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Preparation of Primary Cultures of Embryonic Rat Hippocampal and Cerebrocortical Neurons
胚胎大鼠海马和脑皮质神经元原代培养物的制备   

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本实验方案简略版
Molecular Neurobiology
Jan 2017

Abstract

This protocol aims at standardizing the procedure to obtain primary cultures of hippocampal and cerebrocortical neurons for in vitro experiments. Cultures should be prepared from cells isolated during embryonic development when neuronal precursor cells are not yet fully differentiated. This helps increasing the quality and quantity of cells, while offering minimal cell death that often occurs during dissociation of differentiated neurons. Cells plated under the appropriate conditions, either in Petri-dishes or in multi-well plates, will develop and establish synaptic contacts over time since the neuronal culture medium provides the nutrients and trophic factors required for differentiation. In this protocol we describe the methodology for the preparation of both cortical and hippocampal neuronal cultures.

Keywords: Primary cultures (原代培养), Hippocampal neurons (海马神经元), Cerebrocortical neurons (脑皮质神经元), Cell culture (细胞培养)

Background

The present protocol describes the preparation of primary cultures of rat hippocampal and cerebrocortical neurons, using Neurobasal medium supplemented with NeuroCultTM SM1 (Chen et al., 2008). The composition of NeuroCultTM SM1 is based on the formulation of the B27 supplement (Brewer et al., 1993), but the former cocktail was found to improve the quality of neuronal cultures, in part by replacement of apo-transferrin with holo-transferrin (Chen et al., 2008). Furthermore, the chemical composition of NeuroCultTM SM1 was described in more detail in the original publication, allowing a better control of the experimental conditions. Neuronal cultures prepared with chemically defined culture media are characterized by the presence of a low percentage of astrocytes. The proliferation of astrocytes in cultures maintained for longer periods of time, in order to allow differentiation of neurons, is prevented by adding the chemical inhibitor of mitosis 5-Fluoro-2’-deoxyuridine.

Materials and Reagents

  1. Petri dish, 55 mm Polysterene aseptic non-tissue culture treated (Labbox, catalog number: PDIP-06N-500 )
  2. Petri dish, 150 mm glass soda-lime (DWK Life Sciences, Duran, catalog number: 23 755 52 )
  3. 15 ml conical tube (Corning, Falcon®, catalog number: 352096 )
  4. 5 ml glass pipetes (VWR, catalog number: 612-4124 )
  5. 10 ml glass pipetes (VWR, catalog number: 612-4125 )
  6. 50 ml conical tube (Corning, Falcon®, catalog number: 352070 )
  7. Cell strainer 70 μm (Corning, Falcon®, catalog number: 352350 )
  8. Poly-D-lysine-coated multi-well plate (see step 16 in ‘Procedures’ for instructions)
  9. Coverslips #1.5 (e.g., Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 1014355110NR15 ; for 10 mm coverslips)
  10. Mixed cellulose ester filter, ME Range (ME 24), 0.2 μm pore size for filtration unit (GE Healthcare, Whatman, catalog number: 10406970 )
  11. Acetate cellulose filters of Ø25 mm, 0.20 μm, sterile (FRILABO, catalog number: 1520012 )
  12. Filtration unit (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: DS0320-5033 )
  13. Stericup-GV, 0.20 µm, PVDF, 500/1,000 ml, radio-sterilized (Merck, catalog number: SCGVU10RE )
  14. Pregnant female Wistar rats (E17-E18 days gestation)
  15. Trypan blue solution, 0.4% (Thermo Fisher Scientific, GibcoTM, catalog number: 15250061 )
  16. Poly-D-lysine hydrobomide (Sigma-Aldrich, catalog number: P7886 )
  17. Boric acid (Merck, catalog number: 1001651000 )
  18. Nitric acid (Applichem, catalog number: 133255.1612 )
  19. Ethanol absolute 99.8+% (Fisher Scientific, catalog number: 10342652 )
  20. 5-Fluoro-2’-deoxyuridine (5-FDU) (Sigma-Aldrich, catalog number: F0503 )
  21. Potassium chloride (KCl) (AppliChem, catalog number: 131494.1211 )
  22. Potassium phosphate dibasic (K2HPO4) (Merck, catalog number: 1051041000 )
  23. Sodium chloride (NaCl) (Applichem, catalog number: 131659.1211 )
  24. Sodium bicarbonate (NaHCO3) (Acros Organics, catalog number: 123360010 )
  25. Sodium phosphate dibasic dihydrate (Na2HPO4·2H2O) (Merck, catalog number: 1065800500 )
  26. D(+)-Glucose monohydrate (VWR, catalog number: 24371.297 )
  27. Sodium pyruvate (Sigma-Aldrich, catalog number: P5280 )
  28. HEPES (Fisher Scientific, catalog number: BP310-1 )
  29. Phenol red (Sigma-Aldrich, catalog number: P4758 )
  30. Trypsin (Thermo Fisher Scientific, GibcoTM, catalog number: 27250018 )
  31. Fetal bovine serum (FBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10270106 )
  32. Minimum essential medium Eagle (MEM) (Sigma-Aldrich, catalog number: M0268 )
  33. Neurobasal Medium® (Thermo Fisher Scientific, GibcoTM, catalog number: 21103049 )
  34. NeuroCultTM SM1 Neuronal Supplement (STEMCELL Technologies, catalog number: 05711 )
  35. L-Glutamine (Sigma-Aldrich, catalog number: G8540 ; Thermo Fisher Scientific, GibcoTM, catalog number: 25030024 )
  36. Glutamate (Sigma-Aldrich, catalog number: G1626 )
  37. Horse serum (Thermo Fisher Scientific, GibcoTM, catalog number: 16050122 )
  38. Sodium hydroxide solution
  39. Hanks’ balanced salt solution (HBSS) (see Recipes)
  40. Trypsin solution (2 mg/ml) (see Recipes)
  41. 10% FBS (see Recipes)
  42. Neuronal Plating Medium (see Recipes)
  43. Supplemented neuronal culture medium (see Recipes)
  44. Boric acid solutions (see Recipes)

Equipment

  1. Large and small scissors
  2. Forceps with straight tip (Fine Science Tools, model: Dumont #5 )
  3. Forceps with curved tip (Fine Science Tools, model: Dumont #5 /45 forceps–Dumont standard tip)
  4. Pipetboy (Integra Biosciences, model: PIPETBOY acu 2 )
  5. Magnification glass
  6. Laminar flow hood
  7. Water-bath (Medingen Labortechnik, model: T100 )
  8. Phase contrast inverted microscope equipped with a 10x objective (Nikon Instruments, model: Eclipse TS100 )
  9. Hemocytometer (Marienfeld-Superior, catalog number: 0640010 )
  10. Humidified incubator

Procedure

  1. Remove E18 embryos (to prepare cultures of hippocampal neurons) from a pregnant Wistar rat, anesthetized and sacrificed as previously described, by cesarean-section (Caldeira et al., 2013; Mele et al., 2014; Curcio et al., 2015; Mele et al., 2017).
    Note: To culture cerebrocortical neurons, it is recommended to use E17 embryos. The same procedure also applies.
  2. Transfer the embryos to a 150 mm glass Petri dish filled with ice-cold HBSS (150 ml should be enough; see Recipes).
  3. Sacrifice the embryos by decapitation with scissors and remove the brains (Figure 1). Separate the brain hemispheres and remove the cerebellum with forceps (and other non-essential parts; Figure 2). Carefully remove the meninges under a dissecting microscope using both curved and straight forceps (Video 1).


    Figure 1. Brain of an E18 rat embryo observed at different magnification levels


    Figure 2. Cerebral cortex from the brain of an E18 rat embryo with the hippocampus still enclosed. The meninges can still be observed at the surface of the cerebral cortex.

    Video 1. Initial steps in the dissection of the rat hippocampal

  4. Dissect and isolate the hippocampi and transfer the tissue to a 55 mm plastic Petri dish filled with HBSS (5-7 ml should be enough; see Figure 3 for end result). The use of a dissecting microscope will facilitate this procedure.
    Notes:
    1. We recommend to use the hippocampi from all the embryos obtained from the pregnant rat since the cell yield is low.
    2. To prepare cerebrocortical neurons, dissect the two cortices (see Figure 3 for end result).


    Figure 3. Dissected cerebral cortex (left side) and the isolated hippocampus (right side)

  5. Take the Petri dish with all the hippocampi obtained from the pregnant rat to a laminar flow hood for sterile conditions.
  6. Collect all intact hippocampi (or cortices) by pipetting them to a 15 ml conical tube and bring the volume of HBSS to about 6 ml.
    Notes:
    1. Use a glass pipette of 10 ml. This type of pipette has a wider tip that avoids damaging the tissue.
    2. For cerebrocortical neurons, wash the tissue three times with HBSS (5-8 ml is enough) by allowing the tissue to deposit by gravity; the obtained final volume after the second washing step should be as mentioned in step 6.
    3. Tip: Use four hemispheres for each 15 ml conical tube for preparation of cerebrocortical neuron cultures.
  7. Add 1.5 ml of the trypsin solution (see Recipes) to the medium containing the hippocampi and incubate for 15 min at 37 °C. At the end of the incubation period the tissue should be sedimented.
    Notes:
    1. A water-bath system can be used for this incubation.
    2. For cerebrocortical neurons, this process should last 10 min.
  8. Allow the tissue to deposit, and carefully remove the supernatant in the laminar flow hood.
  9. Add 8 ml of previously warmed (37 °C) 10% FBS solution (see Recipes) to the sedimented hippocampi and agitate gently.
  10. Allow the tissue to sediment and wash out the 10% FBS solution by removing the supernatant and rinsing the hippocampi with 10 ml of previously warmed (37 °C) HBSS.
  11. Remove the supernatant and add 5 ml of previously warmed (37 °C) supplemented neuronal culture medium (see Recipes).
    To prepare cultures of cerebrocortical neurons, steps 10-12 must be substituted by washing the tissue five times with ice-cold HBSS (6-9 ml) and by bringing the volume to about 4 ml of HBSS in the last washing step.
    Note: This volume was found to be optimal for having a final concentration of cells of about 3-5 x 106/ml of solution.
  12. Use mechanical force provided by the Pipetboy to dissociate the tissue with a sterile 5 ml glass pipette, pipetting up and down the hippocampi suspension.
    Notes:
    1. After dissociation of the cells, the suspension becomes cloudy (see Figure 4).
    2. The mechanical dissociation to prepare cultures of hippocampal neurons is performed in supplemented neuronal culture medium, whereas HBSS should be used in the preparation of cerebrocortical neurons.
    3. Tip: The tip of the 5 ml glass pipette should be in close proximity with the bottom of the 15 ml conical tube, keeping only a narrow space to allow the passage of the solution. When the suspension is pipetted up and down, the medium will become cloudy as a result of the cell dissociation.
    4. Since this procedure is rather aggressive to the cells it should not be repeated more than 10 times.


    Figure 4. Cell suspension after dissociation of the cerebrocortical tissue (right). The tube on the left contains the non-dissociated cortical tissue. Similar results are obtained after dissociation of hippocampal cells (not shown).

  13. Collect the cell suspension solution and filter to a sterile 50 ml conical tube through a cell strainer of 70 μm.
    Note: This step allows separating the cells in suspension from the small aggregates of non-dissociated tissue that are retained.
  14. To count the number of cells, dilute equal volumes of the following solutions: HBSS, cell suspension, trypan blue (50 μl of each should be enough).
  15. Add 10 μl of the cell suspension prepared in step 14 to a hemocytometer. Using an inverted phase contrast microscope equipped with a 10x objective, count the cells present in each of the four quadrants located at the corners (area = 0.01 cm2; volume 0.0001 cm3).
    Note: The number of cells present in each ml of solution can be calculated using the following formula: average number of cells/square x 3 (dilution factor in step 14) x 104 = number of cells/ml of solution.
  16. Plate the cells in a poly-D-lysine-coated multi-well plate at the appropriate density.
    1. To prepare the poly-D-Lysine solution, first dilute the 100 mg vial in 100 ml of boric acid (0.1 M, pH 8.2). Aliquots of this solution should be stored at -20 °C until further use. The resulting solution (1 mg/ml) should be further diluted in boric acid (166.3 mM, pH 8.2) and filtered immediately before coating the plates, to obtain a final concentration of 0.1 mg/ml. To coat the plastic dishes, add enough solution to cover the bottom of the wells and incubate for 2 h at 37 °C (overnight incubation in a humidified incubator is recommended). After this coating period, wash each well twice with enough volume of sterile distilled water to cover the bottom of the well, and allow the wells to dry inside the laminar flow hood.
    2. The protocol for coverslip coating is as follows: 1) wash the coverslips in nitric acid overnight with constant agitation, in a closed glass recipient (we use an 11 x 9 x 7 cm; 0.5 cm thick container); 2) after removing carefully nitric acid, with the help of a plastic pipet, wash abundantly with distilled water (5 washes, 20-30 min each); 3) wash the coverslips for 5 min with absolute ethanol; 4) dry the coverslips at 160 °C for 1 h (in a closed glass container); 5) sterilize the coverslips by UV radiation inside a flow chamber, in a glass Petri-dish for about 30 min. To coat the coverslips, transfer them to the appropriate multi-well plates, and follow the instructions mentioned in step 16a.
      Notes:
      1. Tip 1: For a glass container with 11 x 9 x 7 cm we wash a maximum of 100 coverslips. The coverslips may stick onto each other if a higher number is used.
      2. Tip 2: The poly-D-lysine solution used to coat the wells can be recycled once if used in consecutive days.
      3. Tip 3: in a 11 x 9 x 7 cm, 0.5 cm thick glass container we use 50 ml of water/absolute ethanol.
      4. Hippocampal neurons are plated at a density of 90.0 x 103 cells/cm2 whereas cerebrocortical neurons are plated at 92.8 x 103 cells/cm2.
      5. During plating, hippocampal neurons can be diluted and plated directly in supplemented neuronal culture medium containing 25 μM of glutamate (see Recipes), whereas cerebrocortical neurons must be plated in neuronal plating medium (see Recipes). After 2-3 h of incubation at 37 °C in a humidified incubator with 5% CO2/95% air, replace the neuronal plating medium with fresh supplemented neuronal culture medium without glutamate (only valid for the preparation of cerebrocortical neurons).
      6. Feed hippocampal neurons once a week, by changing equal parts of volume (1/3 of the initial volume of culture medium), and cerebrocortical neurons twice a week by changing equal parts of volume (¼ of the initial volume of culture medium). At day 3 replace 1/3 of the culture medium with fresh medium (devoid of glutamate) containing 30 μM 5-FDU (final concentration–10 μM; the stock solution is kept at 10 mM) in order to stop the growth of glia cells. In this step always use supplemented neuronal culture medium lacking glutamate. Subsequent neuronal feeding should be done by using supplemented neuronal culture medium devoid of glutamate and 5-FDU. See Figures 5-8 for final results.


    Figure 5. Cultured hippocampal neurons with 1 DIV


    Figure 6. Example of cultured hippocampal neurons with 15 DIV


    Figure 7. Cultured cerebrocortical neurons with 1 DIV


    Figure 8. Cultured cerebrocortical neurons with 15 DIV

Recipes

  1. Hanks’ balanced salt solution (HBSS)
    5.36 mM KCl
    0.44 mM KH2PO4
    137 mM NaCl
    4.16 mM NaHCO3
    0.34 mM Na2HPO4·2H2O
    5 mM glucose
    1 mM pyruvic acid
    10 mM HEPES
    0.001% phenol red
    Adjust the pH to 7.2 and filter the solution in the laminar flow hood with a filtration unit with a 0.20 μm acetate cellulose filter. Alternatively, use single-use stericups (see Materials and Reagents #10). The solution should be maintained at 4 °C (stable for at least two months)
  2. Trypsin solution (2 mg/ml)
    5 ml HBSS
    10 mg trypsin
    Filter with an acetate cellulose filter of Ø25 mm, 0.20 μm, sterile. Always prepare fresh
  3. 10% FBS
    10 ml heat inactivated FBS stock solution
    90 ml sterile HBSS
    In order to heat inactivate the FBS stock solution, put the original bottle in a water bath pre-warmed at 56 °C for 30 min. After this, FBS can be aliquoted and stored at -20 °C until further usage. 10% FBS solution can be maintained at 4 °C (stable for at least one month)
  4. Neuronal plating medium
    9.50 g/L minimum essential medium Eagle
    0.6% glucose
    1 mM pyruvic acid
    3.64 g/L sodium bicarbonate
    Adjust the pH to 7.2, filter the solution in the laminar flow hood with a filtration unit with a 0.20 μm acetate cellulose filter. Alternatively, use single-use stericups (see Materials and Reagents #10). The solution should be maintained at 4 °C (stable for at least two months)
  5. Supplemented neuronal culture medium (500 ml)
    Neurobasal Medium®
    SM1 supplement (1x; 1:50 dilution)
    0.5 mM glutamine (1.25 ml of 200 mM stock solution [already sterile])
    25 μM glutamate (1.25 ml of 10 mM stock solution [sterile by filtration with an acetate cellulose filter of Ø25 mm, 0.20 μm, sterile])
    0.12 mg/ml gentamycin (1.2 ml of 50 mg/ml stock solution)
    Solution should be maintained at 4 °C (stable for at least one to two months)
  6. Boric acid solutions
    0.1 M boric acid, pH 8.2 (adjust the pH with a sodium hydroxide solution)
    166.3 mM boric acid, pH 8.2 (adjust the pH with a sodium hydroxide solution)
    After adjusting the pH, filter the solution in the laminar flow hood with a filtration unit with a 0.20 μm acetate cellulose filter. Alternatively, use single-use stericups (see Materials and Reagents #10). Both solutions can be kept at 4 °C for two months

Acknowledgments

Work in the authors laboratory was supported by grants from the Portuguese Science and Technology Foundation (FCT), European Union–European Fund for Economic and Regional Development funding (Operational Competitiveness Program [COMPETE] grants, PEst-C/SAU/LA0001/2013-2014, POCI-01-0145-FEDER-007440, UID/NEU/04539/2013, UID/BIM/4501/2013, SFRH/BPD/84593/2012, SFRH/BPD/115546/2016 and CENTRO-01-0145-FEDER-000008: BrainHealth 2020–Early Detection, Neuromodulation and Advanced Therapies to Brain Disorders).

References

  1. Brewer, G. J., Torricelli, J. R., Evege, E. K. and Price, P. J. (1993). Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J Neurosci Res 35: 567-576.
  2. Caldeira, M. V., Curcio, M., Leal, G., Salazar, I. L., Mele, M., Santos, A. R., Melo, C. V., Pereira, P., Canzoniero, L. M. and Duarte, C. B. (2013). Excitotoxic stimulation downregulates the ubiquitin-proteasome system through activation of NMDA receptors in cultured hippocampal neurons. Biochim Biophys Acta 1832(1): 263-274.
  3. Chen, Y., Stevens, B., Chang, J., Milbrandt, J., Barres, B. A. and Hell, J. W. (2008). NS21: re-defined and modified supplement B27 for neuronal cultures. J Neurosci Methods 171: 239-247.
  4. Curcio, M., Salazar, I. L., Inacio, A. R., Duarte, E. P., Canzoniero, L. M. and Duarte, C. B. (2015). Brain ischemia downregulates the neuroprotective GDNF-Ret signaling by a calpain-dependent mechanism in cultured hippocampal neurons. Cell Death Dis 6: e1645.
  5. Mele, M., Aspromonte, M. C. and Duarte, C. B. (2017). Downregulation of GABAA receptor recycling mediated by HAP1 contributes to neuronal death in in vitro brain ischemia. Mol Neurobiol 54(1): 45-57.
  6. Mele, M., Ribeiro, L., Inacio, A. R., Wieloch, T. and Duarte, C. B. (2014). GABA(A) receptor dephosphorylation followed by internalization is coupled to neuronal death in in vitro ischemia. Neurobiol Dis 65: 220-232.

简介

该方案旨在标准化海马和脑皮质神经元原代培养物的体外实验。 培养物应从胚胎发育期间分离的细胞制备,当神经元前体细胞尚未完全分化时。 这有助于提高细胞的质量和数量,同时提供通常在分化的神经元解离期间发生的最小的细胞死亡。 在合适的条件下,在培养皿或多孔板中铺板的细胞将随着时间的推移而发展和建立突触接触,因为神经元培养基提供分化所需的营养和营养因子。 在这个协议中,我们描述了制备皮质和海马神经元培养物的方法。
【背景】本方案描述了使用补充有NeuroCultTM SM1的Neurobasal培养基(Chen等人,2008)的大鼠海马和脑皮层神经元的原代培养物的制备。 NeuroCultTM SM1的组成基于B27补充剂的制剂(Brewer等,1993),但是前者的混合物被发现提高了神经元培养的质量,部分地通过用全转运蛋白替代载脂蛋白转运蛋白 Chen et al。,2008)。 此外,NeuroCultTM SM1的化学成分在原始出版物中有更详细的描述,可以更好地控制实验条件。 用化学确定的培养基制备的神经元培养物的特征在于存在低百分比的星形胶质细胞。 通过添加有丝分裂5-氟-2'-脱氧尿苷的化学抑制剂可以防止维持更长时间的培养物中星形胶质细胞的增殖以允许神经元分化。

关键字:原代培养, 海马神经元, 脑皮质神经元, 细胞培养

材料和试剂

  1. 培养皿,55毫升无菌非组织培养无菌培养物(Labbox,目录号:PDIP-06N-500)
  2. 培养皿,150毫升玻璃钠钙(DWK Life Sciences,Duran,目录号:23 755 52)
  3. 15ml锥形管(Corning,Falcon ®,目录号:352096)
  4. 5毫升玻璃移液器(VWR,目录号:612-4124)
  5. 10毫升玻璃移液器(VWR,目录号:612-4125)
  6. 50ml锥形管(Corning,Falcon ®,目录号:352070)
  7. 细胞过滤器70μm(Corning,Falcon ®,目录号:352350)
  8. 聚-D-赖氨酸涂覆的多孔板(参见"程序"中的步骤16)的说明书)
  9. 盖板#1.5(例如,Thermo Fisher Scientific,Thermo Scientific TM,目录号:1014355110NR15;对于10mm盖玻片)
  10. 混合纤维素酯过滤器,ME范围(ME 24),过滤单元孔径0.2μm(GE Healthcare,Whatman,目录号:10406970)
  11. Ø25mm,0.20μm无菌的醋酸纤维素过滤器(FRILABO,目录号:1520012)
  12. 过滤单元(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:DS0320-5033)
  13. Stericup-GV,0.20μm,PVDF,500 / 1,000ml,无菌灭菌(Merck,目录号:SCGVU10RE)
  14. 孕妇Wistar大鼠(E17-E18天妊娠)
  15. 台盼蓝溶液,0.4%(Thermo Fisher Scientific,Gibco TM,目录号:15250061)
  16. 聚-D-赖氨酸氢溴酸盐(Sigma-Aldrich,目录号:P7886)
  17. 硼酸(Merck,目录号:1001651000)
  18. 硝酸(Applichem,目录号:133255.1612)
  19. 乙醇绝对值为99.8 +%(Fisher Scientific,目录号:10342652)
  20. 5-氟-2'-脱氧尿苷(5-FDU)(Sigma-Aldrich,目录号:F0503)
  21. 氯化钾(KCl)(AppliChem,目录号:131494.1211)
  22. 磷酸氢二钾(K 2)HPO 4(Merck,目录号:1051041000)
  23. 氯化钠(NaCl)(Applichem,目录号:131659.1211)
  24. 碳酸氢钠(NaHCO 3)(Acros Organics,目录号:123360010)
  25. 二水合磷酸氢钠(Na 2 HPO 4•2H 2 O)(Merck,目录号:1065800500)
  26. D(+) - 葡萄糖一水合物(VWR,目录号:24371.297)
  27. 丙酮酸钠(Sigma-Aldrich,目录号:P5280)
  28. HEPES(Fisher Scientific,目录号:BP310-1)
  29. 苯酚红(Sigma-Aldrich,目录号:P4758)
  30. 胰蛋白酶(Thermo Fisher Scientific,Gibco TM ,目录号:27250018)
  31. 胎牛血清(FBS)(Thermo Fisher Scientific,Gibco TM,目录号:10270106)
  32. 最低必需培养基Eagle(MEM)(Sigma-Aldrich,目录号:M0268)
  33. Neurobasal Medium ®(Thermo Fisher Scientific,Gibco TM,目录号:21103049)
  34. NeuroCult TM SM1神经元补充剂(STEMCELL Technologies,目录号:05711)
  35. L-谷氨酰胺(Sigma-Aldrich,目录号:G8540; Thermo Fisher Scientific,Gibco TM,目录号:25030024)
  36. 谷氨酸(Sigma-Aldrich,目录号:G1626)
  37. 马血清(Thermo Fisher Scientific,Gibco TM ,目录号:16050122)
  38. 氢氧化钠溶液
  39. 汉克斯的平衡盐溶液(HBSS)(见食谱)
  40. 胰蛋白酶溶液(2mg / ml)(参见食谱)
  41. 10%FBS(见配方)
  42. 神经电镀培养基(见食谱)
  43. 补充神经元培养基(见食谱)
  44. 硼酸溶液(见配方)

设备

  1. 大而小的剪刀
  2. 镊子直头(精细科学工具,型号:Dumont#5)
  3. 镊子弯曲尖端(精细科学工具,型号:Dumont#5/45镊子 - 杜蒙标准提示)
  4. Pipetboy(Integra Biosciences,型号:PIPETBOY acu 2)
  5. 放大镜
  6. 层流罩
  7. 水浴(Medingen Labortechnik,型号:T100)
  8. 相位倒置显微镜配备了10x物镜(Nikon Instruments,型号:Eclipse TS100)
  9. 血细胞计数器(Marienfeld-Superior,目录号:0640010)
  10. 加湿培养箱

程序

  1. 从怀孕的Wistar大鼠中移除E18胚胎(以制备海马神经元培养物),麻醉并如前所述通过剖宫产(Caldeira等人,2013; Mele等人)处死,2014; Curcio等人,2015; Mele等人,2017)。
    注意:为了培养脑皮质神经元,建议使用E17胚胎。相同的程序也适用。
  2. 将胚胎转移到装有冰冷HBSS的150毫米玻璃培养皿(150毫升应足够;见食谱)。
  3. 用剪刀以斩首牺牲胚胎,并去除大脑(图1)。分离脑半球并用镊子除去小脑(和其他非关键部位;图2)。使用弯曲和直镊子在解剖显微镜下小心地清除脑膜(视频1)。


    图1.以不同放大水平观察到的E18大鼠胚胎的脑部


    图2.来自E18大鼠胚胎大脑的大脑皮质与海马仍然封闭。 脑脊髓仍然可以在大脑皮质表面观察到。

    Video 1. Initial steps in the dissection of the rat hippocampal

    To play the video, you need to install a newer version of Adobe Flash Player.

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  4. 解剖并分离海马,并将组织转移到填充有HBSS的55mm塑料培养皿(5-7ml应足够;参见图3的最终结果)。使用解剖显微镜将有助于此过程。
    注意:
    1. 我们建议使用从怀孕大鼠获得的所有胚胎的海马,因为细胞产量低。
    2. 为了准备脑皮质神经元,要解剖两个皮质(参见图3的最终结果)。


    图3.解剖大脑皮质(左侧)和孤立的海马(右侧)

  5. 将带有从怀孕大鼠获得的所有海马的陪替氏培养皿带至无菌条件的层流罩。
  6. 通过将所有完整的海马(或皮质)吸移到15ml锥形管中并使HBSS的体积达到约6ml,收集所有完整的海马(或皮质)。
    注意:
    1. 使用10毫升的玻璃移液管。这种移液器具有更宽的尖端,避免损坏组织。
    2. 对于脑皮质神经元,通过允许组织通过重力沉积,用HBSS洗涤组织三次(5-8ml)在第二次洗涤步骤之后获得的最终体积应该如步骤6中所述。
    3. 提示:使用每个15毫升锥形管的四个半球来制备脑皮层神经元培养物。
  7. 加入1.5ml胰蛋白酶溶液(参见食谱)到含有海马的培养基中,并在37℃下孵育15分钟。在孵化期结束时,组织应该沉淀 注意:

    1. 可以使用水浴系统进行孵化
    2. 对于脑皮质神经元,这个过程应持续10分钟。
  8. 允许组织沉积,并小心地取出层流罩中的上清液。
  9. 将8ml先前温热(37℃)10%FBS溶液(见食谱)加入到沉淀的海马中,并轻轻搅拌。
  10. 通过去除上清液并用10ml先前温热(37℃)HBSS冲洗海马,使组织沉淀并洗出10%FBS溶液。
  11. 取出上清液,加入5ml先前温热(37℃)的神经元培养液(参见食谱)。
    为了制备脑皮质神经元的培养物,步骤10-12必须用冰冷的HBSS(6-9ml)洗涤组织五次,并在最后的洗涤步骤中将体积加到约4ml的HBSS中。 /> 注意:发现该体积对于具有约3-5×10 -6细胞的最终浓度为最佳浓度 / ml的解决方案。
  12. 使用Pipetboy提供的机械力,用无菌的5ml玻璃移液管分离组织,上下吸收海马悬浮液。
    注意:
    1. 细胞解离后,悬浮液变得混浊(参见图4)。
    2. 在补充的神经元培养基中进行制备海马神经元培养物的机械解离,而HBSS应用于制备脑皮质神经元。
    3. 提示:5毫升玻璃移液器的尖端应紧靠15毫升锥形管的底部,只保留狭窄的空间以允许溶液通过。当悬浮液被上下移动时,由于细胞分解,培养基将变得浑浊。
    4. 因为这个过程对于单元格来说是相当积极的,所以它不应该重复10次以上


    图4.解剖脑皮质组织后的细胞悬浮液(右)。 左侧的管包含非解离的皮质组织。在海马细胞解离后可以获得相似的结果(未显示)
  13. 收集细胞悬浮溶液,并通过70μm的细胞过滤器过滤至无菌的50ml锥形管中 注意:此步骤允许悬浮液中的细胞与保留的非解离组织的小聚集物分离。
  14. 要计数细胞数量,稀释等体积的以下溶液:HBSS,细胞悬浮液,台盼蓝(每个50μl应足够)。
  15. 将10μl在步骤14中制备的细胞悬浮液加入血细胞计数器。使用装有10x物镜的倒置相差显微镜,对位于角部(面积= 0.01cm 2)的四个象限中存在的细胞进行计数,体积为0.0001cm 3 >)。
    注意:可以使用以下公式计算每ml溶液中存在的细胞数:平均细胞数/平方×3(步骤14中的稀释因子)×10 4 =细胞数/ ml溶液。
  16. 以适当的密度将细胞置于聚-D-赖氨酸包被的多孔板中。
    1. 为了制备聚-D-赖氨酸溶液,首先将100mg小瓶稀释在100ml硼酸(0.1M,pH8.2)中。该溶液的等分试样应储存在-20°C直至进一步使用。所得到的溶液(1mg / ml)应进一步稀释于硼酸(166.3mM,pH 8.2)中,并在涂覆平板之前过滤,得到0.1mg / ml的终浓度。为了涂上塑料盘,加入足够的溶液以覆盖孔的底部,并在37℃下孵育2小时(建议在潮湿的培养箱中孵育过夜)。在这个涂层周期后,用足够体积的无菌蒸馏水将每个孔洗两次以覆盖井的底部,并允许孔在层流罩内干燥。
    2. 盖玻片涂层的方案如下:1)在封闭的玻璃接收器(我们使用11×9×7厘米; 0.5厘米厚的容器)中,用硝酸将盖玻片在恒定的搅拌下洗涤过夜。 2)用塑料吸管清除硝酸后,用蒸馏水充分洗涤(5次洗涤,每次20-30分钟); 3)用无水乙醇清洗盖玻片5分钟; 4)将盖玻片在160℃下干燥1小时(在封闭的玻璃容器中); 5)用玻璃培养皿内的紫外线辐射灭菌盖玻片约30分钟。盖上盖玻片,将其转移到相应的多孔板上,并按照步骤16a所述的说明进行操作。
      注意:
      1. 提示1:对于11 x 9 x 7 cm的玻璃容器,我们最多可以洗涤100个盖玻片。如果使用较高的数字,则盖玻片可能会粘在一起。
      2. 提示2:用于涂覆孔的聚-D-赖氨酸溶液可以连续使用一次再循环一次。
      3. 提示3:在11 x 9 x 7厘米,0.5厘米厚的玻璃容器中,我们使用50ml水/无水乙醇。
      4. 将海马神经元以90.0×10 3细胞/ cm 2的密度铺板 ,而脑皮层神经元以92.8×10 3细胞/ cm 3铺平。 sup> 2
      5. 在电镀期间,海马神经元可以稀释并直接铺在补充的含有25μM谷氨酸的神经元培养基中(参见食谱),而脑皮质神经元必须铺板在神经电镀培养基中(参见食谱)。在37℃下,在具有5%CO 2 /空气/ 95%空气的潮湿培养箱中孵育2-3小时后,更换神经元电镀培养基中不含谷氨酸的新鲜补充神经元培养液(仅适用于制备脑皮层神经元)。
      6. 通过改变相等体积的体积(¼培养基的初始体积),通过改变等量的体积(培养基的初始体积的1/3)和脑皮质神经元每周两次,一周一次地引入海马神经元)。在第3天,用含有30μM5-FDU的新鲜培养基(无谷氨酸盐)代替1/3的培养基(终浓度为10μM;储备液保持在10mM),以阻止胶质细胞的生长。在这一步中,总是使用补充的缺乏谷氨酸的神经元培养基。随后的神经元喂养应通过使用不含谷氨酸和5-FDU的补充神经元培养基进行。最终结果见图5-8


    图5.具有1 DIV
    的培养的海马神经元

    图6.具有15 DIV
    的培养海马神经元的实例

    图7.具有1个DIV的培养的脑皮质神经元


    图8.具有15 DIV的培养的脑皮质神经元

食谱

  1. 汉克斯平衡盐溶液(HBSS)
    5.36 mM KCl
    0.44mM KH 2 PO 4
    137 mM NaCl
    4.16mM NaHCO 3
    0.34mM Na 2 HPO 4•2H 2 O 5 mM葡萄糖
    1mM丙酮酸
    10 mM HEPES
    0.001%酚红
    将pH调节至7.2,并用具有0.20μm醋酸纤维素过滤器的过滤单元过滤层流罩中的溶液。或者,使用一次性立体异构体(参见材料和试剂#10)。溶液应保持在4°C(稳定至少两个月)
  2. 胰蛋白酶溶液(2mg / ml)
    5 ml HBSS
    10毫克胰蛋白酶
    用醋酸纤维素过滤器Ø25毫米,0.20微米,无菌。始终准备新鲜的
  3. 10%FBS
    10ml热灭活的FBS储备溶液
    90 ml无菌HBSS
    为了加热灭活FBS储备溶液,将原始瓶子放入预热温度为56℃的水浴中30分钟。此后,FBS可以等分并储存在-20°C直到进一步使用。 10%FBS溶液可以保持在4°C(稳定至少一个月)
  4. 神经电镀培养基
    9.50 g / L最低必需培养基Eagle
    0.6%葡萄糖
    1mM丙酮酸
    3.64克/升碳酸氢钠
    将pH调节至7.2,用带有0.20μm醋酸纤维素过滤器的过滤单元过滤层流罩中的溶液。或者,使用一次性立体异构体(参见材料和试剂#10)。溶液应保持在4°C(稳定至少两个月)
  5. 补充神经元培养液(500 ml) Neurobasal Medium ®
    SM1补充(1x; 1:50稀释)
    0.5mM谷氨酰胺(1.25ml 200mM储备溶液[已经无菌])
    25微克谷氨酸盐(1.25毫升10毫升储备溶液[用乙酸纤维素过滤器,Ø25毫米过滤灭菌,0.20微米无菌])
    0.12mg / ml庆大霉素(1.2ml,50mg / ml储备溶液)
    解决方案应保持在4°C(稳定至少一至两个月)
  6. 硼酸溶液
    0.1M硼酸,pH8.2(用氢氧化钠溶液调节pH)
    166.3mM硼酸,pH8.2(用氢氧化钠溶液调节pH)
    调节pH后,用含有0.20μm醋酸纤维素过滤器的过滤单元过滤层流罩中的溶液。或者,使用一次性立体异构体(参见材料和试剂#10)。两种溶液都可以在4℃保存两个月

致谢

作者实验室的工作由葡萄牙科学技术基金会(FCT),欧盟 - 欧洲经济和区域发展基金(运营竞争力计划[COMPETE])拨款,PEst-C / SAU / LA0001 / 2013- 2014年,POCI-01-0145-FEDER-007440,UID / NEU / 04539/2013,UID / BIM / 4501/2013,SFRH / BPD / 84593/2012,SFRH / BPD / 115546/2016和CENTRO-01-0145- FEDER-000008:BrainHealth 2020-Early Detection,Neuromodulation and Advanced Therapies to Brain Disorders)。

参考

  1. Brewer,GJ,Torricelli,JR,Evege,EK and Price,PJ(1993)。  在补充B27的Neurobasal(一种新的无血清培养基组合)中优化海马神经元的存活。 J Neurosci Res 35:567-576。
  2. Caldeira,MV,Curcio,M.,Leal,G.,Salazar,IL,Mele,M.,Santos,AR,Melo,CV,Pereira,P.,Canzoniero,LM and Duarte,CB(2013)一个class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/23069389"target ="_ blank">兴奋毒素刺激通过激活培养的NMDA受体来下调泛素 - 蛋白酶体系统海马神经元。 Biochim Biophys Acta 1832(1):263-274。
  3. Chen,Y.,Stevens,B.,Chang,J.,Milbrandt,J.,Barres,BA and Hell,JW(2008)。  NS21:用于神经元培养的重新定义和修改的补充B27。神经元方法 171:239- 247.
  4. Curcio,M.,Salazar,IL,Inacio,AR,Duarte,EP,Canzoniero,LM and Duarte,CB(2015)。< a class ="ke-insertfile"href ="http://www.ncbi。 nlm.nih.gov/pubmed/25675305"target ="_ blank">脑缺血通过培养的海马神经元中的钙蛋白酶依赖性机制下调神经保护性GDNF-Ret信号。细胞死亡Dis 6:e1645。
  5. Mele,M.,Aspromonte,MC和Duarte,CB(2017)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/26732589"target = "HIB1"介导的GABA A受体回收的下调有助于体外脑缺血中的神经元死亡。 Mol Neurobiol 54(1):45-57。
  6. Mele,M.,Ribeiro,L.,Inacio,AR,Wieloch,T.and Duarte,CB(2014)。 GABA(A)受体去磷酸化,随后内化与体外缺血中的神经元死亡相结合 Neurobiol Dis 65:220-232。
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引用: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. DOI: 10.21769/BioProtoc.2551.
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Tingting chen
18275112414
7/8/2018 3:46:04 PM Reply