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