brief description on nerve cell culture

2.  By isolating differentiated cell or tissue for
short term nonregenerative cultures
 Isolating precursor cells
 Stem cell culture

3. FASTIDIOUS
Neurite outgrowth is encouraged by a polypeptide
nerve growth factor
Neurons used are hippocampal, cortical, spinal,
cerebellar etc
Disadv : long term culture diffficult

4.  COATED PLATES- POLY L LYSINE OR
COLLAGEN
 NGF
 GLIAL FACTORS
 PROTOCOL CONTRIBUTED BY BERNT
ENGELSEN AND ROLF BJERKVIG

5. Cortical
neurons

6.  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%

7.  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

8.  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

9.  Neuron specific enolase antibody
 Tetanus toxin marker
 Glial fibrillar acidic protein for astrocyte
contamination

10.  3 TYPES
› ASTOCYTES
› MICROGLIAL
› OLIGODENDROCYTES
 Human adult normal astroglial lines from brain
lines express glial fibrillary acidic protein

11.  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

12. 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

13. Labelling with galactocerebroside to distinguish betweeen OEC
and oligodendrocytes
Done prior to cell plating
Primary antibody O4 and secondary antibody anti-GalC

15.  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)

16.  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

17. 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.

19. Glia cell
(astrocyte)

20.  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.

22. 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