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Dec 2017

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A Simple and Efficient Method for Concomitant Isolation and Culture of Enriched Astroglial and Microglial Cells from the Rat Spinal Cord
一种简单有效的大鼠脊髓星形胶质细胞和小胶质细胞联合分离培养方法   

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Abstract

Investigations into glial biology have contributed substantially in understanding the physiology and pathology of the nervous system. However, intricacies of the neuron-glial and glial-glial interactions in vivo present significant challenges while delineating the individual cell-type contributions, thus making the in vitro techniques exceedingly relevant to study glial biology. However, obtaining optimal yield along with high purity has been challenging for microglial cultures. Here we present a simple protocol to establish enriched astroglial as well as microglial cultures from the neonatal rat spinal cord. This method results in highly enriched astroglial and microglial cultures with maximal yield.

Keywords: Primary cultures (原代培养), Microglia (小胶质细胞), Astrocytes (星形胶质细胞), Astroglia (星形胶质), Spinal cord (脊髓)

Background

Astrocytes and microglial cells play a crucial role during the development, as well as in the normal physiology and pathology of the nervous system. These include structural, vascular, metabolic and neuro-regulatory functions like modulation of transmission by forming ‘quad-partite synapses’, release of gliotransmitters and trophic factors, and integration of neuronal-glial networks (Parpura et al., 1994; Araque et al., 1999; Schafer et al., 2013; Michell-Robinson et al., 2015; Rossi, 2015). Microglia, in addition to the canonical immune functions, also plays an important role in adult neurogenesis, differentiation, maturation and integration within the neuronal circuitry (Rezaie and Male, 2002; Polazzi and Monti, 2010; Michell-Robinson et al., 2015; Ransohoff and El Khoury, 2015). In response to a pathological insult, the glial cells react by undergoing morphological and physiological changes and rescue the vulnerable neurons from the insult (Ferraiuolo et al., 2011; Pekny et al., 2014; Mishra et al., 2016; Mishra et al., 2017). At the tissue level, astrocytes undergo morphological transformation from protoplasmic to fibrillary phenotype and move to the site of injury to create a physical barrier between damaged and healthy cells, or by forming glial scars in a process called reactive gliosis. In a similar manner, microglia undergo morphological transformation from the resting/ramified phenotype to the activated/amoeboid phenotype, via intermediate activation stages, (Rezaie and Male, 2002; Michell-Robinson et al., 2015; Ransohoff and El Khoury, 2015). Both the glial cell types thus promote neuronal repair through acute inflammation, which modulates the microenvironment by regulating production and release of trophic factors, gliotransmitters, cytokines and chemokines (Raivich et al., 1999; Parpura et al., 2012; Verkhratsky and Butt, 2013; Pekny et al., 2014). Reactive gliosis is reversible and beneficial during the acute insult. However, in response to prolonged insult or genetic dysregulation, the balance is disrupted and may lead to irreversible and erroneous activation resulting in chronic inflammation and neurodegeneration (Lobsiger and Cleveland, 2007). Therefore, delineating the precise glial response as well as their role in modulating neuronal physiology becomes relevant. However, an in vivo approach may present with a disadvantage owing to the complex neuron-glial and glial-glial interactions, which dynamically modulate these changes at the tissue level. Primary glial cultures help us to overcome this issue, by enabling observations in an isolated cell-type model system. In vitro experimentation also widens the scope for further comparing the effect of neuron-glial/glial-glial interactions through a co-culture model system.

Various approaches have been adopted to culture astrocytes and microglia from animal brains and spinal cords. These methods vary considerably not only in terms of purity and yield, but also with respect to the cell population targeted for isolation (Giulian and Baker, 1986; Saura et al., 2003; Floden and Combs, 2007; Scorisa et al., 2010; Kerstetter and Miller, 2012). For instance, the conventional repeated shaking methods for culturing microglia exploit the microglial layer growing atop astrocytes in the mixed glial cultures (Giulian and Baker, 1986; Scorisa et al., 2010), while the mild trypsinization method targets the ones growing beneath (Saura et al., 2003). We compared all the methods and standardized a procedure to establish exceedingly enriched astroglial and microglial cultures, while maximizing the yield. The current protocol generates enough microglial and astroglial cells per spinal cord to execute parallel experiments in both the cell types, thus minimizing the inter-population variability while comparing cell-specific responses.

Materials and Reagents

  1. Animals
    Wistar strain rat pups with post-natal Day 0-2 (P0-P2), housed and maintained in accordance with the institutional ethics guidelines

  2. Culture products
    1. 0.22 µm membrane filter (Millipore, catalog number‎: ‎GSWP04700)
    2. T-25 ml tissue culture flask (Corning, Sigma, catalog number: CLS430639)
    3. 24-well plate (Thermo Fisher Scientific, catalog number: 142475)
    4. Coverslips, 13 mm, circular (Thermo Fisher Scientific, catalog number: 12-519-21G)
    5. DNase-1 (Sigma-Aldrich, catalog number: 11 284 932 001)
    6. Hanks’ balanced salt solution (HCMF, Thermo Fisher Scientific, catalog number: 14170112)
    7. Poly-L-lysine hydrobromide (PLL, Sigma-Aldrich, catalog number: P5899-5G)
    8. Dulbecco’s modified Eagle’s medium/F-12 (DMEM/F-12) (GIBCO, Invitrogen, USA, catalog number: 12500062)
    9. L-Glutamine, 200 mM solution (Thermo Fisher Scientific, catalog number: 25030081)
    10. Fetal bovine serum-Heat inactivated (FBS-HI) (GIBCO, Invitrogen, USA, catalog number: 10082147)
    11. 0.25% trypsin-EDTA (GIBCO, Invitrogen, USA, catalog number: 25200)
    12. Penicillin-streptomycin (Pen/Strep; 100x solution; 10,000 units/ml each) (Thermo Fisher Scientific, catalog number: 15140122)
    13. HEPES (1 M) (Thermo Fisher Scientific, catalog number: 15630080)
    14. MilliQ water
    15. HCl (Sigma-Aldrich, catalog number: 7647-01-0)
    16. Ethanol (Sigma-Aldrich, catalog number: 64-17-5)
    17. HBSS
    18. Glucose powder
    19. NaCl
    20. KCl
    21. Na2HPO4
    22. KH2PO4
    23. PFA
    24. Growth medium (500 ml) (see Recipes)
    25. 50 ml FBS-HI (10% v/v) (see Recipes)
    26. Dissection medium (500 ml) (see Recipes)
    27. Dissociation medium (10 ml) (see Recipes)
    28. DMEM-trypsin-EDTA (1:4) (10 ml) (see Recipes)

  3. Immunostaining
    1. 0.1 M PBS, freshly prepared
    2. Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A2153)
    3. Anti-ChAT (rabbit polyclonal Abcam, catalog number: ab18736; 1:500)
    4. Anti-GFAP (mouse monoclonal, Abcam, catalog number: ab24345; 1:500)
    5. Anti-IBA1 (Rabbit polyclonal Abcam, catalog number: ab153696; 1:500)
    6. Anti-mouse IgG (Cy3-conjugated; 1:200, Sigma-Aldrich)
    7. Anti-rabbit IgG (FITC-conjugated; 1:200, Chemicon)
    8. PVA-DABCO anti-fade mounting medium (Sigma-Aldrich, catalog number: 10981)
    9. PBS 1x (pH-7.4) (see Recipes)
    10. PFA, 4% (see Recipes)

Equipment

  1. Fine forceps, Dumont #5 (2), curved (1) (Fine Science Tools)
  2. Scissors (1), 51/2 in., straight, operating (Fine Science Tools)
  3. Scissors (2), 4 in., straight, micro dissecting (Fine Science Tools)
  4. Dissecting microscope (Leica Microsystems)
  5. Phase contrast microscope (Leica Microsystems)
  6. Confocal laser-scanning microscope (Leica Microsystems)
  7. CO2 incubator (Eppendorf)
  8. Orbital Shaker (New Brunswick, Eppendorf)
  9. Centrifuge (Thermo Scientific)
  10. Fire polished glass pipette (Fisherbrand)
  11. Light source
  12. Laminar flow hood
  13. 4 °C refrigerator
  14. -20 °C freezer
  15. -80 °C freezer
  16. Hemocytometer

Software

  1. Photoshop CS2 (Adobe)
  2. Microsoft Office

Procedure

  1. Coating the coverslips
    1. Wash, dry and sterilize the coverslips by autoclaving and/or ethanol treatment (95% to absolute ethanol) prior to coating.
    2. Incubate the dried, sterile coverslips with PLL (0.1 mg/ml, final concentration in PBS) for 30 min, followed by rinsing twice for 30 s with PBS to prevent the toxicity associated with excess PLL.
    3. Let the coverslips dry completely before use. We recommend using freshly coated coverslips. Alternatively, the pre-coated coverslips can be stored at 4 °C for up to 3 weeks.

  2. Dissection of neonatal spinal cords
    1. Prepare ice-cold dissection medium (see Recipes).
    2. Carefully anesthetize the pups. Wipe the body with 70% ethanol and swiftly decapitate with the help of a scalpel.
    3. Make an incision in the lower back and expose the vertebral column. The skin and vertebral column tissue of the neonatal rats are soft and can be dissected out with relative ease when compared to the adults.
    4. With the help of spring scissors and fine forceps, carefully cut open the vertebral column to expose the spinal cord, moving from the caudal to rostral end. Gently but swiftly remove the spinal cord and place it into ice-cold dissection medium. For a description of the dissection process, we recommend the protocol previously published by Gal et al. (2016).
    5. Thoroughly separate the meninges from the spinal cords using the tips of fine forceps and minimizing tissue damage.
    6. With the help of a scalpel, cut the tissue into small pieces and proceed with trituration in a Laminar Air Flow hood (LAF).

  3. Mixed glial cultures
    1. Transfer the tissue to 1 ml of the dissociation medium per spinal cord, at 37 °C for 15 min. Trypsin helps in dissociation of the tissue while addition of DNase aids by digesting the viscous DNA released from the damaged cells.
    2. Stop the reaction by adding pre-warmed growth media (DMEM/F-12 supplemented with 10% FBS-HI) and centrifuge at 400 x g for 3 min.
    3. Replace the supernatant with growth media, and mechanically triturate by passing through 1 ml fire polished glass pipette 8-10 times gently. Care should be taken to avoid bubbles.
    4. Allow the undissociated tissue to settle down and transfer the supernatant to another vial. Add fresh media and repeat the process of mechanical trituration 2 more times or till the tissue is dissociated. The optimal shear stress causes the tissues to dislodge into a single-cell suspension. However, harsh trituration may lead to loss of yield due to cell death.
    5. Pellet down the single-cell suspension thus obtained at 400 x g for 3 min.
    6. Resuspend in fresh media.
    7. Count the cells using a hemocytometer and plate with a seeding density of 2.5 x 104 cells/ml in T-25 tissue culture flask. Incubate at 37 °C in a sterile cell culture incubator with 5% CO2 and 95% humidity.
      Note: Coating the flasks with PLL prior to seeding is not necessary, as glial cells adhere robustly. In our experience, seeding on uncoated flasks further ensured enrichment.
    8. Change the media every day for 3-4 days to ensure the proper removal of residual debris. Afterward, replenish the media as per the requirement. By day in vitro (DIV) 7, the cultures start attaining confluence, and by DIV 12, a population of microglia with fringent, phase-bright soma can be seen growing on the top of astrocytes (Figure 1).


      Figure 1. Mixed glial cultures isolated from the rat spinal cord. A and A’ represent the growing (DIV 7) and confluent (DIV 12) cultures, respectively. Panel B depicts the presence of distinct microglial marker, Iba-1 positive cells growing on top of the astroglial marker, GFAP labeled cells. Scale bars are indicated.

  4. Astroglial cultures
    1. Establish and allow the mixed cultures to attain confluence (8-10 DIV). Replace the media every alternate day. On the 11th DIV, place the culture flasks on an orbital shaker at 200 rpm for 3-4 h maintained at 37 °C. Aspirate the resultant media that contains the less adherent microglial cells dislodged from the top of astrocytes.
    2. The media containing microglial cells obtained at this stage can be further centrifuged at 400 x g for 3 min to pellet microglia. This microglial population can be resuspended in DMEM/F-12 and plated for future experiments in appropriate culture dishes with the desired plating density. However, care must be taken to avoid the dislodged non-microglial cells, including astrocytes, as it can easily lead to cross-contamination. Moreover, we adopted another, more efficient method to culture microglia (Saura et al., 2003), that provided better yield and reduced contamination, as described later.
    3. Thoroughly wash the intact astroglial layer with PBS, followed by incubation in 0.25% trypsin-EDTA for 1-2 min, to ensure proper detachment of the cells.
    4. Monitor the rounding of cells and stop the trypsinization with an equal amount of growth media when the detachment is complete.
    5. Pellet the cells at 400 x g for 3 min and resuspend in fresh growth media. Replate at a density of 2.5 x 104 cells/ml on the circular coverslips in a 24-well plate and proceed with the experiments (Figure 2).


      Figure 2. Enriched astroglial cultures derived from the mixed cultures. A, A’, C, C’. The cultures mainly consist of a flat/protoplasmic morphology. B, D-E’. Fibrous morphology can be seen occasionally in unstimulated cultures but frequently in cultures provided with inflammatory stimulus. B, B’. Astrocytes further subcultured (20-25 DIV) do not show a significant change in their viability or reactive ability. The cultures stain negative for microglia as shown by Iba-1 staining (F-F”) and neurons, as shown by ChAT staining (G-G”). Scale bars are indicated.

  5. Microglial cultures
    1. Derive the mixed glial cultures from the Wistar rat pups as described above and proceed for establishing microglial cultures using mild trypsinization method.
    2. After the cultures reach confluence, allow the cultures to mature for a few more days while constantly replenishing one-half of the media with fresh medium every alternate day. Don’t completely remove the old media conditioned with glial factors, as these factors allow for the optimal growth of microglia.
    3. After the intact astroglial monolayer has matured for a while, subject the glial cultures to mild trypsin-EDTA treatment. Add 4-6 ml of Trypsin EDTA-DMEM/F-12, in a ratio of 1:4 per flask, enough to cover the cells. The mild trypsinization step removes the astroglial monolayer, revealing the microglial layer growing beneath, while DMEM/F-12 provides nourishment throughout the process. The process usually takes 10-15 min. Periodically check for the detachment of the monolayer under a phase contrast microscope and standardize the time for optimal detachment.
    4. Once the astroglial layer is fully detached, discard the media containing the astroglial layer and wash the culture dish thoroughly with DMEM/F-12 to remove traces of non-microglial cells.
    5. Check the cultures for purity. Afterward, use the cells directly, or replate on the circular coverslips in a 24-well plate, or as per the requirement of the experiment (Figure 3).


      Figure 3. Enriched microglial cultures derived from the mixed cultures. A-A’’, B-B’’. Healthy cultures predominantly display process bearing morphology in the unstimulated state. However, intermediate (A’’’, B’’’) and reactive, phagocytotic (A’’’’, B’’’’) stages can be observed as the microglial response to external stimuli. The cultures stain negative for astroglial contamination (C-C’’). Scale bars are indicated.

  6. Fixation and immunostaining
    1. Wash the coverslips containing cells with sterile PBS and follow with fixation using 4% PFA for 15 min at RT. Aspirate PFA and thoroughly wash with PBS before proceeding for immunostaining.
    2. Antigen retrieval step is optional and should be standardized depending upon the guidelines for each antibody. For the antibodies used in the present study, antigen retrieval was neither needed nor performed.
    3. Proceed for blocking with 3% BSA for an hour, followed by the overnight incubation at 4 °C with the primary antibody of interest. After primary incubation, subject the coverslips to 3 washes of 5 min each before adding appropriate, fluorescently labeled secondary antibodies. After incubation for 2 h at RT, wash the coverslips with PBS thrice for 5 min each and mount them on a clean slide with PVA-DABCO anti-fade mounting medium. Once the slide has dried completely, proceed for imaging with a confocal microscope.

Data analysis

The mixed glial cultures, as well as the primary astroglial and microglial cultures obtained using these protocols were investigated and compared for their purity and individual responses through a series of assays. GFAP, Iba-1 and DAPI staining demonstrated > 98% enrichment of both the astroglial as well as microglial cultures (Mishra et al., 2016 and 2017). However, the analysis is beyond the scope of this protocol and has already been published. Therefore, the data analysis has not been discussed.

Notes

  1. Supplementation with muscle extract from the gastrocnemius muscles of the wistar rats promotes the growth of motor neurons at the Step D7, and should be done if motor neuronal survival is desired in the mixed cultures (Shobha et al., 2007).
  2. The mixed cultures can be further subcultured by trypsinization prior to isolation of astrocytes and /or microglia, to further enhance the yield. In that case, the cultures should be standardized and characterized prior to planning the experiments. Moreover, all the experiments must be conducted and replicated at the same passage status to avoid inducing any phenotypic variability that might arise due to subculturing.
  3. Subculturing further ensures the purity of the cultures. The astroglial cultures can be subcultured 3-4 times without significant loss of characteristics. Plating at a high density further obliterates cross contamination.
  4. At the Step F2 it is crucial that the astroglial monolayer is allowed to mature and remain intact. An immature layer may not detach efficiently, resulting in improper isolation and/or cross-contamination of microglial cultures. Usually 5-7 days after the cultures have attained confluence, it is optimal to proceed with mild trypsinization.
  5. In our hands the trypsin-EDTA-DMEM/F-12 ratio of 1:4 used at the Step F3 efficiently removed the astroglial monolayer for 15-20 DIV cultures. However, the ratio can be further increased to 1:3 to 1:2 depending on the culture conditions and must be standardized. Moreover, EDTA is essential to ensure the selective removal of astrocytes, while leaving the microglial layer intact. For further insights, we recommend reading the article by Saura et al. (2003), where the protocol was first described.
  6. In view of achieving purity, the washing step is critical at the Step F4, as the astrocytes remaining in the culture dish can efficiently reattach and proliferate, leading to astroglial contamination.
  7. The microglia thus obtained can be propagated overnight in serum free DMEM/F-12 and plated for the experiment in the following day. Microglia could be grown in mixed glial/astroglial conditioned media for a longer duration. However, microglial cells are dynamic and very quickly respond to the slightest changes in their environment in vitro. Therefore, for the best, unbiased results, we recommend using the cultures at the earliest.
  8. The astroglial monolayer detached during the Step F3 can be further subcultured. However, since the confluent astroglial layer has a high nutritional demand, the media must be frequently replenished to ensure a healthy subculture. One way of determining the health of the cultures is to monitor the detachment of the layer, as unhealthy or starved astroglial layer detaches very quickly with or without mild trypsinization. We recommend standardizing and checking the integrity of the subcultured astrocytes before using them for further experimentation. In our hands, a second subculture yielded viable, protoplasmic astrocytes that actively responded to inflammatory stimulus (Figures 1B-B’). Further, replating the cultures at a high density ensured healthy and enriched astroglial cultures. Unlike astrocytes, microglial cells cannot be subcultured.

Recipes

Note: The media preparation should be done under a laminar flow hood to ensure sterility.

  1. Media preparation
    DMEM/F-12 powdered media package into 800 ml of autoclaved MilliQ
    0.11% HEPES and sodium bicarbonate, with pH set to 7.4 using 1 N HCl
    10 ml of 1x antibiotic mixture (100,000 IU penicillin, 0.05% streptomycin and 0.002% amphotericin B)
    Make the final volume up to 1 L and Sterile-filter the media using a 0.22 µm membrane filter store at 4 °C until further use
  2. Growth medium (500 ml)
    450 ml DMEM/F-12
    50 ml FBS-HI (10% v/v)
    Filter and store at 4 °C
  3. Dissection medium (500 ml)
    487 ml 1x HBSS
    5 ml 1 M HEPES (0.1 M solution)
    3 g glucose powder (6 mg/ml)
    5 ml Pen/Strep solution (100 U/ml)
    Filter and store at 4 °C
  4. Dissociation medium (10 ml)
    HBSS 10 ml
    0.25% trypsin-EDTA
    DNase 100 μg/ml
  5. DMEM-trypsin-EDTA (1:4) (10 ml)
    2 ml of 2.5% trypsin EDTA dissolved in 8 ml of DMEM/F-12, freshly prepared
  6. PBS 1x (pH-7.4)
    NaCl 8 g (0.137 M)
    KCl 200 mg (0.0027 M)
    Na2HPO4 1.44 g ( 0.01 M)
    KH2PO4 240 mg (0.0018 M)
    Make up the volume to 1 L with milliQ water, adjust the pH
  7. PFA 4% (50 ml)
    1. 2 mg PFA in 20 ml Double Distilled water
    2. Adjust the pH to 7.4
    3. Make the volume 50 ml using water and 25 ml 2x PBS

Acknowledgments

The protocols were standardized with the help of grants from DBT and ICMR, govt. of India, respectively. PM was a Junior/senior research fellow under UGC/CSIR fellowship program by govt. of India. The protocols were used in two separate studies published by Mishra et al. (2016 and 2017). The authors are further grateful to Dr. Josep Saura and Dr. Kumarasamy Murali for troubleshooting and standardization of the enriched microglial cultures.

Competing interests

The authors declare no competing interest.

Ethics

All the animal procedures used in establishing the protocols were approved by the Institutional Animal Ethics Committee (AEC/44/264/NP and AEC/55/343/NP).

References

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简介

[ 摘要] 对神经胶质生物学的研究对理解神经系统的生理学和病理学做出了重大贡献。然而,体内神经元-神经胶质和神经胶质-胶质相互作用的复杂性在描述单个细胞类型的贡献时提出了重大挑战,因此使得体外技术与研究神经胶质生物学极为相关。然而,对于小胶质细胞培养而言,获得最佳产量和高纯度一直是挑战。在这里,我们提出了一个简单的协议,可以从新生大鼠中建立丰富的星形胶质细胞和小胶质细胞培养物 脊髓。此方法可产生高度富集的星形胶质细胞和小胶质细胞,且产量最高。
[ 背景技术] 星形胶质细胞和小神经胶质细胞p 铺设一个神经系统的发育过程中的关键作用,以及在正常的生理和病理。这些包括结构,血管,代谢和神经调节功能等传输的调制通过形成“四三方突触”,gliotransmitters和营养因子的释放和神经元的集成- 胶质网络(Parpura 等人,1994;阿拉克等人; 1999; Schafer 等,2013 ;Michell-Robinson 等,2015; Rossi,2015)。除典型的免疫功能外,小胶质细胞还在成人神经元回路中的神经发生,分化,成熟和整合中起重要作用(Rezaie和Male,2002; Polazzi和Monti,2010; Michell-Robinson 等,2015;M。Ransohoff和El Khoury,2015年)。响应于病理损伤,神经胶质细胞通过从损伤发生形态和生理变化和救援的脆弱的神经元反应(Ferraiuolo 等人,2011; Pekny 等人,2014;米什拉等人,2016;米什拉等人。,2017)。在组织水平,星形胶质细胞从原质表型转变为原纤维表型,并移动到损伤部位,在受损细胞与健康细胞之间形成物理屏障,或者在称为反应性神经胶质增生的过程中形成胶质瘢痕。小胶质细胞以类似的方式通过中间激活阶段经历从静止/分支表型到激活/类猿表型的形态转化(Rezaie and Male,2002; Michell-Robinson et al 。,2015; Ransohoff and El Khoury,2015)。因此,两种神经胶质细胞类型均通过急性炎症促进神经元修复,并通过调节营养因子,神经胶质递质,细胞因子和趋化因子的产生和释放来调节微环境(Raivich 等,1999; Parpura 等,2012; Verkhratsky 和Butt, 2013; Pekny 等人,2014;)。在急性损伤期间,反应性神经胶质增生是可逆的并且是有益的。然而,响应于长期的侮辱或遗传失调,平衡被破坏并且可能导致不可逆和错误的激活,从而导致慢性炎症和神经退行性变(Lobsiger和Cleveland,2007)。因此,描述精确的神经胶质反应及其在调节神经元生理学中的作用变得很重要。ħ H但是,一个体内方法可以呈现与一个缺点由于复杂的神经元的神经胶质和神经胶质细胞-胶质细胞的相互作用,其动态地调节这些变化在组织水平。通过在孤立的细胞类型模型系统中进行观察,初级神经胶质细胞培养可以帮助我们克服这一问题。体外实验还拓宽了通过共培养模型系统进一步比较神经元-神经胶质/神经胶质-胶质相互作用的作用的范围。

已经采用了各种方法来培养动物脑和脊髓中的星形胶质细胞和小胶质细胞。这些方法不仅在纯度和产量上有很大差异,而且在分离目标细胞方面也有很大差异(Giulian和Baker,1986; Saura 等,2003; Floden和Combs,2007; Scorisa 等,2010)。 ; Kerstetter and Miller,2012)。例如,用于培养小胶质细胞的常规重复摇动方法利用混合胶质细胞培养物中星形胶质细胞顶部生长的小胶质层(Giulian和Baker,1986; Scorisa 等,2010),而温和的胰蛋白酶消化法则针对生长在胶质细胞下方的小胶质细胞(Saura)。等人,2003)。我们比较了所有方法并标准化了建立极其丰富的星形胶质和小胶质细胞培养物的程序,同时使产量最大化。当前的协议每个脊髓产生足够的小神经胶质细胞和星形胶质细胞,以在两种细胞类型中执行并行实验,从而在比较细胞特异性反应的同时最大程度地减少了种群间差异。

关键字:原代培养, 小胶质细胞, 星形胶质细胞, 星形胶质, 脊髓

材料和试剂


 


动物
出生后第0-2天(P0-P2)的Wistar品系幼崽,并根据机构道德准则进行饲养和维护


 


文化产品
1. 0.22μm膜滤器(Millipore,目录号: GSWP04700)       


2. T-25 ml组织培养瓶(Corning,Sigma ,目录号:CLS430639)       


3. 24 - 孔板(赛默飞世尔科技,产品目录号:142475)       


4. 13毫米圆形盖玻片(Thermo Fisher Scientific,目录号:12-519-21G)       


5. DNase-1 (Sigma-Aldrich,目录号:11 284 932 00)       


6. Hanks的平衡盐溶液(HCMF ,Thermo Fisher Scientific,目录号:14170112)       


7. 聚-L-赖氨酸氢溴酸盐(PLL ,Sigma-Aldrich,目录号:P5899-5G)       


8. Dulbecco改良的Eagle培养基/ F-12(DMEM / F-12)(GIBCO,美国Invitrogen ,目录号:12500062)       


9. L-谷氨酰胺,200 mM溶液(Thermo Fisher Scientific,目录号:25030081)       


10. 胎牛血清热灭活(FBS-HI)(GIBCO,美国Invitrogen ,商品目录号:10082147)   


11. 0.25%胰蛋白酶-EDTA(GIBCO,美国,Invitrogen,目录号:25200)   


12. 青霉素链霉素(笔/链球菌; 100x溶液;每个10,000单位/ ml)(Thermo Fisher Scientific,目录号:15140122)   


13. HEPES(1 M)(赛默飞世尔科技公司,目录号:15630080)   


14. MilliQ水   


15. HCl (Sigma-Aldrich,目录号:7647-01-0)   


16. 乙醇(西格玛奥德里奇,目录号:64-17-5)   


17. HBSS   


18. 葡萄糖粉   


19. 氯化钠   


20. 氯化钾   


21. Na 2 HPO 4   


22. KH 2 PO 4   


23. PFA   


24. 生长培养基(500毫升)(请参阅食谱)   


25. 50毫升FBS-HI(10%v / v)(请参阅食谱)   


26. 解剖培养基(500毫升)(请参阅食谱)   


27. 解离介质(10毫升)(请参阅食谱)   


28. DMEM-胰蛋白酶-EDTA(1:4)(10毫升)(请参阅食谱)   


 


免疫染色
新鲜配制的0.1 M PBS
牛血清白蛋白(BSA)(Sigma-Aldrich,目录号:A2153)
Anti-ChAT(兔多克隆Abcam,目录号:ab18736; 1:500)
抗GFAP(小鼠单克隆,Abcam,目录号:ab24345; 1:500)
抗IBA1(兔多克隆Abcam,目录号:ab1536961:500)
抗小鼠IgG(Cy3偶联; 1:200,Sigma-Aldrich)
抗兔IgG(FITC偶联; 1:200,Chemicon)
PVA-DABCO防褪色固定介质(Sigma-Aldrich,目录号:10981)
P BS 1x (pH-7.4)(请参阅食谱)
PFA,4%(请参阅食谱)
 


设备


 


细镊子,Dumont#5(2),弯曲的(1)(Fine Science Tools)
剪刀(11/2),51/2 in。,直的,可操作的(Fine Science Tools)
剪刀(2个),4 英寸,直的,微解剖的(精细科学工具)
解剖显微镜(Leica Microsystems)
相衬显微镜(Leica Microsystems)
共焦激光扫描显微镜(Leica Microsystems)
CO 2 培养箱(Eppendorf)
轨道振荡器(新不伦瑞克,埃彭多夫)
离心机(Thermo Scientific)
火抛光玻璃移液器(Fisherbrand)
光源
层流罩
4°C冰箱
-20°C冷冻室
-80°C冷冻室
血细胞计数器
 


软件


 


Photoshop CS2(Adobe)
微软办公软件
 


程序


 


盖玻片
w ^ 灰,干燥和steriliz Ë通过高压灭菌和/或乙醇处理(95%无水乙醇)之前涂覆的盖玻片。
将干燥的无菌盖玻片与PLL(0.1 mg / ml,在PBS中的终浓度)孵育30分钟,然后用PBS漂洗两次30 s,以防止与过量PLL相关的毒性。
使用前,让盖玻片完全干燥。我们建议使用新涂的盖玻片。或者,可以将预涂的盖玻片在4°C下最多保存3周。
 


DIS 新生儿脊髓节
准备冰- 冷解剖培养基(请参阅食谱)。
仔细麻醉幼崽。用70%的乙醇擦拭身体,并在手术刀的帮助下迅速断头。
在下背部切开切口,露出椎骨柱。与成年动物相比,新生大鼠的皮肤和椎骨组织很柔软,可以相对容易地解剖。
在弹簧剪刀和细镊子的帮助下,小心地将脊柱切开以暴露脊髓,从尾端到头端。轻轻但迅速地取下脊髓,然后将其放入冰中- 冷解剖介质。对于解剖过程的描述,我们建议先前由Gal 等人发表的方案。,(2016年)。
使用细镊子的尖端将脊髓的脊髓彻底分开,并最大程度地减少组织损伤。
在手术刀的帮助下,将组织切成小块,然后在层流气流罩(LAF)中进行研磨。
 


混合的胶质文化
将组织转移到每毫升1 ml的离解培养基中,在37°C下放置15分钟。胰蛋白酶有助于组织的解离,而DNase的添加则可通过消化从受损细胞释放的粘性DNA来辅助。
通过添加预热的生长培养基(补充有10%FBS-HI的DMEM / F-12)并以400 xg离心3分钟来终止反应。
用生长培养基代替上清液,并轻轻地通过1 ml火抛光的玻璃移液器8-10次以进行机械研磨。应注意避免气泡。
让未分离的组织沉淀下来,并将上清液转移到另一个小瓶中。添加新鲜的培养基,并重复机械研磨过程2次以上,直到组织解离为止。最佳剪切应力使组织以移去成一个单- 细胞悬浮液。但是,严酷的磨碎可能会由于细胞死亡而导致产量下降。
沉淀下来的单- 从而在400获得的细胞悬浮液XG 3分钟。
重悬于新鲜培养基中。
使用血细胞计数器和平板在T-25组织培养瓶中接种密度为2.5 x 10 4 细胞/ ml 的细胞进行计数。在无菌的细胞培养箱中于37°C,5%CO 2 和95%湿度下孵育。
注意:由于神经胶质细胞牢固粘附,因此在播种前无需用PLL覆盖烧瓶。根据我们的经验,在未包被的烧瓶中撒种可进一步确保富集。


每天更换介质3-4天,以确保正确清除残留的碎屑。然后,根据需要补充介质。由天体外(DIV)7,将培养开始达到汇合时,和由DIV 12,与fringent,相明亮的体细胞小胶质细胞的群体可以看出在星形胶质细胞的顶部生长(图URE 1)
 






图1 。从大鼠脊髓分离出的混合胶质细胞培养物。A和A'分别代表生长的(DIV 7)和融合的(DIV 12)文化。图B描绘了存在于星形胶质标记GFAP标记的细胞之上的不同的小胶质标记Iba-1 阳性细胞的存在。标有比例尺。


 


星形胶质文化
         建立并允许混合文化达到融合(8-10 DIV)。每隔一天更换一次介质。在第11个DIV上,将培养瓶以200 rpm的速度放置在定轨振荡器上3-4 h,保持在37 °C 。吸出含有从星形胶质细胞顶部脱落的粘附较少的小神经胶质细胞的所得培养基。
         可以将在此阶段获得的包含小胶质细胞的培养基进一步以400 xg离心3分钟,以沉淀小胶质细胞。可以将此小胶质细胞群体重悬于DMEM / F-12中,并以所需的接种密度接种在合适的培养皿中,以备将来实验。但是,必须小心避免脱落的非小胶质细胞,包括星形胶质细胞,因为它很容易导致交叉污染。此外,我们采用了另一种更有效的方法来培养小胶质细胞(Saura 等人,2003年),该方法可提供更高的产量并减少污染,如下所述。
         用PBS彻底清洗完整的星形胶质层,然后在0.25%胰蛋白酶-EDTA中孵育1-2分钟,以确保细胞正确分离。
         监视的单元的舍入和与停止胰蛋白酶消化的等量的生长培养基的分离完成时。
         将细胞以400 xg 沉淀3分钟,然后重悬于新鲜的生长培养基中。Replate在2.5×10密度4 在24上的圆形盖玻片细胞/ ml - 孔板中并与实验(图进行URE 2) 。
 






图2 。来自混合文化的丰富的星形胶质文化。A,A',C,C'。培养物主要由扁平/原生质形态组成。B,D-E'。纤维形态在非刺激性培养物中偶见,但在具有炎症刺激的培养物中则常见。B,B ' 。一个ST rocytes进一步传代(20-25 DIV)没有表现出他们的生存能力和反应能力的显著变化。如Iba-1染色(FF“)和ChAT染色(GG”)所示,培养物对小胶质细胞呈阴性。标有比例尺。


 


小胶质细胞培养
              如上所述,从Wistar大鼠幼崽中获得混合的神经胶质细胞培养物,然后使用温和的胰蛋白酶消化法进行小胶质细胞培养。
              在培养物达到汇合后,允许培养成熟了几天,同时不断补充一个- 半媒体用新鲜培养基每隔一天。不要完全卸下旧媒体与胶质因素的限制,因为这些因素允许的小胶质细胞的最佳生长。
              完整的星形胶质单层细胞成熟一段时间后,对神经胶质培养物进行温和的胰蛋白酶-EDTA处理。以每瓶1:4的比例添加4-6毫升的胰蛋白酶EDTA-DMEM / F-12,足以覆盖细胞。温和的胰蛋白酶消化步骤去除了星形胶质细胞单层,揭示了其下方生长的小胶质细胞层,而DMEM / F-12在整个过程中提供了营养。该过程通常需要10-15分钟。在相衬显微镜下定期检查单层的分离情况,并标准化时间以实现最佳分离。
              一旦星形胶质细胞层完全脱离,丢弃含有星形胶质细胞层的培养基,并用DMEM / F-12彻底清洗培养皿,以去除痕量的非微胶质细胞。
              检查培养物的纯度。然后,使用直接在圆形盖玻片的细胞,或replate在24 - 孔板中,或者作为每requiremen 实验T(图URE 3) 。
 






图URE 3 。来自混合培养物的丰富的小胶质细胞培养物。A-A'',B-B''。健康的文化主要表现出处于未刺激状态的过程形态。然而,作为对外部刺激的小胶质细胞反应,可以观察到中间阶段(A''',B''')和反应性吞噬阶段(A'''',B'''')。培养物染色为星形胶质细胞污染(C-C'')。标有比例尺。


 


固定和免疫染色
用无菌PBS洗涤含有细胞的盖玻片,然后在室温下使用4%PFA固定15分钟。抽吸PFA并用PBS 彻底清洗后再进行免疫染色。
抗原回收步骤是可选的,应根据每种抗体的指南进行标准化。对于本研究中使用的抗体,既不需要也不进行抗原修复。
继续用3%BSA封闭1小时,然后在4°C与所需的一抗孵育过夜。初步孵育后,将盖玻片分别洗涤3次,每次5分钟,然后添加适当的荧光标记的二抗。在室温下孵育2小时后,将盖玻片用PBS洗涤三次,每次5分钟,然后将其安装在带有PVA-DABCO防褪色固定介质的干净载玻片上。幻灯片完全干燥后,请使用共聚焦显微镜进行成像。
 


数据分析


 


研究了混合胶质细胞培养物,以及使用这些方案获得的主要星形胶质和小胶质细胞培养物,并通过一系列测定比较了它们的纯度和个体反应。GFAP,Iba-1和DAPI染色显示星形胶质细胞和小胶质细胞培养物均富集> 98%(Mishra 等人,2017 和2016)。 但是,该分析超出了该协议的范围,并且已经发布。因此,没有讨论数据分析。


 


笔记


 


补充来自wistar大鼠腓肠肌的肌肉提取物在步骤D7促进运动神经元的生长,并且如果需要在混合培养物中运动神经元的存活,则应该这样做(Shobha 等,2007)。
混合培养物可以通过胰蛋白酶消化进一步继代培养或分离星形胶质细胞和/或小胶质细胞,以进一步提高产量。在这种情况下,应在计划实验之前对文化进行标准化和特征化。此外,所有实验都必须在相同的传代状态下进行和重复,以避免引起任何因传代培养而产生的表型变异。
继代培养进一步确保了培养物的纯度。可将星形胶质细胞传代培养3-4次,而不会明显丧失特性。高密度电镀进一步消除了交叉污染。
在步骤F2中,至关重要的是使星形胶质单分子层成熟并保持完整。未成熟的层可能无法有效分离,从而导致小胶质细胞培养物的不正确分离和/或交叉污染。通常在培养物达到汇合后的5-7天,最好进行轻度的胰蛋白酶消化。
在我们的手中,步骤F3中使用的胰蛋白酶-EDTA-DMEM / F-12比例为1:4,可有效去除15-20 DIV培养的星形胶质单层。然而,取决于培养条件,该比例可以进一步增加至1:3至1:2,并且必须标准化。此外,EDTA 对于确保选择性去除星形胶质细胞,同时保持完整的小胶质细胞层至关重要。为了获得更多见解,我们建议阅读Saura 等人的文章。(2003),该协议首次被描述。
考虑到获得纯度,洗涤步骤在步骤F4是至关重要的,因为保留在培养皿中的星形胶质细胞可以有效地重新附着和增殖,从而导致星形胶质细胞污染。
如此获得的小胶质细胞可以在无血清的DMEM / F-12中繁殖过夜,并在第二天铺板用于实验。小胶质细胞可以在胶质/星形胶质混合条件培养基中生长更长的时间。如何以往,小胶质细胞是动态的,并很快在他们的环境丝毫的变化做出反应体外。因此,为了获得最佳,公正的结果,我们建议尽早使用这些文化。
在步骤F3中分离的星形胶质单分子膜可以进一步传代培养。但是,由于汇合的星形胶质层具有很高的营养需求,因此必须经常补充培养基以确保健康的亚培养。确定培养物健康状况的一种方法是监视层的分离,因为不健康或饥饿的星形胶质层会在有或没有轻度胰蛋白酶消化的情况下非常迅速地分离。我们建议标准化和检查继代培养的星形胶质细胞的完整性,然后再将其用于进一步的实验。在我们手中,第二传代培养产生可行的,星形胶质细胞原生质即积极响应炎症刺激(图小号1和B-B')。此外,以高密度重新培养可确保健康和丰富的星形胶质细胞培养。与星形胶质细胞不同,小胶质细胞不能继代培养。
 


菜谱


 


Ñ OTE :媒体准备工作应在层流罩下进行,以确保无菌。


培养基制备将
DMEM / F-12粉末培养基包装到800毫升高压灭菌的MilliQ中
0.11%HEPES 和碳酸氢钠,使用1 N HCl将pH设置为7.4


10 ml的1x抗生素混合物(100000 IU青霉素,0.05%链霉素和0.002%两性霉素B)


使最终体积最大至1 L,并使用0.22 µm的膜滤器在4°C下无菌过滤介质,直至进一步使用


生长培养基(500毫升)45 0 毫升DMEM / F-12 50毫升FBS-HI(10%v / v)过滤并储存在4°C




解剖培养基(500 ml)
487 ml 1x HBSS
5 ml 1 M HEPES(0.1 M溶液)3 g葡萄糖粉(6 mg / ml)5 ml Pen / Strep溶液(100 U / ml)过滤并储存在4°C




解离培养基(10 ml)
HBSS 10 ml 0.25%胰蛋白酶-EDTA DNase 100μg/ ml
             
DMEM-胰蛋白酶-EDTA(1:4)(10 ml)将
2 ml的2.5%胰蛋白酶EDTA溶于8 ml DMEM / F-12,新鲜制备
PBS 1 x ,(pH-7.4)
NaCl 8 g (0.137 M)KCl 200 mg (0.0027 M)Na 2 HPO 4 1.44 g(0.01 M)KH 2 PO 4 240 mg (0.0018 M)补足1 L用MilliQ水调节pH             
                           
             
                           
PFA 4%(50毫升)
20毫升双蒸馏水中2毫克PFA
调节pH至7.4
用水和25 ml 2x PBS稀释50 ml
 


致谢


 


在DBT和ICMR(政府)的资助下,对这些协议进行了标准化。印度。PM是政府在UGC / CSIR奖学金计划下的初级/高级研究员。印度。该协议用于Mishra 等人发表的两项独立研究中。(201 6和201 7 )。作者还要感谢Josep Saura博士和Kumarasamy Murali博士对丰富的小胶质细胞培养进行故障排除和标准化。


 


利益争夺


 


作者宣称没有竞争利益。


 


伦理


 


建立规程所用的所有动物程序均得到机构动物伦理委员会的批准(AEC / 44/264 / NP和AEC / 55/343 / NP)。


 






参考文献


 


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引用:Mishra, P. S. and Raju, T. R. (2020). A Simple and Efficient Method for Concomitant Isolation and Culture of Enriched Astroglial and Microglial Cells from the Rat Spinal Cord. Bio-protocol 10(2): e3501. DOI: 10.21769/BioProtoc.3501.
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