Neuronal culture

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brief description on nerve cell culture

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  • .o25% trypsin for 15min at 37
  • Inhibits the growth of other cell types
  • Neuronal culture

    1. 1.  By isolating differentiated cell or tissue for short term nonregenerative cultures Isolating precursor cells Stem cell culture
    2. 2. FASTIDIOUSNeurite outgrowth is encouraged by a polypeptidenerve growth factorNeurons used are hippocampal, cortical, spinal,cerebellar etcDisadv : long term culture diffficult
    3. 3.  COATED PLATES- POLY L LYSINE OR COLLAGEN NGF GLIAL FACTORS PROTOCOL CONTRIBUTED BY BERNT ENGELSEN AND ROLF BJERKVIG
    4. 4. Corticalneurons
    5. 5.  DMEM with › Glucose 30mM › L- glutamine 2mM › KCl 24.5mM › Insulin 100mU/L › P- amino benzoic acid 7μM › Gentamycin 100μg/ml › Fetal calf serum 10%
    6. 6.  Dissect cerebella aseptically and place in HBSS Mince to 0.5 cubic mm Wash with HBSS three times Trypsinisation Add growth medium: stop the action of trypsin Trituration to obtain single cell suspension
    7. 7.  Centrifuge at 200g for 5 min.. Resuspend the pellet in growth medium and seed the cells at a conc of 2.5- 3×106 cells/plate After 2-4 days incubate the culture with 5-10μM cytosine arabinoside for 24 hrs Change to regular culture medium
    8. 8.  Neuron specific enolase antibody Tetanus toxin marker Glial fibrillar acidic protein for astrocyte contamination
    9. 9.  3 TYPES › ASTOCYTES › MICROGLIAL › OLIGODENDROCYTES Human adult normal astroglial lines from brain lines express glial fibrillary acidic protein
    10. 10.  DMEM containing › Glucose 25mM › Gentamycin 25 μg/ml › BSA pathocyte 0.0286% › Glutamine 2mM › Bovine pancreas insulin 10μg/ml › Human transferrin 100 μg/ml › Progestrone 0.2 μM › Putrescine 0.10 μM › Selenium 0.224 μM › Triiodo thyronine 0.49 μM › Thyroxine 0.45 μM
    11. 11. PROTOCOL- OLFACTORY ENSHEATHING CELLS CULTURE Collect olfactory lobes Mince well Collaginase treatment for 30-45 min at 37°C Centifuge 100g 5 min Resuspend pellet in Ca and Mg free HBSS Centrifuge and culture 5×106 cells/ml of DMEM
    12. 12. Labelling with galactocerebroside to distinguish betweeen OECand oligodendrocytesDone prior to cell platingPrimary antibody O4 and secondary antibody anti-GalC
    13. 13.  Isolate cerebrum Peel off the meninges and transfer cortex to a tube containing cold dissection buffer placed on ice Pour tissue into a dish and wash with modified DMEM/F12 culture medium with 10% FBS, 1% glutamine, and gentamicin Mechanically disintegration Trypsinization and DNase treatment- Incubate at 37ºC for 25 minutes.swirl tube every 5 minutes Wash tissue with Glial Medium twice Dilute suspended cells in 10 mL of Glial Medium, and pass the solution through a 40 uM strainer Centrifuge cells at 1700 rpm for 5 minute Resuspend pellet with 10 mL Glial Medium Seed 2 x106 cells/T75 in 15 ml Glial medium Incubate the flasks at 37oC in 5% CO2 for 2-3 days without disruption. Change the medium in each flask every 2-3 days by aspirating and adding 15 mL fresh Glial Medium until confluency is achieved (after approximately 6-7 days)
    14. 14.  Once the primary cultures are confluent, change the medium and tighten flask caps. Wrap flasks in plastic and place on shaker platform horizontally with medium covering the cells Shake at 350 rpm for 6 hours at 37°C to separate oligodendrocytes from astrocytes Change medium (10mL) and replace flasks on shaker for 18 more hours Remove flasks from shaker, and aseptically pour contents into a new T75. Incubate Change medium in flasks (10mL), tighten caps, cover in plastic, and shake, again, for 24 more hours (change medium in 6 hr) Aseptically pour contents into a new T75 and incubate until confluent Reseed at 3 x 105 cells in each T75 flask fresh culture must be prepared every three weeks
    15. 15. Passage Sterilize petri dishes by coating with 1 mg/ml of PureCol Collagen, washing with sterile ddH20, and allowing to dry in culture hood for 30 minutes The next day. wash glial cells with PBS once Add 3-5 mL of trypsin to the culture flask; incubate at 37°C for 5 minutes Add 5-7 mL of Glial Medium to the culture flask, and then transfer cells to a 50 mL tube Centrifuge cells at 1700 rpm for 5 minutes Remove the supernatant and resuspend the cells in 10 mL of Glial Medium Seed cells at 7.5 X1O4 cells/6cm dish in 6mL Glial Medium.
    16. 16. Glia cell(astrocyte)
    17. 17.  to study the membrane properties of Schwann cells axon-Schwann cell communication how these alter in neuropathic conditions to use Schwann cells for the repair of lesioned peripheral nerve to exploit their potential for regeneration in CNS lesions.
    18. 18. o USE AS A MODEL  Advantages Neuronal activity in controlled env Observation possible at several points and methods  Disadvantages Inter Connectivity is lost Lacks body PATHOLOGICAL STUDIES CELL BASED THERAPIES › Glial cell used in spinal cord injuries
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