NERVE CELL
CULTURE
METHODS OF ACHIEVING SPECIALISED CELL CULTURE
By isolating differentiated cell or tissue for short term nonregenerative cultures
Isolating precursor cells
Stem cell culture
NEURONS
FASTIDIOUS
Neurite outgrowth is encouraged by a polypeptide nerve growth factorNeurons used are hippocampal, cortical, spinal, cerebellar etcDisadv : long term culture diffficult
PREREQUSITES FOR NEURONAL CULTURE
COATED PLATES- POLY L LYSINE OR COLLAGEN
NGF
GLIAL FACTORS
PROTOCOL CONTRIBUTED BY BERNT ENGELSEN AND ROLF BJERKVIG
CORTICAL NEURONS
Cortical neurons
MEDIUM USED
DMEM with› Glucose 30mM› L- glutamine 2mM› KCl 24.5mM› Insulin 100mU/L› P- amino benzoic acid7μM› Gentamycin 100μg/ml› Fetal calf serum 10%
PROTOCOL
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
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
Characterization of cultures
Neuron specific enolase antibody
Tetanus toxin marker
Glial fibrillar acidic protein for astrocyte contamination
GLIA
3 TYPES› ASTOCYTES› MICROGLIAL› OLIGODENDROCYTES
Human adult normal astroglial lines from brain lines express glial fibrillary acidic protein
GLIAL CELLCULTURE MEDIUM
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
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
FACS
Labelling with galactocerebroside to distinguish betweeen OEC and oligodendrocytesDone prior to cell platingPrimary antibody O4 and secondary antibody anti-GalC
ASTROCYTE CULTURE 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)
Purify culture 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
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.
Immunocytochemical staining of Astrocytes in culture using an antibody against glial fibrillary acidic protein
ASTROCYTE
Glia cell (astrocyte)
SCHWANN CELL CULTURE
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.
Phase photos of normal adult rat and human Schwann cell cultures in serum-free, hormone-supplemented medium. The rat cultures on the left are shown at passage 20 and continued dividing to establish a cell line. The human cultures are shown at passage 3. Their growth rate slowed appreciably at passage 5.
APPLICATIONSo USE AS A MODEL
AdvantagesNeuronal activity in controlled envObservation possible at several points and methods
DisadvantagesInter Connectivity is lostLacks body
PATHOLOGICAL STUDIES CELL BASED THERAPIES
› Glial cell used in spinal cord injuries
THANK YOU
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