TITLE: Optimized Derivation of Midbrain Dopaminergic Neurons from iPSCs for research application
AUTHORS: Elisangela Bressan, Ashutosh Dhingra, Stella Donato, Peter Heutink

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>The derivation of human induced pluripotent stem cells (iPSCs) into midbrain dopaminergic (mDA) neurons presents an exciting opportunity to access a large number of patients-specific cells in vitro, to model disease, perform target screenings, and test drug candidates. Previously published small molecule-based protocols are straightforward, implementable in automated cell culture systems </span><a href="https://dx.doi.org/10.3791/61525" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">(Dhingra et al. J Vis Exp. 162, 2020)</span></a><span>, and suitable for the differentiation of large sets of cell lines </span><a href="https://dx.doi.org/10.17504/protocols.io.bfpzjmp6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">(Bressan et al. Protocols.io, 2020)</span></a><span> at relatively low cost and working time. However, the derivation of mDA from a large set of human iPSC lines shows variations in differentiation efficiency between lines. In addition, the current protocols produce heterogeneous cell populations in which only a small subset represents the cells of interest. To address these issues, we optimized a previously established mDA neuron differentiation protocol </span><a href="https://doi.org/10.1038/nature10648" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">(Kriks et al., Nature 480, 547–551, </span></a><a href="https://doi.org/10.1038/nature10648" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">2011</span></a><span>) by: (1) adjusting the SMAD inhibition to improve and achieve more homogeneous neuron conversion between iPSC lines; (2) reinforcing the WTN signaling activation to trigger more efficient midbrain floor plate induction and conversion into mDA neurons, and (3) applying the DNA cross-linker Mitomycin-C to eliminate remaining proliferating cells. The optimized mDA neuron derivation protocol presented here resulted in more homogeneous differentiation between iPSC lines, higher yields of neurons with higher proportion of mDA neurons, and completely elimination of contaminating proliferative cells.                                                                   </span></div><style>
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[STEPS]
?. [Maintenance of iPSCs before start of differentiation]
Grow iPSCs on Matrigel hESC-Qualified Matrix-coated 6-well plates in Essential 8 Flex (E8) medium for 4-5 days. Cells can be passaged once they reach 70-80% confluency (i.e., 70-80% of plate growth area is covered by a cell monolayer) with Gentle Dissociation Reagent (GDR) followed by seeding at a ratio of 1:6 (i.e., 1 well of a 6-well plate passaged into 6-wells). Note: Grow thawed iPSC lines for at least five passages before the start of differentiation.
?. [Passaging of iPSCs with Gentle Differentiation Reagent (GDR)]
Before starting: Coat 6-well plates withMatrigel hESC-Qualified Matrix atfor following manufacturer dilution and preparation instructions.
[per well]
37 °C
?. [Passaging of hiPSC with Gentle Differentiation Reagent (GDR)]
?. [Passaging of iPSCs with Gentle Differentiation Reagent (GDR)]
Warm PBS and GDR to.
37 Room temperature
?. [Passaging of iPSCs with Gentle Differentiation Reagent (GDR)]
Discard old culture medium and wash cells once withof PBS.
[per well]
?. [Passaging of iPSCs with Gentle Differentiation Reagent (GDR)]
Discard PBS and addof GDR.
[per well]
?. [Passaging of iPSCs with Gentle Differentiation Reagent (GDR)]
Bring the plate back to and discard GDR very gently.Critical step: Do not shake or tap the plate after incubation with GDR to avoid cell colony dislodgement and loss of cells during removal of the dissociation reagent.
37 Room temperature
?. [Passaging of iPSCs with Gentle Differentiation Reagent (GDR)]
Flush the wells once using a P1000 pipette filled withof E8 medium to dislodge cell colonies.Critical step: do not pipette up and down cell colonies to avoid single cell dissociation.
1 mL
?. [Passaging of iPSCs with Gentle Differentiation Reagent (GDR)]
Collect cell colonies using a P1000 pipette and seed at a ratio of 1:6 on Matrigel-coated recipient plates prepared in advance.
?. [Passaging of iPSCs with Gentle Differentiation Reagent (GDR)]
Perform a quick microscope inspection of the parent plate to check if all cell colonies were dislodged. If cell colonies are still attached to the plate,, repeat the procedure, and seed the remaining cell colonies in the recipient plate.
?. [Passaging of iPSCs with Gentle Differentiation Reagent (GDR)]
After seeding, rock the plate back-forth and side-to-side forto achieve an even spread of cell colonies in the plate well.
?. [Differentiation of iPSCs into midbrain dopaminergic (mDA) neurons]
Note: This protocol is optimized for mDA neuron differentiation in 6-well plate format.Medium change schema:Day 0-20:Day 21-65:Day 0-15: daily media changesDay 16-20: media changes every 2 daysDay 21-65: media changes every 2-3 daysBefore starting:Perform a quick microscope inspection of the iPSCs. Ensure that iPSC colonies appear healthy (homogeneous colonies with clear borders and absence of differentiating cells; Figure 1). A manual removal of spontaneous differentiating cells might be required.
[per well]
[per well]
?. [Passaging of iPSCs with Gentle Differentiation Reagent (GDR)]
Perform medium change after overnight recovery from passaging and, subsequently, every two days until cells reach 70-80% confluency.
?. [Day -1: Single cell dissociation and seeding for differentiation]
Discard GFR Matrigel solution and addof E8 medium supplemented withY-27632 avoiding the coating to dry out.
[per well]
?. [Day -2: Coating of 6-well plates with Growth Factor Reduced (GFR) Matrigel]
Coat 6-well culture plates withof GFR Matrigel solutionat.
[per well]
37 °C
[CO2]
?. [Day -1: Single cell dissociation and seeding for differentiation]
Keep the plate atforbefore seeding cells.
37 °C
?. [Day -1: Single cell dissociation and seeding for differentiation]
Warm PBS, Accutase and E8 medium to.
37 Room temperature
?. [Day -1: Single cell dissociation and seeding for differentiation]
Discard old culture media and wash cells once withof PBS.
[per well]
?. [Day -1: Single cell dissociation and seeding for differentiation]
Discard PBS and addof Accutase supplemented with Y-27632.
[per well]
?. [Day -1: Single cell dissociation and seeding for differentiation]
Addof E8 medium supplemented withY-27632.
[per well]
?. [Day -1: Single cell dissociation and seeding for differentiation]
Dissociate cells with a P1000 pipette by pipetting the cell suspension up and down for 3-6 times.
?. [Day -1: Single cell dissociation and seeding for differentiation]
Perform a quick microscope inspection of the cells. If still cell colonies are visible,and repeat the procedure. A single cell suspension should be obtained.
?. [Day -1: Single cell dissociation and seeding for differentiation]
Transfer the cell suspension to a 15 mL conical centrifuge tube. A total volume ofshould be obtained from a full 6-well plate.
12 mL
?. [Day -1: Single cell dissociation and seeding for differentiation]
Centrifuge the cell suspension at.
Centrifuge: 200 34, 5 min, 23 10
?. [Day -1: Single cell dissociation and seeding for differentiation]
Discard the supernatant carefully and resuspend the cell pellet inE8 medium supplemented withY-27632 by gently pipetting the cell suspension up and down for 3-6 times with a P1000 pipette to obtain a homogeneous cell suspension.
1 mL
?. [Day -1: Single cell dissociation and seeding for differentiation]
Adjust the volume with E8 medium supplemented withY-27632  to. Mix well by 10x full inversions of the conical tube.
12 mL
?. [Day -1: Single cell dissociation and seeding for differentiation]
Perform two separate live-cell counts using a hemocytometer or an automated cell counter. Critical step: Adjust the cell suspension volume accordingly to obtain an accurate counting. Calculate the mean achieved from the two counts and determine the concentration of live cells per milliliter. Note: Working with a different brand of Accutase might impact the cell viability when following the dissociation protocol described above. If cell viability is lower than expected, reduce the incubation time with Accutase toand the number of pipetting to dissociate the cell colonies to 3-4 times.
A cell viability of 95-99% should be obtained.
?. [Day -1: Single cell dissociation and seeding for differentiation]
Seed 200,000 cells per cm2 in a total volume ofon 6-well plates coated with GFR Matrigel (i.e., to start a differentiation in one well of a 6-well plate, seed 1.9 x 10^6 cells per well, considering the growth area of one well equal to 9.5 cm2).
[per well]
?. [Day 0: Start differentiation]
Before starting: a) Prepare enough amount of Knockout Serum Replacement (KSR) medium. For of KSR medium, add: Knockout DMEM/F-12 medium Knockout Serum Replacement MEM Non-Essential Amino Acids GlutaMAX  2-mercaptoethanol  Penicillin-Streptomycin  Storage: KSR medium can be stored for 5 days ator for up to one month at. b) Reconstitute lyophilized reagents following the instructions and stock concentrations indicated in Materials (1.1.4 Reconstitution of reagents). Use the following final concentrations:  LDN193189 SB431542 SHH Purmorphamine FGF8 CHIR99021 BDNF Ascorbic acid GDNF db-cAMP TGFβ3or DAPTStorage: Once thawed, the stocks of small molecules and growth factors can be stored for up to 5 days at. Notes: Small molecules and growth factors must be freshly added immediately before each medium change. It is strongly advised to avoid mixing different lots of reagents in the same differentiation.
500 mL
413.5 mL
75 mL
5 mL
5 mL
500 µl
1 mL
4 °C
-20 °C
4 °C
?. [Day -1: Single cell dissociation and seeding for differentiation]
Keep cells intactat.
37 °C
[CO2]
?. [Day 0: Start differentiation]
Perform a quick microscope inspection to the cells to check confluency.Critical step: Start the differentiation with a 100% confluent cell culture, meaning that the culture area of the plate should be completely covered with a cell monolayer. Not confluent cell cultures might affect differentiation efficiency. If cell lines did not achieve 100% confluency on day 0, the number of cells seeded per cm2 on day -1 should be adjusted accordingly.
?. [Day 0: Start differentiation]
Prepare differentiation medium: Warm KSR medium at.Add: LDN193189 SB431542Mix well by 20x full inversions of the conical tube or flask. Critical step: Do not add small molecules to cold medium to avoid inadequate dissolution.
37 °C
500
10
?. [Day 0: Start differentiation]
Perform full media change:Discard old culture medium and addof differentiation medium very carefully avoiding touching the bottom of the well. Critical step: To prevent cells from drying out during full media changes, change the medium of one 6-well plate at each time. Add differentiation medium very gently (dropwise) to avoid perturbation of the cell layer. Cell detachments might affect the differentiation efficiency. In case of cell detachment, a confluency above 95% is desired to continue the differentiation.
[per well]
?. [Media changes during differentiation]
From day 1, change 75% of the differentiation medium daily until day 15, and then, every 2 days until day 20.Note: to perform 75% medium change of a working volume of, discard of old medium and addof fresh prepared differentiation medium.
[per well]
3 mL
[per well]
?. [Day 1 and 2]
Warm KSR medium at.Add: LDN193189 SB431542 SHH Purmorphamine FGF-8bMix well by 20x full inversions of the conical tube or flask. Perform medium change: .Place cells back at.
37 °C
[per well]
37 °C
[CO2]
?. [Day 3 and 4]
Warm KSR medium at.Add: LDN193189 SB431542 SHH Purmorphamine FGF-8b CHIR99021Mix well by 20x full inversions of the conical tube or flask. Perform medium change: .Place cells back at.
37 °C
[per well]
37 °C
[CO2]
?. [Day 5 and 6]
Before starting:Prepare enough amount of N2 medium.For of N2 medium, add: Neurobasal medium B27 supplement without vitamin A  N2 supplement  GlutaMAX  Penicillin-Streptomycin  Storage: N2 medium can be stored for 5 days ator for up to one month at. Warm KSR and N2 medium at.Mix:Add: LDN193189 SB431542 SHH Purmorphamine FGF-8b CHIR99021Mix well by 20x full inversions of the conic tube or flask. Perform medium change: .Place cells back at.
500 mL
479 mL
10 mL
5 mL
5 mL
1 mL
4 °C
-20 °C
37 °C
[KSR medium]
[N2 medium]
[per well]
37 °C
[CO2]
?. [Day 7 and 8]
Warm KSR and N2 medium at.Mix:Add: LDN193189 SB431542 SHH CHIR99021Mix well by 20x full inversions of the conical tube or flask. Perform medium change: .Place cells back at.
37 °C
[KSR medium]
[N2 medium]
[per well]
37 °C
[CO2]
?. [Day 9 and 10]
Warm KSR and N2 medium at.Mix:Add: LDN193189 SB431542 SHH CHIR99021Mix well by 20x full inversions of the conical tube or flask. Perform medium change: .Place cells back at.
37 °C
[KSR medium]
[N2 medium]
[per well]
37 °C
[CO2]
?. [Day 11 and 12]
Before starting:Prepare enough amount of NB/B27 medium.For NB/B27 medium, add: Neurobasal medium B27 supplement without vitamin A GlutaMAX Penicillin-StreptomycinStorage: NB/B27 medium can be stored for 5 days ator for up to one month at. Warm NB/B27 medium at.Add: CHIR99021 BDNF Ascorbic acid GDNF db-cAMP TGFβ3 DAPTMix well by 20x full inversions of the conical tube or flask. Perform medium change: .Place cells back at.
500 mL
484 mL
10 mL
5 mL
1 mL
4 °C
-20 °C
37 °C
[per well]
37 °C
[CO2]
?. [Day 13 -15, 17 and 19]
Warm NB/B27 medium at.Add: BDNF Ascorbic acid GDNF db-cAMP TGFβ3 DAPTMix well by 20x full inversions of the conical tube or flask. Perform medium change: .Place cells back at.
37 °C
[per well]
37 °C
[CO2]
?. [Day 20: Replating of mDA neuron precursors at high cell density]
Note: At day 20 of differentiation, mDA neuron precursors can be replated as describe below or cryopreserved. Before starting:Coating of 6-well plates step 1Coat 6-well culture plates withPoly-L-Ornithine (PLO) in PBS. Incubate platesat. Wash plates three times with PBS. Discard PBS and proceed to coating step 2.Coating of 6-well plates step 2Coat 6-well culture plates withLaminin plus Fibronectin, both diluted in PBS. Incubate platesat. Do not store coated plates. Proceed with preparation of plates for seeding cells.Preparation of 6-well plates for seeding cellsWarm NB/B27 medium at.Make NB/B27 complete medium by adding: BDNF Ascorbic acid GDNF db-cAMP TGFβ3 DAPT Y-27632Discard coating reagents and addof NB/B27 complete medium.Keep the plate atforbefore seeding cells.
[go to Step 12]
[per well]
37 °C
[per well]
37 °C
37 °C
[per well]
37 °C
?. [Day 20: Replating of mDA neuron precursors at high cell density]
Warm PBS, Accutase and NB/B27 medium to.
37 Room temperature
?. [Day 20: Replating of mDA neuron precursors at high cell density]
Discard old culture media and wash cells once withof PBS.
[per well]
?. [Day 20: Replating of mDA neuron precursors at high cell density]
Discard PBS and addof Accutase supplemented withY-27632.
[per well]
?. [Day 20: Replating of mDA neuron precursors at high cell density]
After incubation, block Accutase withNB/B27 medium supplemented withY-27632.
[per well]
?. [Day 20: Replating of mDA neuron precursors at high cell density]
Perform a quick microscope inspection of the cells. If a significant number of cell clumps are visible, and repeat the procedure to dissociate remaining cell clumps. A single cell suspension should be obtained.
?. [Day 20: Replating of mDA neuron precursors at high cell density]
Transfer the cell suspension to a conical tube. A total volume ofshould be obtained from a full 6-well plate.
12 mL
?. [Day 20: Replating of mDA neuron precursors at high cell density]
Centrifuge the cell suspension at.
Centrifuge: 200 34, 5 min, 23 10
?. [Day 20: Replating of mDA neuron precursors at high cell density]
Discard the supernatant carefully and addNB/B27 complete medium. Resuspend the cell pellet very gently with a P1,000 pipette by pipetting the cell suspension up and down for 3-6 times.
1 mL
?. [Day 20: Replating of mDA neuron precursors at high cell density]
Complete the volume towith NB/B27 complete medium and mix well by 10ix full inversions of the conical tube.
12 mL
?. [Day 20: Replating of mDA neuron precursors at high cell density]
Perform two separate live-cell counts using a hemocytometer or an automated cell counter. Critical step: Adjust the cell suspension volume accordingly to obtain an accurate counting. Calculate the mean achieved from the two counts and determine the concentration of live cells per milliliter.
A cell viability of 90-99% should be obtained. If lower, reduce the number of pipetting to dissociate the cell clumps to 3-4 times.
?. [Day 20: Replating of mDA neuron precursors at high cell density]
Seed 300,000 cells per cm2 in a total volume ofon 6-well plates coated with PLO/Laminin/Fibronectin (i.e., to make one well of a 6-well plate, seed 2.85 x 10^6 cells per well, considering the growth area of one well equal to 9.5 cm2).
[per well]
?. [Day 21, 23 and 25: Medium change]
From day 21, change 75% of the differentiation medium every 2-3 days.Warm NB/B27 medium at.Add: BDNF Ascorbic acid GDNF db-cAMP TGFβ3 DAPTMix well by 20x full inversions of the conical tube or flask. Perform medium change: .Place cells back at.
37 °C
[per well]
37 °C
[CO2]
?. [Day 27: Treatment with Mitomycin-C]
Before starting: Warm NB/B27 medium at.Make NB/B27 complete medium by adding: BDNF Ascorbic acid GDNF db-cAMP TGFβ3 DAPTPrepare enough amount of NB/B27 complete medium supplemented with Mitomycin-C. Mix well by 20x full inversions of the conical tube or flask.
37 °C
?. [Day 27: Treatment with Mitomycin-C]
Discard old culture medium and addof NB/B27 complete medium supplemented with Mitomycin-C.
[per well]
?. [Day 27: Treatment with Mitomycin-C]
Incubate cells forat.
37 °C
?. [Day 29 and 31: Medium change]
Warm NB/B27 medium at.Add: BDNF Ascorbic acid GDNF db-cAMP TGFβ3 DAPTMix well by 20x full inversions of the conical tube or flask. Perform medium change: .Place cells back at.
37 °C
[per well]
37 °C
[CO2]
?. [Day 32: Replating of mDA neurons at low cell density (optional)]
A second replating of the mDA neurons at low cell density in 96-well plate format can be performed for imaging assays requiring single cell analysis.Before starting:Coating of 96-well plates step 1Coat 96-well culture plates withPoly-L-Ornithine (PLO) in PBS. Incubate platesat. Wash plates three times with PBS. Discard PBS and proceed to coating step 2.Coating of 96-well plates step 2Coat 96-well culture plates withLaminin plusFibronectin, both diluted in PBS. Incubate platesat. Do not store coated plates. Proceed with preparation of plates for seeding cells.Preparation of 96-well plates for seeding cellsWarm NB/B27 medium at.Make NB/B27 complete medium by adding: BDNF Ascorbic acid GDNF db-cAMP TGFβ3 DAPT Y-27632Cool down NB/B27 complete medium to.Add: Laminin FibronectinDiscard coating reagents and addof NB/B27 complete medium supplemented with Laminin and Fibronectin.Keep the plate atforbefore seeding cells.
[per well]
37 °C
[per well]
37 °C
37 °C
0 Room temperature
[per well]
37 °C
?. [Day 32: Replating of mDA neurons at low cell density (optional)]
Dissociate cells following the steps described for replating mDA neurons on day 20.
?. [Day 32: Replating of mDA neurons at low cell density (optional)]
Seed 100,000 cells per cm2 in a total volume ofin 96-well plates (i.e., to make one well of a 96-well plate, seed 32,000 cells per well, considering the growth area of one well equal to 0.32 cm2).
[per well]
?. [Day 32: Replating of mDA neurons at low cell density (optional)]
Keep cells intact for at.
37 °C
[CO2]
?. [Day 34: Treatment with Mitomycin-C]
Before starting: Warm NB/B27 medium at.Make NB/B27 complete medium by adding: BDNF Ascorbic acid GDNF db-cAMP TGFβ3 DAPTPrepare enough amount of NB/B27 complete medium supplemented withMitomycin-C. Mix well by 20x full inversions of the conical tube or flask.
37 °C
?. [Day 34: Treatment with Mitomycin-C]
Discard old culture medium and addof NB/B27 complete medium supplemented with Mitomycin-C.
[per well]
?. [Day 34: Treatment with Mitomycin-C]
Incubate cells forat.
37 °C
?. [Day 34: Treatment with Mitomycin-C]
Discard medium containing Mitomycin-C and addof NB/B27 complete medium.
[per well]
?. [Day 34: Treatment with Mitomycin-C]
Place cells back at.
37 °C
[CO2]
?. [Day 27: Treatment with Mitomycin-C]
Discard medium containing Mitomycin-C and addof NB/B27 complete medium.
[per well]
?. [Day 27: Treatment with Mitomycin-C]
Place cells back at.
37 °C
[CO2]
?. [Every 2-3 days: Change medium for terminal differentiation of mDA neurons]
Warm NB/B27 medium at.Add: BDNF Ascorbic acid GDNF db-cAMP TGFβ3 DAPTMix well by 20x full inversions of the conical tube or flask. Perform medium change: .Place cells back at.
37 °C
[per well]
37 °C
[CO2]
?. [Every 10 days: Supplement differentiation medium with Laminin and Fibronectin]
Warm NB/B27 medium at.Make NB/B27 complete medium by adding: BDNF Ascorbic acid GDNF db-cAMP TGFβ3 DAPTCool down NB/B27 complete medium to.Add: Laminin FibronectinMix well by 20x full inversions of the conical tube or flask.
37 °C
0 Room temperature
?. [Every 10 days: Supplement differentiation medium with Laminin and Fibronectin]
Discard old culture medium. Perform medium change as following: in 6-well plates in 96-well plates
[per well]
[per well]
?. [Every 10 days: Supplement differentiation medium with Laminin and Fibronectin]
Images of mDA neurons differentiated until day 65.
?. [Day 20: Replating of mDA neuron precursors at high cell density]
Keep cells intact at.
37 °C
[CO2]
?. [Cryopreservation of mDA neurons on day 20]
Before starting:Supplement Synth-a-Freeze cryopreservation medium withY-27632.Place the cryopreservation medium and a CoolCell LX container on ice.
?. [Cryopreservation of mDA neurons on day 20]
Perform cells dissociation and previously described.
?. [Cryopreservation of mDA neurons on day 20]
After determining the number of live cells, centrifuge the cell suspension at.
Centrifuge: 200 34, 5 min, 23 10
?. [Cryopreservation of mDA neurons on day 20]
Resuspend the cell pellet very gently in ice cold Synth-a-Freeze medium to 3-5x10^6 cells/mL.
?. [Cryopreservation of mDA neurons on day 20]
Distributeof the cell suspension in cryogenic vials.
1 mL
?. [Cryopreservation of mDA neurons on day 20]
Transfer cryogenic vials to a pre-cooled CoolCell LX container.
?. [Cryopreservation of mDA neurons on day 20]
Transfer cells to the vapor phase of a liquid nitrogen storage facility.
?. [Thawing cryopreserved mDA neurons]
One day before thawing:Coat 6-well culture plates withGFR Matrigel diluted in ice cold DMEM/F-12 mediumat.
37 °C
?. [Day of thawing]
Before startingPrepare: Wash mediumWarm NB/B27 medium at.Add: Y-27632.Recovery mediumWarm NB/B27 medium at.Make NB/B27 complete medium by adding: BDNF Ascorbic acid GDNF db-cAMP TGFβ3 DAPT Y-27632Cool down NB/B27 complete medium to.AddGFR Matrigel and mix well by 20x full inversions of the conical tube or flask. 6-well platesDiscard coating reagent and addof recovery medium.Keep plates atforbefore seeding cells.Conical centrifuge tubes Label 15-mL conical tubes and fill withwash medium. Keep at .
37 °C
37 °C
0 Room temperature
[per well]
37 °C
5 mL
0 Room temperature
?. [Day of thawing]
Thaw cryopreserved mDA neurons by placing the cryogenic vial containing cells in awater bath for approximatelyor until no ice is visible but the liquid is still cold.
37 °C
?. [Day of thawing]
Fill a 5-mL serological pipette withwash medium and collect the thawed cell suspension very carefully.
4 mL
?. [Day of thawing]
Transfer the cell suspension dropwise to the 15-mL conical tube containingof wash medium.
5 mL
?. [Day of thawing]
Wash the cryogenic vial withwash medium to collect residual cells and transfer to the 15-mL conical tube.
1 mL
?. [Day of thawing]
Centrifuge the cell suspension at.
Centrifuge: 200 34, 5 min, 23 10
?. [Day of thawing]
Discard the supernatant carefully and resuspend the cell pellet withrecovery medium by gently pipetting up and down 3-6 times to obtain a homogeneous cell suspension.
1 mL
?. [Day of thawing]
Complete the volume with recovery medium toand mix well by 10x full inversions of the tube.
12 mL
?. [Day of thawing]
Perform two separate live-cell counts using a hemocytometer or an automated cell counter. Critical step: Adjust the cell suspension volume accordingly to obtain an accurate counting. Calculate the mean achieved from the two counts and determine the concentration of live cells per milliliter.
A cell viability of 85-98% should be obtained. A lower cell viability than expected might require more gentle manipulation of the cells during thawing. Reduce the number of pipetting and thaw less cells lines at once to speed the process and seed cells quickly.
?. [Day of thawing]
Seed 400,000 cells per cm2 in a total volume ofon 6-well plates coated with GFR Matrigel (i.e., to make one well of a 6-well plate, seed 3.8 x 10^6 cells per well, considering the growth area of one well equal to 9.5 cm2).
[per well]
?. [Day of thawing]
Keep cells intact at.
37 °C
[CO2]
?. [Day 1, 3 and 5 after thawing: medium change]
Warm NB/B27 medium at.Add: BDNF Ascorbic acid GDNF db-cAMP TGFβ3 DAPTMix well by 20x full inversions of the conical tube or flask. Perform medium change: .Place cells back at.
37 °C
[per well]
37 °C
[CO2]
?. [Day 5 after thawing: replating mDA neurons]
For replating of mDA neurons on day 5.Seed cells at desired densities. Change differentiation mediumevery 2-3 days
?. [Day 5 after thawing: replating mDA neurons]
and seed cells at desired densities according your experimental design.
?. [Day 5 after thawing: replating mDA neurons]
From now on, change differentiation mediumevery 2-3 days.
?. [Passaging of iPSCs with Gentle Differentiation Reagent (GDR)]
Discard coating and addof E8 medium. Keep the plate atforbefore seeding cells.
[per well]
37 °C
?. [Day -2: Coating of 6-well plates with Growth Factor Reduced (GFR) Matrigel]
Thaw GFR Matrigeland dilute toin ice cold DMEM/F-12.
on ice
?. [Day -1: Single cell dissociation and seeding for differentiation]
Incubate cells atfor.
37 °C
?. [Day 20: Replating of mDA neuron precursors at high cell density]
Dissociate cells with a P1,000 pipette by pipetting the cell suspension up and down for 3-6 times.
?. [Day -1: Single cell dissociation and seeding for differentiation]
Rock the plate back-forth and side-to-side forto achieve an even spread of cells in the plate well.
?. [Passaging of iPSCs with Gentle Differentiation Reagent (GDR)]
Place the plate back toand keep intactallowing cells to recover from passaging.
37 °C
[CO2]
?. [Day 20: Replating of mDA neuron precursors at high cell density]
Rock the plate back-forth and side-to-side forto achieve an even spread of cells in the plate well.
?. [Day 32: Replating of mDA neurons at low cell density (optional)]
Rock the plate back-forth and side-to-side forto achieve an even spread of cells in the plate well.
?. [Day of thawing]
Rock the plate back-forth and side-to-side forto achieve an even spread of cells in the plate well.
?. [Day 7 after thawing: Replate mDA neurons]
Replate mDA neurons 7 days after thawing following the procedure described for replating mDA neurons on day 32.
?. [Day 7 after thawing: Replate mDA neurons]
Treat cells with Mitomicyn-Cafter replating following the same procedure described on day 34 of differentiation.
?. [Day 7 after thawing: Replate mDA neurons]
Images of thawed mDA neurons differentiated until day 65.
?. [Day 7 after thawing: Replate mDA neurons]
Seed cells at desired cell density and plate format.Examples:High cell density in 6-well plates: seed 300,000 cells per cm2.Low cell density in 96-well plates: seed 100,000 cells per cm2.
?. [Day 20: Replating of mDA neuron precursors at high cell density]
Incubate cells atfor.
37 °C
?. [Day 7 after thawing: Replate mDA neurons]
For long-term culture of mDA neurons, change differentiation medium every 2-3 days as described previously.
?. [Every 10 days: Supplement differentiation medium with Laminin and Fibronectin]
Place cells back at.
3 °C
[CO2]
?. [Day 21, 23 and 25: Medium change]
Images of mDA neurons differentiated until day 26.
?. [Cryopreservation of mDA neurons on day 20]
Keep the CoolCell LX container at.
-80 °C
?. [Passaging of iPSCs with Gentle Differentiation Reagent (GDR)]
Incubate cells atfor.
37 °C