Oct 20, 2020
Generation of iPSC-derived dopaminergic neurons
- Julie Jerber1,
- James Haldane1,
- Juliette Steer1,
- Daniel Pearce1,
- Minal Patel1
- 1Wellcome Sanger Institute
- Cellular Generation and Phenotyping
Protocol Citation: Julie Jerber, James Haldane, Juliette Steer, Daniel Pearce, Minal Patel 2020. Generation of iPSC-derived dopaminergic neurons. protocols.io https://dx.doi.org/10.17504/protocols.io.bjpgkmjw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 13, 2020
Last Modified: October 20, 2020
Protocol Integer ID: 40392
Keywords: Dopaminergic, Differentiation, iPSC, Human, Neurons, dopaminergic neurons from human ipsc, derived dopaminergic neuron, dopaminergic neurons this protocol, dopaminergic neuron, generation of ipsc, human ipsc, ipsc, day long differentiation method, long differentiation method, single 12 well plate,
Abstract
This protocol describes a method for the production of dopaminergic neurons from human iPSCs, using a 52-day long differentiation method adapted from doi.org/10.1038/nature10648.
Steps in this protocol assume the use of a single 12 well plate for the purposes of differentiation; adjust volumes accordingly depending on labware used.
Guidelines
Unless otherwise stated, all steps should be performed under sterile conditions in a CL2 biological safety cabinet.
Below are example images showing a neuronal culture undergoing a 52-day differentiation to dopaminergic neurons.
Materials
MATERIALS
SB431542 10 mg
STEMCELL Technologies Inc.Catalog #72234
Penicillin-Streptomycin (10,000 U/mL)Thermo Fisher ScientificCatalog #15140122
Falcon™ 15mL Conical Centrifuge TubesFisher ScientificCatalog #14-959-53A
DMEM/F-12, GlutaMAX™ supplementThermo FisherCatalog #10565018
MEM Non-Essential Amino Acids Solution (100X)Thermo FisherCatalog #11140050
UltraPure™ 0.5M EDTA, pH 8.0Thermo FisherCatalog #15575020
DMEM/F-12, no phenol redThermo FisherCatalog #21041025
Neurobasal™ MediumThermo FisherCatalog #21103049
2-Mercaptoethanol (50 mM)Thermo FisherCatalog #31350010
StemPro™ Accutase™ Cell Dissociation ReagentThermo FisherCatalog #A1110501
Essential 8™ MediumThermo FisherCatalog #A1517001
KnockOut™ DMEMThermo Fisher ScientificCatalog #10829018
Knockout serum replacement (KSR)Gibco - Thermo Fisher ScientificCatalog #10828028
Y-27632 dihydrochlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #Y0503
DPBSInvitrogen - Thermo FisherCatalog #14190
Vitronectin XF™STEMCELL Technologies Inc.Catalog ##07180
DNase Vial (D2)Worthington Biochemical CorporationCatalog #LK003170
PDS Kit Papain VialWorthington Biochemical CorporationCatalog #LK003176
PluriStrainer Mini 40µmpluriSelect Life ScienceCatalog #43-10040-60
1.5 ml TubeOne® Microcentrifuge Tubes Natural (Sterile)StarLabCatalog #S1615-5510
Bovine Serum AlbuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #A0281
12-well Falcon™ Polystyrene Microplates Fisher ScientificCatalog #10489482
Recombinant Human TGF-β3peprotechCatalog #100-36E
SAG hydrochloride Smo activatorAbcamCatalog #ab145866
RotenoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #R8875
Recombinant Human FGF-8apeprotechCatalog #100-25A
LDN193189 hydrochlorideCatalog #SML0559
Human BDNF (powder)Cambridge UniversityCatalog #T10583
L-Ascorbic acidMerck MilliporeSigma (Sigma-Aldrich)Catalog # A4544
PurmorphamineTocrisCatalog #4551
Water (for embryo transfer sterile-filtered BioXtra suitable for mouse embryo cell culture)Merck MilliporeSigma (Sigma-Aldrich)Catalog #W1503
Dibutyryl cAMP sodium saltMerck MilliporeSigma (Sigma-Aldrich)Catalog #https://www.sigmaaldrich.com/cat
Citric AcidMerck MilliporeSigma (Sigma-Aldrich)Catalog #C2404
Equipment:
- Centrifuge (for both 15mL & 1.5mL tubes)
- Pipette boy
- Sterile 5/10/25/50mL stripettes
- P1000, P200, P20, P10 pipettes and filter tips
- Vacuum aspirator and tips
- Microbiology Safety Cabinet (MSC)
- Light Microscope
- Scale (for making up BSA)
- Method of Cell Counting (NucleoCounter®NC-200™)
- 37 °C , 5% CO2 incubator
Safety warnings
- Rock inhibitor (Y-27632) - Harmful if swallowed, in contact with skin or inhaled.
- CHIR99021 - Powder is Toxic by inhalation and ingestion.
- Citric acid powder - Irritant
- 2-Mercaptoethanol solution - Irritant
- Chemicals dissolved in DMSO (CHIR99021, DAPT, LDN193189, Purmorphamine, SAG, SB431542) - Increased risk of absorption through skin.
Before start
iPSCs should be prepared prior to use of this protocol; cultured for at least 2 passages post-thaw in Essential 8 medium on vitronectin (VN-XF)-coated plates.
Supplement Preparation
Supplement Preparation:
Supplements to be added to media (on day of use) in this protocol are listed in the table below, with recommended stock concentrations for aliquots, concentrations to be used on cells and the diluent needed for each reagent. It is recommended to aliquot supplements in volumes for single use.
Aliquots should be stored at -80 °C .
| Supplement | Recommended Stock Concentration | Final Concentration to be used on cells | Diluent | |
| Ascorbic Acid | 200 mM | 0.2 mM | Water for Cell Culture | |
| hBDNF | 100 µg/mL | 20 ng/mL | 0.1% BSA in Water for Cell Culture | |
| CHIR99021 | 10 mM | 3 µM | DMSO | |
| DAPT | 50 mM | 10 µM | DMSO | |
| dibutyryl cAMP | 100 mM | 0.5 mM | Water for Cell Culture | |
| hFGF8a | 100 µg/mL | 100 ng/mL | 0.1% BSA in Water for Cell Culture | |
| hGDNF | 100 µg/mL | 20 ng/mL | 0.1% BSA in Water for Cell Culture | |
| LDN193189 | 1 mM | 100 nM | DMSO | |
| Purmorphamine | 20 mM | 1 µM | DMSO | |
| SAG | 1 mM | 100 nM | DMSO | |
| SB431542 | 10mM | 10µM | DMSO | |
| TGF-β3 | 25µg/mL | 1ng/mL | 10mM Citric Acid & 0.1% BSA in Water for Cell Culture (1:3)* |
List of media supplements used in the protocol.
* When reconstituting, add 1 part 10mM Citric acid first, followed by 3 parts 0.1% BSA.
10% BSA stock solution (100 mg/mL):
Dissolve Bovine Serum Albumin powder in sterile Water for Cell Culture at 100 mg/mL. Filter sterilise with a 0.2 µm sterilising grade filter. Aliquot into sterile microcentrifuge tubes. Store at -20 °C. Thaw at room temperature and resuspend prior to use.
0.1% BSA Solution (1mg/mL):
Dilute a thawed aliquot of 10% BSA solution 100-fold with sterile Water for Cell Culture to create a 0.1% BSA solution; e.g. add 100µL 10% BSA to 9.9mL water.
Plate Preparation (Day -2 & 19)
Preparing Geltrex-coated Plate(s) (Day -2 & 19):
This section covers the preparation of the 12 well Geltrex-coated plate(s) that:
- iPSCs will be seeded onto for neural induction (Day -1)
- Early neural progenitors will be seeded onto for neuronal maturation (Day 20)
We recommend preparing the coated plates 1 day prior to the day of use.
Prepare 10% Geltrex stock aliquots:
- Thaw a vial of Geltrex at 4 °C overnight.
- Dilute 1:10 with ice-cold DMEM:F12 w/o phenol red.
- Aliquot into sterile tubes in single use volumes. Ensure the temperature does not exceed 15 °C while working with Geltrex.
- Store aliquots at -80 °C
Prior to coating plates, thaw 10% Geltrex aliquots at 4 °C overnight or on the day required.
Prepare 1% Geltrex by adding 1.25 mL of 10% Geltrex to 11.25 mL of ice-cold DMEM:F12 w/o phenol red, keeping the Geltrex on ice during the process.
Note: if lumps are observed in the Geltrex aliquot then it should not be used (to avoid premature gelling).
Add 1 mL of cold 1% Geltrex to each well of the 12 well plate.
Incubate the plate at 37 °C for at least 02:00:00 , but not more than 24 hours.
Equilibrate plates to room temperature for 1 hour before use.
Seeding iPSCs for neural induction (Day -1)
Seeding iPSCs for Neural Induction (Day -1):
This section covers the seeding of iPSCs onto a prepared 12 well Geltrex-coated plate for neural induction.
Note: Our lab carried out Day -1 steps on a Friday and any days mentioned in the protocol assume this to be the case.
Cells are seeded at a density of 200,000 cells/cm2 or 760,000 cells per well in a 3.8cm2 12 well. The total number of cells required to seed a 12 well plate is at least 9.2x106 cells.
Select an adequate number of high quality iPSC wells/ plates/ flasks at a suitable density for harvesting. As a general guide a 60-80% confluent well on a 6 well plate (9.6cm2) should yield 2-4 million cells.
Prepare E8-ROCKi medium by supplementing E8 with ROCK inhibitor (Y-27632) to a final concentration of 10μM.
Aspirate the media from the well(s) of the iPSC culture and add 2 mL of DPBS (-/-) to wash.
Aspirate the DPBS (-/-) and add 1 mL of warm Accutase to each well.
Incubate cells for 00:10:00 at 37 °C
Inspect the cultures after incubation to confirm cell detachment.
If cells are still partially adhered, incubate for a further 3 minutes.
Add 2 mL of E8-ROCKi to each well and repeatedly wash the media over the well(s) (with a P1000 pipette) to detach the cells and dissociate them to a single-cell suspension.
Confirm a majority single cell suspension has been obtained under the microscope before proceeding to the next step.
If there are a lot of cell aggregates still present then triturate the cell suspensions further with a pipette. Alternatively, centrifuge the cell suspension at 250xg for 3 minutes and resuspend the cell pellet with 1mL E8-ROCKi using a P1000 pipette.
Collect the cell suspension from each dissociated well and transfer into 15mL centrifuge tube(s) capped with a 40µm mini cell strainer.
Centrifuge cells at 250 x g, Room temperature, 00:03:00 .
Note: These next steps describe a 1/10 dilution to prepare a 500uL suspension for cell counting with a Nucleocounter. Adjust as necessary for your chosen method of counting.
Prepare a 1.5mL tube for cell counting. Add 450 µL E8-ROCKi to the tube.
Remove supernatant and gently re-suspend the cell pellet(s) in 1 mL of E8-ROCKi medium with a P1000 pipette. Pipette up and down several times to ensure a single cell suspension is created.
If there are mutiple tubes then combine the cell suspensions together in one tube.
Top up to 5 mL with E8-ROCKi and mix well.
Immediately transfer 50 µL of the re-suspended cell suspension into the 1.5mL tube prepared in step 15 and perform a viable cell count.
Prepare a cell suspension of 9.9x106 viable iPS cells in 13 mL E8-ROCKi. This is enough to seed 12 wells at a seeding density of 760,000 cells/well (or 2x105 cells/cm2).
If seeding a different number of wells always prepare a slightly higher volume than required to account for volume loss during processing.
Prepare the Geltrex-coated 12 well plate for seeding by aspirating excess Geltrex and adding 1 mL of E8-ROCKi into each coated well to be used. Do not let the coated wells dry out.
Thoroughly mix and resuspend the cell suspension prepared in step 18 by pipetting up and down with a stripette and then transfer 1 mL to each well of the 12 well plate.
Transfer the plate to a tissue culture incubator set at 37 °C and 5% CO2. Agitate plate gently on the incubator shelf to ensure even distribution of cells across wells and then leave undisturbed overnight.
Neural Induction (Day 0):
Inspect the plate(s) and verify that the cells are well-distributed and ~90-100% confluent.
From this point, carry out a daily media change as described in the 'Media Change' section until Day 20 when the culture is passaged.
Media Change (Day 0-52)
Media Change (Day 0-52):
Confirm how much media you will need for the next few days before making up base media (or for the day in the case of supplemented media).
Base media should be warmed to between Room temperature and 37 °C before adding supplements. Do not put complete supplemented media in the water bath.
Perform media change very gently, especially later on into the differentiation process, as cells can detach very easily. We recommend tilting plates and dispensing media down the side of each well to reduce force applied to the cell layer.
Refer to the 'Media Composition' section for details on each media type and which media to use for Day 0-12.
| Day | Media Change | Media Type | Media feed volume per well | |
| 0-12 | Daily | Differs (See 'Media Composition') | 2 mL | |
| 13-19 | Daily | Maturation Medium | 2 mL | |
| 20 | Passage | Maturation Medium | - | |
| 21-22 | Daily | Maturation Medium | 1 mL | |
| 23-33 | Every other day | Maturation Medium | 2 mL | |
| 34-52 | Every 2 (*or 3) days | Maturation Medium | 2mL (*or 3mL) |
Media change instructions across the differentiation process.
* Our lab fed cells with 3mL of media on Fridays from Day 34 onward to avoid media change during weekends.
Media Composition
Base Media:
Listed below are the reagent quantities needed for composition of the three types of base media used throughout this protocol. All of these base media require further supplements prior to media change; supplements needed are listed in the Neural Induction Media and Maturation Medium steps.
Note: Use-within dates are only for the base media, not for media with added supplements. Supplemented media (i.e. Neural Induction and Maturation media) should only be used on the day of preparation.
| KOSR (use within 1 week) | 100mL | 1mL | [Final] | |
| Knockout DMEM | 81.98 mL | 819.8 µL | - | |
| Knockout Serum Replacement (KSR) | 15 mL | 150 µL | 15% | |
| Glutamax (100X) | 1 mL | 10 µL | 1X | |
| MEM Non Essential Amino Acids (NEAA) (100X) | 1 mL | 10 µL | 1mM | |
| 2-mercaptoethanol (50mM) | 20 µL | 0.2 µL | 0.01mM | |
| Penicillin-Streptomycin (10,000 U/mL) | 1 mL | 10 µL | 100 U/mL |
A base media used for days 0-10 of the differentiation process. Use of Penicillin-Streptomycin is optional.
| NNB (Use within 3 days) | 100mL | 1mL | [Final] | |
| Neurobasal medium | 96.5 mL | 965 µL | - | |
| N2 (100X) | 0.5 mL | 5 µL | 0.5X | |
| B27 (50X) | 1 mL | 10 µL | 0.5X | |
| Glutamax (100X) | 1 mL | 10 µL | 1X | |
| Penicillin-Streptomycin (10,000 U/mL) | 1 mL | 10 µL | 100 U/mL |
A base media used for days 5-10 of the differentiation process. Use of Penicillin-Streptomycin is optional.
| NB/B27 (Use within 3 days) | 100mL | 1mL | [Final] | |
| Neurobasal medium | 96 mL | 0.96 mL | - | |
| B27 (50X) | 2 mL | 20 µL | 1X | |
| Glutamax (100X) | 1 mL | 10 µL | 1X | |
| Penicillin-Streptomycin (10,000 U/mL) | 1 mL | 10 µL | 100 U/mL |
A base media used for days 11-52 of the differentiation process. Use of Penicillin-Streptomycin is optional.
Neural Induction Media:
This is the media that will be used for the neural induction stage (Day 0-12). Each complete medium must be used only on the day of preparation, though the unsupplemented base media (varies) can be prepared earlier and used within the associated timespan listed in the 'Base Media' tables.
During neural induction, the media is gradually altered from 100% KOSR to 25% KOSR/75% NNB.
Days listed in the table assume that iPSC seeding (Day -1) takes place on a Friday.
| Day 0 | Neural Induction Medium-1 | 28 mL | 1mL | [Final] | |
| (Sat) | KOSR | 28mL | 1 mL | 100% | |
| LDN193189 (stock 1 mM) | 2.8 µL | 0.1 µL | 100 nM | ||
| SB431542 (stock 10 mM) | 28 µL | 1 µL | 10 µM | ||
| Day 1-2 | Neural Induction Medium-2 | 28 mL | 1 mL | ||
| (Sun/Mon) | KOSR | 28mL | 1 mL | 100% | |
| LDN193189 (stock 1 mM) | 2.8 µL | 0.1 µL | 100 nM | ||
| SB431542 (stock 1 0mM) | 28 µL | 1 µL | 10 µM | ||
| SAG (stock 1 mM) | 2.8 µL | 0.1 µL | 100 nM | ||
| Purmorphamine (stock 20 mM) | 2.8 µL | 0.1 µL | 2 µM | ||
| FGF8a (stock 100 µg/mL) | 28 µL | 1 µL | 100ng/mL | ||
| Day 3-4 | Neural Induction Medium-3 | 28 mL | 1 mL | [Final] | |
| (Tue/Wed) | KOSR | 28mL | 1 mL | 100% | |
| LDN193189 (stock 1 mM) | 2.8 µL | 0.1 µL | 100 nM | ||
| SB431542 (stock 10 mM) | 28 µL | 1 µL | 10 µM | ||
| SAG (stock 1 mM) | 2.8 µL | 0.1 µL | 100 nM | ||
| Purmorphamine (stock 20 mM) | 2.8 µL | 0.1 µL | 2 µM | ||
| FGF8a (stock 100 µg/mL) | 28 µL | 1 µL | 100ng/mL | ||
| CHIR99021 (stock 10 mM) | 8.4 µL | 0.3 uL | 3 µM | ||
| Day 5-6 | Neural Induction Medium-4 | 28 mL | 1 mL | [Final] | |
| (Thu/Fri) | KOSR | 21 mL | 0.75 mL | 75% | |
| NNB | 7 mL | 0.25 mL | 25% | ||
| LDN193189 (stock 1 mM) | 2.8 µL | 0.1 µL | 100 nM | ||
| SAG (stock 1mM) | 2.8 µL | 0.1 µL | 100 nM | ||
| Purmorphamine (stock 20mM) | 2.8 µL | 0.1 µL | 2 µM | ||
| FGF8a (stock 100 µg/mL) | 28 µL | 1 µL | 100ng/mL | ||
| CHIR99021 (stock 10 mM) | 8.4 µL | 0.3 uL | 3 µM | ||
| Day 7-8 | Neural Induction Medium-5 | 28 mL | 1 mL | [Final] | |
| (Sat/Sun) | KOSR | 14 mL | 0.5 mL | 50% | |
| NNB | 14 mL | 0.5 mL | 50% | ||
| LDN193189 (stock 1 mM) | 2.8 µL | 0.1 µL | 100 nM | ||
| CHIR99021 (stock 10 mM) | 8.4 µL | 0.3 uL | 3 µM | ||
| Day 9-10 | Neural Induction Medium-6 | 28 mL | 1 mL | [Final] | |
| (Mon/Tue) | KOSR | 7 mL | 0.25 mL | 25% | |
| NNB | 21 mL | 0.75 mL | 75% | ||
| LDN193189 (stock 1 mM) | 2.8 µL | 0.1 µL | 100 nM | ||
| CHIR99021 (stock 10 mM) | 8.4 µL | 0.3 uL | 3 µM | ||
| Day 11-12 | Neural Induction Medium-7 | 28 mL | 1 mL | [Final] | |
| (Wed/Thu) | Maturation Medium | 28 mL | 1 mL | 100% | |
| CHIR99021 (stock 10 mM) | 8.4 µL | 0.3 µL | 3 µM |
Various base media supplemented for days 0-12 of the differentiation process. Note that volumes are based on recommended stock concentrations (column 'B'). Column 'C' gives recommended volumes to prepare, assuming use of a single 12 well plate for this protocol (2mL per well plus excess).
Maturation Medium:
This is the medium that will be used for the majority of this protocol (Day 13 onward). The complete supplemented medium must be used only on the day of preparation, though the base medium (NB/B27) can be prepared earlier and used within 3 days.
| Maturation Medium (Use within 1 day) | 28mL | 1mL | [Final] | |
| NB/B27 | ||||
| Neurobasal medium | 26.88 mL | 960 µL | - | |
| B27 (50X) | 560 µL | 20 µL | 1X | |
| Glutamax (100X) | 280 µL | 10 µL | 1X | |
| Penicillin-Streptomycin (10,000 U/mL) | 280 µL | 10 µL | 100 U/mL | |
| Supplements | ||||
| BDNF (stock 100 µg/mL) | 5.6 µL | 0.2 µL | 20 ng/mL | |
| DAPT (stock 50 mM) | 5.6 µL | 0.2 µL | 10 uM | |
| GDNF (stock 100 µg/mL) | 5.6 µL | 0.2 µL | 20 ng/mL | |
| TGFB3 (stock 25 µg/mL) | 1.12 µL | 0.04 µL | 1 ng/mL | |
| Dibutyryl cAMP (stock 100mM) | 140 µL | 5 µL | 0.5 mM | |
| Ascorbic acid (stock 200mM) | 28 µL | 1 µL | 0.2 mM |
The NB/B27 base media supplemented for days 13-52 of the differentiation process. Note that volumes are based on recommended stock concentrations (column 'A'). Use of Penicillin-Streptomycin is optional. Column 'B' gives recommended volume to prepare for most days, assuming use of a single 12 well plate for this protocol (2mL per well plus excess).
Passage (Day 20)
Passaging of Neuro-epithelial sheet (Day 20):
This section covers the passaging of the neuro-epithelial sheet on Day 20. Cells are lifted and re-seeded for neuronal maturation at 3.5x105 cells/cm2 on a 12 well Geltrex-coated plate (prepare prior to these steps as described in the 'Plate Preparation' section). This is the only passage during the differentiation process.
Note: These steps assume seeding back onto one 12 well plate, however we found that we consistently had more than enough cells to seed two 12 well plates after this passage; adjust volumes as needed.
Prepare 30mL of Wash Buffer 1 and 12mL Wash Buffer 2 for every 12 well plate to be dissociated.
Supplement 42 mL DMEM:F12 + GlutaMAX with ROCK inhibitor (Y-27632) to a final concentration of 10 micromolar (µM) .
Split this into two tubes of 30 mL (for Wash Buffer 1) and 12 mL (Wash Buffer 2).
Wash Buffer 1 will be further supplemented with DNase1 in step 34.
1 vial DNase (D2) should be added for every 15mL of Wash Buffer 1 prepared. However, DNase should only be added immediately prior to use of the buffer when the cells are nearing the end of their dissociation. Keep the DNase at 4 °C until use.
Prepare Dissociation Buffer by supplementing 10 mL Accutase (warmed) with 2 vials Papain (PDS Kit).
Reconstitute the lyophilised papain with the Accutase, replace the lid on the vial and invert several times ensuring all powder in the vial and on the lid is dissolved. Transfer solution back into the Dissociation Buffer tube.
0.5mL Dissociation Buffer is required per well to be harvested. 5 wells of a 12 well plate (2.5mL Dissociation Buffer) will provide enough cells to seed onto one 12 well plate; adjust volumes as needed.
Prepare Seeding Medium by supplementing 30 mL of Maturation Medium with ROCK inhibitor (Y-27632) to a final concentration of 10μM.
Aspirate the media from the well(s) of the neuronal culture and gently add 1 mL of DPBS (-/-) without disturbing the cell layer.
Aspirate the DPBS (-/-) and add 0.5 mL of Dissociation Buffer to each well.
Transfer cells to a 37 °C 5% CO2 tissue culture incubator. Incubate for 00:20:00 .
Complete the preparation of Wash Buffer 1 within the last 5 minutes of incubation of the cells.
- Retrieve the DNase (D2) vial(s) from 4 °C storage.
- Reconstitute the vial(s) by adding 250 µL Wash Buffer 1 to each vial to make a 2 mg/mL solution. Make sure to reconstitute all the powder (check the lid). Avoid vigorous mixing - do not vortex.
- Transfer the entire contents of the reconstituted vial(s) back into the Wash Buffer 1 tube. Use 250µL (1 vial) for every 15 mL buffer.
Following incubation inspect the cells under a microscope.
The cells should be detaching from the plate and the cell layer should have a darkened appearance. Cells will have a rounded appearance as they dissociate and individual cells should be visible at the edges or in gaps in the cell layer.
Optional: Test for ease of dissociation by gently pipetting ~100 µL of the Dissociation Buffer against the cells with a P1000 pipette. If cells do not dissociate easily, extend digestion by 00:03:00 and repeat this step, but do not exceed 30 mins.
Add 2 mL of Wash Buffer 1 per well.
Repeatedly wash the buffer over the well(s) with a P1000 pipette to detach the cells and dissociate them to a single-cell suspension.
Sufficient dissociation is critical at this step. It is recommended to check the cell suspension under a microscope and if necessary pipette the cell suspension up and down further until the majority is single cells. Try to avoid over-pipetting the cells as this could affect viability.
Transfer the cell suspension into a 15mL centrifuge tube capped with a 40µm cell strainer.
Optional: If there are residual cells in the plate, wash well(s) with 0.5 mL of Wash Buffer 1 and transfer this suspension into the tube from the previous step.
Centrifuge cells at 150 x g, Room temperature, 00:03:00
Aspirate supernatant and gently re-suspend the cell pellet in 12 mL (~1mL per well harvested) of Wash Buffer 2.
Centrifuge cells at 150 x g, Room temperature, 00:03:00 .
Note: These next steps describe a 1/10 dilution to prepare a 500uL suspension for cell counting with a Nucleocounter. Adjust as necessary for your chosen method of counting.
Prepare a 1.5mL tube for cell counting. Add 450 µL Wash Buffer 2 to the tube.
Aspirate supernatant and gently re-suspend the cell pellet in 5 mL of Seeding Medium.
Immediately transfer 50 µL of the re-suspended cell suspension into the 1.5mL tube prepared in step 44 and perform a viable cell count.
Prepare a cell suspension of 1.729x107 viable cells in 13 mL Seeding Medium. This is enough to seed 12 wells at a density of 1.33x106 cells per well.
If seeding a different number of wells always prepare a slightly higher volume than required to account for volume loss during processing.
Aspirate excess Geltrex from the prepared 12 well plate and immediately add 1 mL of Seeding Medium into each coated well to be used. Do not let the coated wells dry out.
Thoroughly mix and resuspend the cell suspension prepared in step 47 by pipetting up and down with a stripette. Transfer 1 mL of the cell suspension to each well of the prepared Geltrex 12 well plate.
Transfer the plate to a tissue culture incubator set at 37 °C and 5% CO2. Agitate the plate gently on the incubator shelf to ensure even distribution of cells across wells and then leave undisturbed overnight.
On Day 21 inspect the plate(s) and verify that the cells are attached and well distributed.
Continue to maintain the culture with media changes as described in the 'Media Change' section up to Day 52 (or beyond, according to your requirements)