TITLE: 3.1 Human iPSC Culture
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 3.1 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 cells (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]
?. [3.1 Human iPSC Culture]
For the coating of cell culture plates, resuspend one aliquot of Matrigel in the appropriate amount of cold coating medium (see Note 1). Add the Matrigel solution to the cell culture plates and distribute equally so that the entire well is covered (see Note 6).
?. [3.1 Human iPSC Culture]
To thaw iPSCs, get the frozen vials from the liquid nitrogen tank and keep on dry ice. Thaw carefully in a  water bath or alternatively with the ThawSTAR™ Automated Cell Thawing System (BioCision™) until only a small ice cube remains.
37 °C
?. [3.1 Human iPSC Culture]
In order to passage iPSCs, aspirate the culture medium and wash the cells once with . Dissociate the cells by adding TrypLE and place in the incubator for approximately –.
[PBS w/o Ca2+ and Mg2+]
?. [3.1 Human iPSC Culture]
To freeze iPSCs, dissociate the cells with TrypLE and collect in , spin down and resuspend the pellet in mFreSR™ medium. If necessary, count the cells and aliquot the appropriate amount into cryotubes (see Note 10).
[PBS w/o Ca2+ and Mg2+]
?. [3.1 Human iPSC Culture]
Transfer the cell solution to a 15 ml Falcon tube and add dropwise –.
2 mL
[1× PBS w/o Ca2+ and Mg2+]
?. [3.1 Human iPSC Culture]
Spin down at  and aspirate the supernatant.
Centrifuge: 400 34, 4 min
?. [3.1 Human iPSC Culture]
Resuspend the cell pellet in mTeSR™1 with ROCKi and transfer to a cell culture plate coated with Matrigel and place in the incubator (, ).
37 °C
[CO2]
?. [3.1 Human iPSC Culture]
After , wash the cells once with  and change the medium to mTeSR™1 w/o ROCKi. Change the medium every day until the next passaging (see Note 7).
[PBS w/o Ca2+ and Mg2+]
?. [3.1 Human iPSC Culture]
Add  and pipet up and down to collect all cells.
[PBS w/o Ca2+ and Mg2+]
?. [3.1 Human iPSC Culture]
Transfer the cell solution to a 15 ml Falcon tube and spin down at . Aspirate the supernatant and resuspend the cell pellet in mTeSR™1 with ROCKi.
Centrifuge: 400 34, 4 min
?. [3.1 Human iPSC Culture]
Count the cells using Trypan Blue (e.g., with the Countess™ II FL Automated Cell Counter or hemocytometer) and seed the appropriate number of cells in Matrigel- or poly-L-lysine + laminin-coated cell culture plates (see Note 8). Mix well and place in the incubator (, ).
37 °C
[CO2]
?. [3.1 Human iPSC Culture]
After , wash the cells once with  and change the medium to mTeSR™1 w/o ROCKi. Change the medium every day until next passaging (see Note 7 and Fig. 1). It is recommended to check the iPSCs regularly for mycoplasma contamination (see Note 9).
[PBS w/o Ca2+ and Mg2+]
?. [3.1 Human iPSC Culture]
Put the tubes in a freezing container and store at  for at least . Subsequently, store in liquid nitrogen.
-80 °C
?. [3.1 Human iPSC Culture]
Incubate at  for at least . Use immediately or store at  for a maximum of 2 weeks. Prior to use, simply aspirate the coating medium and add the cell suspension. No washing step is required.
0 Room temperature
4 °C