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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

FP Francesca Pischedda
Giovanni Piccoli Giovanni Piccoli

发布: 2019年06月20日第9卷第12期 DOI: 10.21769/BioProtoc.3270 浏览次数: 5482

评审: Alessandro DidonnaDenise DrongitisAnonymous reviewer(s)

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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) search Cryopreservation (冷冻保存) search Long-term storage (长期保存) search Reproducibility (再生性) search Specimen sharing (样品共享) search

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

English
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文章信息

版权信息

© 2019 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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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分类

神经科学 > 基础技术 > 低温贮藏

细胞生物学 > 细胞分离和培养 > 低温贮存

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