Jan 12, 2023
Version 2
Preparing MEF-cultured hPSCs for nucleofection V.2
- 1University of California, Berkeley;
- 2Albert Einstein College of Medicine
Protocol Citation: Hanqin Li, Oriol Busquets, Steven Poser, Dirk Hockemeyer, Frank Soldner 2023. Preparing MEF-cultured hPSCs for nucleofection. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l611ddvqe/v2Version created by Devin E Snyder
Manuscript citation:
Hanqin Li, Oriol Busquets, Yogendra Verma, Khaja Mohieddin Syed, Nitzan Kutnowski, Gabriella R Pangilinan, Luke A Gilbert, Helen S Bateup, Donald C Rio, Dirk Hockemeyer, Frank Soldner (2022) Highly efficient generation of isogenic pluripotent stem cell models using prime editing eLife 11:e79208
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: January 12, 2023
Last Modified: February 17, 2023
Protocol Integer ID: 75228
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's
Grant ID: ASAP-000486
Abstract
This protocol describes the procedure or preparing MEF-cultured human pluripotent stem cells (hPSCs) for the delivery of plasmids, mRNA or ribonucleoprotein (RNP) using nucleofection.
General notes
1. Throughout this protocol, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.
2. This protocol is to prepare cells for protocol nucleofection of hPSCs. Before starting, familiarize yourself with the protocol and the required preparations. A detailed protocol on maintaining MEF-cultured hPSCs can be found in the collection "Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell culture;" doi:
3. Detailed protocols for preparing plasmids, RNA, and RNP for nucleofection can be found in the collection "Nucleofection (Amaxa) and electroporation (Biorad) of hPSCs." A link to this collection can be found in the title section of this protocol, located above
Materials
| Item | Vendor | Catalog # | |
| DMEM/F12 | Thermo Fisher | 11320082 | |
| DPBS w/o calcium and magnesium | Corning | MT21031CV | |
| Fetal Bovine Serum (FBS) | Corning | 35-011-CV | |
| Knockout Serum Replacement | Thermo Fisher | 10828-028 | |
| L-Glutamine | Sigma | G8540 | |
| Penicillin & Streptomycin (100X) | Thermo Fisher | 15140163 | |
| MEM Non-Essential Amino Acids (100X) | Thermo Fisher | 11140050 | |
| Heat Stable Recombinant Human FGF2 | Thermo Fisher | PHG0360 | |
| Collagenase type IV | Thermo Fisher | 17104019 | |
| 2-Mercaptoethanol | Sigma | M3148 | |
| mTeSR-plus | STEMCELL Technologies | 100-0276 | |
| StemFlex | Thermo Fisher | A3349401 | |
| Vitronectin (VTN-N) Recombinant Human Protein, Truncated | Thermo Fisher | A14700 | |
| Accutase | Thermo Fisher | SCR005 | |
| Dispase | STEMCELL Technologies | NC9995391 | |
| Y-27632 | Chemdea | CD0141 | |
| Cas9, purified protein, 40uM | Macrolab, QB3 UC Berkeley | ||
| Synthetic pegRNAs | IDT or Synthego | ||
| Synthetic sgRNAs | Synthego | ||
| P3 primary Cell 4D X kit S | Lonza | V4XP-3032 | |
| Countess™ Cell Counting Chamber Slides | Thermo Fisher | C10228 | |
| pCMV-PE2 | Addgene | 132775 | |
| 4D-Nucleofector TM Core + X Unit | Lonza | AAF-1002B, AAF-1002X | |
| 5 ml polystyrene round-bottom tube with cell-strainer cap | Corning | 352235 | |
| Cell-strainer (70 µm) | Fisher | 07201431 | |
| Gene Pulser Xcell Eukaryotic System | Bio-Rad | 1652661 | |
| Gene Pulser Electroporation Cuvettes, 0.4 cm gap | Bio-Rad | 1652081 | |
| Exact N Amp Blood PCR Kit | Sigma | XNAB2-1TK |
Note: This protocol makes reference to other protocols. Please check for any materials found in those protocols, which might not be listed here
When MEF-cultured hPSCs reach 50% confluency, change medium to hPSCs medium + Rock inhibitor, preparing for nucleofection the next day. For each 20 µl nucleofection reaction, prepare half to 1 well of cells.
A detailed protocol on maintaining MEF-cultured hPSCs can be found in the collection "Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cells for human pluripotent stem cell culture;" dx.doi.org/10.17504/protocols.io.b4pbqvin
hPSCs medium
| Reagent | Volume | |
| DMEM/F12 | 385 ml | |
| Fetal Bovine Serum (FBS) | 75 ml | |
| Knockout Serum Replacement | 25 ml | |
| L-Glutamine (100X) | 5 ml | |
| Penicillin & Streptomycin (100X) | 5 ml | |
| MEM Non-Essential Amino Acids (100X) | 5 ml | |
| 2-Mercaptoethanol (10,000X) | 50 µl | |
| Heat Stable Recombinant Human FGF2 (25µg/ml)* | 80 µl |
*While we prefer Heat Stable Recombinant Human FGF2, we also have used regular FGF2. Final volume: 500 ml
L-Glutamine (100X)
| L-Glutamine, powder | 14.6 g | |
| MilliQ H2O | 500 ml |
Final volume: 500 ml
2-Mercaptoethanol (10,000X)
| 2-Mercaptoethanol | 0.78 ml | |
| MilliQ H2O | 9.22 ml |
Final volume: 10 ml
Heat Stable Recombinant Human FGF2 (25µg/ml)
| A | B | |
| Heat Stable Recombinant Human FGF2 | 500 µg | |
| 0.1% BSA | 20 ml |
Final volume: 20 ml
Y-27632 (1,000X)
| A | B | |
| Y-27632 | 5 mg | |
| DMSO | 1.56 ml |
hPSCs medium + Rock inhibitor, 500ml
| A | B | |
| hPSCs medium | 500 ml | |
| Y-27632 (1,000X) | 500 µl |
Final volume: 500 ml
Prepare feeder plate at least 1 day earlier as depicted in the collection "Maintenance and inactivation of mouse embryonic fibroblasts (MEFs) as feeder cell for human pluripotent stem cell culture," dx.doi.org/10.17504/protocols.io.b4pbqvin
Wash MEF-cultured hPSCs with DPBS
Add 1 ml Dissociating Solution to each well
Collagenase solution (10mg/ml)
| A | B | |
| Collagenase type IV | 100 mg | |
| KSR medium | 10 ml |
Final volume: 10 ml
KSR medium
| A | B | |
| DMEM/F12 | 385 ml | |
| Knockout Serum Replacement | 100 ml | |
| L-Glutamine (200 mM) | 5 ml | |
| Penicillin & Streptomycin (100X) | 5 ml | |
| MEM Non-Essential Amino Acids (100X) | 5 ml |
Final volume: 500 ml
Dissociating solution, 10ml
| A | B | |
| Collagenase solution (10mg/ml) | 1 ml | |
| Dispase (1U/ml) | 5 ml | |
| DMEM/F12 | 4 ml |
Final volume: 10 ml
Incubate 00:30:00 37 °C . Watch for edge curling of the colonies as indication collagenase incubation is complete.
30m
Add 2 ml DMEM/F12 to each well
Pipette repeatedly with 5 ml pipette to lift colonies, careful not to carry over too many MEFs.
Collect into 15 ml conical tube.
Add 7 ml DMEM/F12.
Centrifuge at 200-300 x g, 00:05:00
5m
Aspirate supernatant
Re-suspend cell pellet in 1 ml pre-warmed Accutase
Incubate 37 °C 00:05:00
5m
Add 9 ml DMEM/F12, invert to mix
Centrifuge at 200-300 x g, 00:05:00
5m
Aspirate supernatant
Resuspend cell pellet in 1 ml DMEM/F12, triturate to single cells using P1000 tips
Take two 10 µl sets of the cell suspension.. Mix each set with 10 µl trypan blue dye which comes with the Countess™ Cell Counting Chamber Slides
Count cells with Countess automated cell counter or hemocytometer, average the counts from the two sets. Continue with re-suspending the cell pellet in 20 µl nucleofection solution as described in the protocol "Nucleofection of hPSCs" (Step 2)
The protocol "Nucleofection of hPSCs" can be found in the collection "Nucleofection (Amaxa) and electroporation (Biorad) of hPSCs." A link to this collection can be found in the title section of this protocol, located above
Mix the cell suspension in the conical tube, take 500,000 cells per nucleofection reaction and transfer to a new conical tube
Centrifuge at 200-300 x g, 00:05:00
5m
Aspirate supernatant
Resuspend cell pellet in 10 ml DPBS
Centrifuge at 200-300 x g, 00:05:00
5m
Aspirate supernatant as much as possible, to minimize the interference to the nucleofection buffer system.
To proceed with the nucleofection process, refer to the protocol "Nucleofection of hPSCs;" dx.doi.org/10.17504/protocols.io.b4pcqviw