TITLE: 3.6 Coculturing with Astrocytes
AUTHORS: Evelyn J. Sauter, Lisa K. Kutsche, Simon D. Klapper, Volker Busskamp

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is part 3.4 of the "</span><a href="https://www.protocols.io/private/C65DB81838BC11EBA61A0A58A9FEAC2A/protocols" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Induced Neurons for the Study of Neurodegenerative   and Neurodevelopmental Disorders</span></a><span>" collection of protocols.</span></div><br/><div class = "text-block"><span style = "font-weight:bold;">Collection Abstract:</span><span> Patient-derived or genomically modified human induced pluripotent stem cell s (iPSCs) offer the opportunity to study neurodevelopmental and neurodegenerative disorders. Overexpression of certain neurogenic transcription factors (TFs) in iPSCs can induce efficient differentiation into homogeneous populations of the disease-relevant neuronal cell types. Here we provide protocols for genomic manipulations of iPSCs by CRISPR/Cas9. We also introduce two methods, based on lentiviral delivery and the piggyBac transposon system, to stably integrate neurogenic TFs into human iPSCs. Furthermore, we describe the TF-mediated neuronal differentiation and maturation in combination with astrocyte cocultures.</span></div></div>

[STEPS]
?. [3.6 Coculturing with Astrocytes]
In order to increase the maturation of neurons for electrophysiological measurements, coculturing with astrocytes is highly recommended [4, 15]. We adapted the protocol from Kaech and Banker [16] to our cell culture.
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Rat primary cortical astrocytes are cultured in astrocyte medium at  and  according to the manufacturer’s instructions.
37 °C
[CO2]
?. [3.6 Coculturing with Astrocytes]
For the coculture with neurons, prepare astrocytes to be ~80% confluent at day 4 of neuronal differentiation. One day before the reseeding of neurons, wash the astrocytes three times with  and add BrainPhys™ medium with minimal supplements.
[PBS w/o Ca2+ and Mg2+]
?. [3.6 Coculturing with Astrocytes]
Thoroughly clean the coverslips in a big glass petri dish. First, rinse the coverslips in ddH2O for  and then shake in .
[1 M HCl]
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Autoclave ~ in a 500 ml bottle. Melt in a boiling water bath (1 l beaker with  on a heat plate with ). Use a 2 ml aspiration pipette with a 200 μl pipette tip attached and soak it in melted paraffin. Shake off extra drops and place small drops on coverslips to create paraffin feet as spacer (see Note 28). Coat the coverslips with PLL and laminin (see protocol 3.5) on the side with the paraffin dots.
[low melting paraffin]
[H2O]
350 °C
?. [3.6 Coculturing with Astrocytes]
Differentiate the iPSCs on Matrigel-coated cell culture dishes using  for 4 days (see protocol 3.5). Add  to the culture for 1 day to remove occasionally undifferentiated cells.
[doxycycline in mTeSR™1 medium]
[Ara-C]
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At day 5, reseed predifferentiated neurons on PLL and laminin coated coverslips with paraffin feet. Collect the old medium from the culture well and wash the cells very carefully with prewarmed .
[PBS w/o Ca2+, Mg2+]
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After , place the coverslips with the differentiated iPSCs upside down into culture wells containing 80% confluent rat astrocytes. Every 7 days, exchange 50% of the BrainPhys™ medium and compensate the volume loss due to evaporation with ddH2O (see Note 30).
?. [3.6 Coculturing with Astrocytes]
For passaging, aspirate the culture medium and store it in a Falcon tube as a washing solution (see Note 26). Rinse the cells once with .
[PBS w/o Ca2+ and Mg2+]
?. [3.6 Coculturing with Astrocytes]
Add prewarmed Accutase and incubate the cells at  until all of them are detached (usually  are sufficient).
37 °C
?. [3.6 Coculturing with Astrocytes]
Stepwise add the cell culture medium stored in step 3 to flush cells and collect all cells to a prerinsed 15 ml Falcon tube.
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Centrifuge at . Aspirate the supernatant and resuspend the pellet in prewarmed astrocyte growth medium.
Centrifuge: 400 34, 5 min
?. [3.6 Coculturing with Astrocytes]
Count the cells using Trypan Blue and seed the appropriate amount in uncoated tissue-culture treated dishes at a seeding density of approximately 5000 cells per cm2. Change the growth medium every 3–4 days.
?. [3.6 Coculturing with Astrocytes]
Dissociate the cells by adding Accutase and place in the incubator for approximately  until the neuronal network detaches. Add the cell culture medium stored in step 3 and transfer the cell solution to a 15 ml Falcon tube.
?. [3.6 Coculturing with Astrocytes]
Rinse the well 1–2 times with  to collect all cells and centrifuge at .
[BSA]
Centrifuge: 400 34, 5 min
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Aspirate the supernatant and resuspend the cell pellet in  slowly and carefully (this step is crucial for the survival of single cells).
[0.2% BSA]
?. [3.6 Coculturing with Astrocytes]
Add  to a total volume of .
[0.2% BSA]
1 mL
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Centrifuge at  and collect  supernatant in a fresh tube (this is the single cell suspension).
Centrifuge: 20 34, 1 min
800 µl
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Repeat the dissociation of the pellet for a maximum of five times until no pellet is visible any more.
?. [3.6 Coculturing with Astrocytes]
Centrifuge the single cell suspension at  and resuspend the pellet in . Count the cells if necessary and seed on coated coverslips on the side with the paraffin feet (see Note 29).
Centrifuge: 400 34, 5 min
[BrainPhys™ medium]
?. [3.6 Coculturing with Astrocytes]
Rinse three times with ddH2O by shaking for , and rinse another two times with ddH2O by shaking for . Shake three times in  for  and one time .
[ethanol]
?. [3.6 Coculturing with Astrocytes]
Sterilize the coverslips at  for – (can be done overnight) (see Note 27) [16].
225 °C