedure A–Neuronal cells preparation and freezing procedure, Step A15).
      Note: Be fast in cell counting to reduce cellular stress. We suggest evaluating cellular viability by Trypan Blue staining.
    10. Seed the desired number of cells on proper culture support in Neuronal complete medium.
      Notes:
      1. We suggest coating culture support with Poly-D-Lysine coating solution (see Recipe 6). Incubate o/n at room temperature or 2 h at 37 °C followed by 3 x ultra-pure water washes.
        For 12-well plates, we suggest adding a volume of 500 μl/ well of Poly-D-Lysine coating solution. We suggest scaling up or down the volume of the poly-D-Lysine coating according to the diameter of the plate wells.
      2. We suggest seeding 750-1,000 cells/mm2. Mix the proper amount of cells and Neuron complete medium to reach the desired number of cell. Add the correct volume of the mix into each well.

Data analysis

Note: We refer to results and figures reported in our original publication (Pischedda et al., 2018. doi: 10.3389/fncel.2018.00081).

  1. To verify the possibility to cryopreserve cortical neurons, we characterized E15.5 mouse cortical cells upon freezing and thawing. We thawed and seeded cultures after one week at -80 °C. After 14-days in culture, we noticed that cultures frozen in Neurostore generated a complex network of processes (Figure 1A).
  2. Using MTT assay, we noticed comparable viability among fresh and cultures stored in Neurostore at -80 °C for 2 or 4 weeks (Figure 1B). Physiologically, neuronal cultures respond to pharmacological stimulation by triggering intracellular pathways. Cryopreserved cultures and acutely dissociated ones answered comparably in terms of AKT (Protein Kinase B) or ERK1/2 (Extracellular signal-regulated kinases 1/2) phosphorylation upon FGFb (Basic fibroblast growth factor) or EGF (Epidermal growth factor) stimulation (Figures 1F-1H). Cryopreservation procedure may induce oxidative stress. However, ROS (Reactive oxygen species) production resulted similar between fresh and cryopreserved cultures (Figures 2C and 2F).
  3. Next, we infected cell with GFP expressing virus and analyzed the morphology of the culture at DIV14 (Figure 3A). The analysis of neurite number, total length, and average length did not reveal any significant difference among acutely dissociated and cryopreserved cultures (Figures 3B-3D).
  4. To appreciate synaptic architecture, we stained acutely dissociated and 2- and 4-week cryopreserved cultures for synapsin and PSD-95 at DIV14 (Figure 4A). We could not detect any major differences among the experimental groups under analysis (Figures 4B-4D). To complement this analysis, we investigated by Western-blotting the expression of pre- and postsynaptic proteins in acutely dissociated and 2- and 4-week cryopreserved cortical cultures at DIV14 (Figures 5A-5C).
  5. Lastly, we analyzed the electrical features of acutely dissociated and 2- and 4-week cryopreserved cortical cultures by whole-cell patch clamp recording. The quantification of all parameters demonstrated no significant differences between frozen cultures and fresh neurons (Figure 6 and Table 2).

Notes

Neuronal mortality in Neurostore medium is estimated in 20%-30% for both cortical and hippocampal neurons if cells are stored at the concentration of 5 x 106 cells/ml Neurostore medium. It is extremely important to count cells after thawing to reduce variability between different neuronal preparations.

Recipes

  1. Dissection medium
    HBSS 1x
    6 mM MgSO4
    10 mM HEPES, pH 7.4
    10 μg/ml gentamicin
  2. Disruption medium
    HBSS 1x
    6 mM MgSO4
    10 mM HEPES pH 7.4
    10 μg/ml gentamicin
    0.25% Trypsin
  3. Neutralization medium
    HBSS 1x
    6 mM MgSO4
    10 mM HEPES, pH 7.4
    10 μg/ml gentamicin
    10% FBS
  4. Neuronal complete medium
    Neurobasal 1x
    B-27 supplement 1x
    0.5 mM L-glutamine
    10 μg/ml gentamicin
  5. Neurostore medium (under patent consideration, available upon request. Please contact Prof. Giovanni Piccoli giovanni.piccoli@unitn.it)
  6. Poly-D-Lysine coating solution
    Resuspended poly-D-Lysine powder in ultra-pure water at final concentration 50 μg/ml

Acknowledgments

Funding source: GP is a Dulbecco Telethon Scientist (Grant No. TDPG00514TA). The procedure was adapted from Pischedda et al., 2018.

Competing interests

Neurostore recipe is under patent consideration.

Ethics

All procedures involving animals were approved by Body for the Protection of Animals at the University of Trento and National Agency (autorizzazione 793/2016-PR).

References

  1. Ichikawa, J., Yamada, R. X., Muramatsu, R., Ikegaya, Y., Matsuki, N. and Koyama, R. (2007). Cryopreservation of granule cells from the postnatal rat hippocampus. J Pharmacol Sci 104(4): 387-391.
  2. Ma, X. H., Shi, Y., Hou, Y., Liu, Y., Zhang, L., Fan, W. X., Ge, D., Liu, T. Q. and Cui, Z. F. (2010). Slow-freezing cryopreservation of neural stem cell spheres with different diameters. Cryobiology 60(2): 184-191.
  3. Paynter, S. J. (2008). Principles and practical issues for cryopreservation of nerve cells. Brain Res Bull 75(1): 1-14.
  4. Pischedda, F., Montani, C., Obergasteiger, J., Frapporti, G., Corti, C., Rosato Siri, M., Volta, M. and Piccoli, G. (2018). Cryopreservation of primary mouse neurons: The benefit of neurostore cryoprotective medium. Front Cell Neurosci 12: 81.
  5. Salazar, I. L., Mele, M., Caldeira, M., Costa, R. O., Correia, B., Frisari, S. and Duarte, C. B. (2017). Preparation of primary cultures of embryonic rat hippocampal and cerebrocortical neurons. Bio-protocol 7(18): e2551.

简介

来自啮齿动物的原代神经元培养是神经生物学的关键工具。 然而,从动物交配开始,原代培养物的制备需要精确的计划。 此外,每种制剂都会产生大量细胞,最终会被浪费掉。 冷冻保存原代神经细胞的可能性代表了体外研究的资源,并显着减少了动物的牺牲。 在这里我们描述Neurostore缓冲液支持原代神经元的冷冻保存。
【背景】初级啮齿动物培养是任何神经生物学实验室的主要工具。 原代培养持续2-3周。 因此,通常在必要时制备细胞,并在分离后立即用于实验。 冷冻保存是允许长期储存哺乳动物细胞的常规程序。 实现神经细胞冷冻保存的方案将使动物牺牲并避免细胞的浪费。 此外,细胞储存可能允许创建文化档案,支持实验的可重复性,并改善不同实验室之间的标本共享。 一些研究已经提出了实现神经元冷冻保存的方案(Ichikawa et al。,2007; Paynter,2008; Ma et al。,2010)。 在这里,我们描述Neurostore,一种新型缓冲液,可促进原代小鼠神经元的长期储存,并确保解冻后的高活力。

关键字:神经元, 冷冻保存, 长期保存, 再生性, 样品共享

材料和试剂

  1. P1000提示(VWR,目录号:GREI740280_60)
  2. 15毫升猎鹰管(EuroClone,目录号:ET5050B)
  3. 培养皿100毫米(EuroClone,目录号:ET2100)
  4. Cryovials(康宁,目录号:430659)
  5. E 15.5小鼠胚胎(Charles River,C57BL / 6 Mouse,Strain Code:027)
  6. 胎牛血清欧盟批准(FBS)(欧洲克隆,目录号:ECS0180L),储存于-20°C
  7. 胰蛋白酶2.5%在HBSS(Euro Clone,目录号:ECB3051D)中,储存在-20°C
  8. 庆大霉素(Euro Clone,目录号:ECM0011B0),储存在4°C
  9. HBBS没有钙和钙镁(Euro Clone,目录号:ECB4007L),储存在4°C
  10. 硫酸镁(MgSO 4 )(Sigma-Aldrich,目录号:M7506),在室温下储存
  11. HEPES(Sigma-Aldrich,目录号:H3375),在RT存储
  12. L-谷氨酰胺100x(200 mM)(Euro Clone,目录号:ECB3000D-20),储存于-20°C
  13. Neurobasal TM 培养基(Thermo Fisher Scientific,目录号:21103049),储存于4°C
  14. 聚-D-赖氨酸氢溴酸盐(Sigma-Aldrich,目录号:P6407),储存于-20°C
  15. B-27 TM 补充剂(50x),无血清(Thermo Fisher Scientific,目录号:17504044)
  16. 液氮储存系统(Midsci,目录号:38K-CS200)
  17. 超纯水系统Milli-Q ® Direct(Merck Millipore,目录号:C85358)
  18. 解剖介质(见食谱)
  19. 破坏媒介(见食谱)
  20. 中和介质(见食谱)
  21. 神经元完全培养基(见食谱)
  22. Neurostore培养基(见食谱)
  23. 聚D-赖氨酸涂料溶液(见食谱)

设备

  1. 10毫升移液器(EuroClone,目录号:EPS10N)
  2. 电池冷却器(Thermo Fisher Scientific,目录号:5100-0001)
  3. 水浴(Thermo Fisher Scientific,目录号:TSGP10)
  4. Carl Zeiss TM Stemi TM 带LED照明的DV4系列立体显微镜(蔡司,产品目录号:0000001036143)
  5. SL 8小型台式离心机(赛默飞世尔科技,目录号:75007220)
  6. CO 2 培养箱(Thermo Fisher Scientific,目录号:51030992)
  7. 自动CO 2 交付单位(Vet-Tech.co.uk,目录号:AN044)
  8. CO 2 商会(Vet-Tech.co.uk,目录号:AN045BR)
  9. 直剪112 mm(VWR,目录号:233-0011)
  10. 直剪式剪刀150 mm(VWR,目录号:233-0013)
  11. 解剖钳,超细尖,直(VWR,目录号:82027-402)
  12. 镊子直,钝105毫米(VWR,目录号:232-2111)
  13. Bürker计数室,0.1 mm(VWR,目录号:HECH40423001)

程序

  1. 神经细胞制备和冷冻程序
    1. 用过量二氧化碳(CO 2 )对怀孕小鼠实施安乐死,用解剖剪刀取出子宫,从胚胎袋中取出单个胎儿。将胚胎放入100毫升培养皿中的10毫升无菌解剖培养基(见配方1)中,并将其置于冰上。
      注意:用70%的乙醇清洁腹部,然后用剪刀剪去皮肤,以免污染胚胎。
    2. 用解剖剪刀剔除小鼠胚胎,去除皮肤和头骨,将大脑置于含有10毫升冷解剖培养基的无菌100毫米培养皿中。
      注意:使用尖锐的超细尖端解剖钳去除皮肤和颅骨。
    3. 在立体显微镜下,使用超细尖镊子从小鼠脑中丢弃嗅球,小脑和中脑。然后去除脑膜和血管。
      注意:在脑膜切除过程中要小心。由于海马被脑膜包围,你可能会无意中移除并丢弃它。
    4. 使用超细尖镊子解剖每个半球的海马和皮质。
      注意:海马可以很容易地识别为“香蕉”形状的组织。您还可以在Salazar中找到图像和解剖海马的程序 等。 ,2017。
    5. 将标本收集在装有冰的15毫升Falcon管中的12毫升冷处理培养基中。
      注意:在整个过程中将隔离的标本存放在冰上。在组织分离后立即使用超细镊子将切开的组织从培养皿移至15 ml Falcon管。
    6. 在解剖过程结束时,让组织通过重力沉降在Falcon管的底部。用10ml新鲜的解剖培养基洗涤组织各3次,所述新鲜的解剖培养基预先在37℃在水浴中加热。
      注意:
      1. 从猎鹰顶部加入解剖培养基,让组织通过重力沉淀在猎鹰管的底部
      2. 在每次洗涤之间尽可能多地去除培养基,但在每个步骤中保持组织被培养基覆盖。使用10 ml移液器轻轻取出培养基。避免吸尘。 
    7. 丢弃培养基,然后在15 ml Falcon试管中向组织标本中加入10 ml破碎培养基(参见配方2)。在37°C温暖15分钟,每隔2分钟倒入Falcon进行混合。
    8. 让组织通过重力沉淀在Falcon管的底部。然后丢弃破碎培养基并用10ml中和培养基清洗组织(参见方法3)。
    9. 让组织通过重力沉淀在Falcon管的底部。然后用预先在37℃加热的10ml新鲜解剖培养基洗涤组织三次。
    10. 让组织通过重力沉淀在Falcon管的底部。丢弃培养基,然后向组织中加入5ml神经元完全培养基(见配方4),预先在37℃加热。
    11. 用10ml移液管机械破坏组织,并将组织重悬15-20次或直至几乎组织块溶解。 
    12. 在室温下,在15 ml Falcon台式离心机中以600 x g 旋转细胞10分钟。 
    13. 用10ml移液管丢弃所有培养基并加入1ml Neuronal完全培养基。用P1000移液管重悬细胞10-20次。
      注意:避免形成细胞团块!如果残留一些组织碎片,让它通过重力沉淀在猎鹰的底部。然后收集含有所有解离细胞的培养基并将其转移到新的15ml Falcon管中。丢弃旧的15毫升管与组织碎片。
    14. 加入4ml Neuronal完全培养基。
    15. 计数神经细胞。平均产量是从E15.5小鼠胚胎获得的约8×10 6个细胞/皮质,2.5×10 5个细胞/海马体。细胞计数在Neuronal完全培养基中稀释少量细胞悬液(20μl)1:4,并在Bürker计数室中计数细胞。
      注意:稀释细胞有助于细胞计数。包括圆形和明亮的细胞,并在计数中排除不规则形状和暗的细胞。计算后,请记住应用稀释因子。
    16. 温热的Neurostore培养基(参见配方5)和37℃的细胞冷却器在水浴中。
    17. 计数后,将细胞在600 x g 下旋转10分钟。
    18. 用10毫升移液管丢弃所有神经元完全培养基。
    19. 用P1000移液管轻轻地将细胞沉淀重悬于1ml温热的Neurostore培养基中(约10次)。一旦除去所有细胞团块,加入温热的Neurostore培养基以达到所需的细胞浓度。
      注意:建议浓度为5 x 106 cells / ml Neurostore培养基。 
    20. 在每个冷冻管中储存1毫升含有神经元的Neurostore培养基。
      注意:用P1000尖端等分细胞,5×106个细胞/冷冻。
    21. 立即将电池冷却器移至-80°C。长期储存(一个月以上,一年以上)将液体储存在液氮罐中。

  2. 解冻程序
    1. 温浴,解剖培养基和神经元完全培养基在37°C。
    2. 将9毫升温热的解剖介质放入15毫升Falcon管中。
    3. 将冷冻管移至37°C浴槽。通常,完全解冻需要2-3分钟。
    4. 使用P1000移液管将含有细胞的1ml含有神经细胞的细胞加入15ml Falcon管中,使用温热的Dissection培养基。
    5. 在600 x g 下沉淀细胞10分钟。
    6. 丢弃媒体。
    7. 用P1000移液器轻轻地将细胞重悬于1 ml温热神经元完全培养基中(约10次)。
      注意:重新悬浮直到细胞团块消失。
    8. 加入4ml温热神经元完全培养基。
    9. 计数细胞(如程序A-神经细胞制备和冷冻程序,步骤A15中所述)。
      注意:快速进行细胞计数以减少细胞压力。我们建议通过台盼蓝染色评估细胞活力。
    10. 在Neuronal完全培养基中在适当的培养支持物上培养所需数量的细胞。
      注意:
      1. 我们建议使用Poly-D-Lysine涂层溶液进行涂层培养支持(参见配方6)。在室温下孵育o / n或在37°C孵育2小时,然后进行3次超纯水洗涤。
        对于12孔板,我们建议添加500μl/孔的Poly-D-Lysine涂层溶液。我们建议根据板孔的直径放大或缩小聚D-赖氨酸涂层的体积。
      2. 我们建议接种750-1,000个细胞/ mm 2 。混合适量的细胞和Neuron完全培养基以达到所需数量的细胞。在每个孔中加入正确体积的混合物。

数据分析

注意:我们参考原始出版物中报告的结果和数据(Pischedda 等。 ,2018。doi:10.3389 / fncel.2018.00081)。

  1. 为了验证冷冻保存皮层神经元的可能性,我们在冷冻和解冻时表征了E15.5小鼠皮质细胞。我们在-80℃下一周后解冻并接种培养物。在培养14天后,我们注意到在Neurostore中冷冻的培养物产生了复杂的过程网络(图1A)。
  2. 使用MTT测定,我们注意到在-80℃下存储在Neurostore中的新鲜培养物和培养物之间相当的生存力2周或4周(图1B)。在生理学上,神经元培养物通过触发细胞内途径来响应药理学刺激。冷冻保存的培养物和急性解离的培养物在AKT(蛋白激酶B)或ERK1 / 2(细胞外信号调节激酶1/2)磷酸化对FGFb(碱性成纤维细胞生长因子)或EGF(表皮生长因子)刺激方面的回答相当(图1F-1H)。冷冻保存程序可能会诱发氧化应激。然而,ROS(活性氧物质)产生在新鲜和冷冻保存的培养物之间产生类似的结果(图2C和2F)。
  3. 接下来,我们用表达GFP的病毒感染细胞并在DIV14分析培养物的形态(图3A)。神经突数,总长度和平均长度的分析未显示急性解离和冷冻保存的培养物之间的任何显着差异(图3B-3D)。
  4. 为了理解突触结构,我们在DIV14染色急性解离的2周和4周冷冻保存的突触蛋白和PSD-95培养物(图4A)。我们无法检测到所分析的实验组之间的任何主要差异(图4B-4D)。为了补充该分析,我们通过Western-blotting在DIV14的急性解离的和2周和4周的冷冻保存的皮质培养物中的突触前和突触后蛋白的表达进行了研究(图5A-5C)。
  5. 最后,我们通过全细胞膜片钳记录分析了急性解离的和2周和4周冷冻保存的皮质培养物的电学特征。所有参数的定量表明冷冻培养物和新鲜神经元之间没有显着差异(图6和表2)。

笔记

如果细胞以5×10 6 细胞/ ml Neurostore培养基的浓度储存,则对于皮质和海马神经元,Neurostore培养基中的神经元死亡率估计为20%-30%。解冻后计数细胞以减少不同神经元制剂之间的变异性是非常重要的。

食谱

  1. 解剖媒体
    HBSS 1x
    6mM MgSO 4
    10 mM HEPES,pH 7.4
    10μg/ ml庆大霉素
  2. 中断媒介
    HBSS 1x
    6mM MgSO 4
    10 mM HEPES pH 7.4
    10μg/ ml庆大霉素
    0.25%胰蛋白酶
  3. 中和介质
    HBSS 1x
    6mM MgSO 4
    10 mM HEPES,pH 7.4
    10μg/ ml庆大霉素
    10%FBS
  4. 神经元完全中等
    Neurobasal 1x
    B-27补充1x
    0.5mM L-谷氨酰胺
    10μg/ ml庆大霉素
  5. Neurostore培养基(根据专利考虑,可根据要求提供。请联系Giovanni Piccoli教授 giovanni.piccoli@unitn.it )
  6. 聚D-赖氨酸涂料溶液
    重悬的聚-D-赖氨酸粉末在超纯水中,终浓度为50μg/ ml

致谢

资金来源:GP是Dulbecco Telethon Scientist(Grant No. TDPG00514TA)。该程序改编自Pischedda 等人,,2018。

利益争夺

Neurostore配方正在申请专利。

伦理

涉及动物的所有程序均由特伦托大学和国家机构(autorizzazione 793/2016-PR)的动物保护机构批准。

参考

  1. Ichikawa,J.,Yamada,R.X.,Muramatsu,R.,Ikegaya,Y.,Matsuki,N。和Koyama,R。(2007)。 从出生后大鼠海马中冷冻保存颗粒细胞。 J Pharmacol Sci 104(4):387-391。
  2. Ma,X.H.,Shi,Y.,Hou,Y.,Liu,Y.,Zhang,L.,Fan,W.X.,Ge,D.,Liu,T.Q。和Cui,Z.F。(2010)。 慢速冷冻保存不同直径的神经干细胞球。 低温生物学 60(2):184-191。
  3. Paynter,S.J。(2008)。 冷冻保存神经细胞的原则和实际问题。 Brain Res Bull 75(1):1-14。
  4. Pischedda,F.,Montani,C.,Obergasteiger,J.,Frapporti,G.,Corti,C.,Rosato Siri,M.,Volta,M。和Piccoli,G。(2018)。 原代小鼠神经元的低温保存:神经外膜冷冻保护介质的益处。 前Cell Neurosci 12:81。
  5. Salazar,I.L。,Mele,M.,Caldeira,M.,Costa,R.O.,Correia,B.,Frisari,S。和Duarte,C.B。(2017)。 制备胚胎大鼠海马和脑皮层神经元的原代培养物。 生物协议 7(18):e2551。
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引用:Pischedda, F. and Piccoli, G. (2019). Neurostore: A Novel Cryopreserving Medium for Primary Neurons. Bio-protocol 9(12): e3270. DOI: 10.21769/BioProtoc.3270.
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