Transfection of Pluripotent Stem Cells with Lipofectamine Stem Transfection Reagent in StemFlex Medium

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PSC growth medium, passaging reagents, and complexation medium

Starting with undifferentiated human pluripotent stem cells (PSCs), expanded in a feeder-free culture system such as Gibco StemFlex Medium on Gibco Geltrex matrix or on a defined substrate such as rhLaminin-521, is ideal for efficient transfection.

Passaging

• Maintain PSCs in the format of your choice, such as 6-well plates, 60 cm dishes, or T-75 flasks coated with Geltrex matrix or rhLaminin-521, in StemFlex Medium. Propagating PSCs in 6-well plates and transfecting in 24-well plates are convenient formats used in this protocol.
• Passage PSCs every 3 to 5 days, before they reach ~85% confluence.
• Tip: For routine passaging of PSCs with Gibco Versene Solution for expansion, the replating of large clumps of 5–10 cells promotes reattachment and survival in StemFlex Medium without the need to add Gibco RevitaCell Supplement. PSCs can be expanded in StemFlex Medium for subsequent transfection on Geltrex matrix or rhLaminin-521.

Precoating 24-well plates with Geltrex matrix or rhLaminin-521

Coating with Geltrex matrix

1. Prepare a 1:100 dilution of Geltrex matrix in cold Gibco DMEM/F-12 Medium with GlutaMAX Supplement (Cat. No. 10565).
2. Add 300 μL of diluted Geltrex matrix to each well of a 24-well plate and incubate at 37°C for ≥1 hour, before use.

Coating with rhLaminin-521

1. Prepare a 1:40 dilution of rhLaminin-521 by adding 300 μL of rhLaminin-521 stock solution (0.5 mg/mL) to 12 mL of DPBS, for a final concentration of 2.5 μg/mL.
2. Add 400 μL of diluted rhLaminin-521 to each well of a 24-well plate, and incubate at 37°C for ≥2 hours to coat the wells with 0.5 μg/cm² of rhLaminin-521.

• Important: The optimal coating concentration of rhLaminin-521 can depend on the PSC line and ranges from 0.5 to 2 μg/cm². Increase the concentration if you observe areas of incomplete cellular attachment.
• Tip: Plates coated with Geltrex matrix or rhLaminin-521 can be prepared ahead of time and stored for up to 2 weeks at 4°C. Equilibrate at room temperature for 1 hour before plating cells.

Seeding cells for transfection

1. To maximize transfection efficiency, seeding a single-cell suspension of PSCs prepared with Gibco TrypLE Select Enzyme is recommended.
   Important: As the plating efficiency of PSCs dissociated into single cells is lower than the passaging efficiency of clumped cells, we recommend adding RevitaCell Supplement for overnight replating in StemFlex Medium onto Geltrex matrix or rhLaminin-521 for transfecting the following day.
2. When feeder-free PSC cultures are less than 85% confluent, remove the StemFlex Medium and gently wash the cells twice with 2 mL of DPBS per well in a 6-well plate.
3. Add 1 mL of TrypLE Select Enzyme to each well, swirl to evenly coat the PSCs, and incubate at 37°C for 3–5 minutes.
4. Using a 1 mL pipette, gently triturate the cell suspension 5–10 times to dissociate into single cells.
5. Transfer the cell suspension into a 15 mL conical tube containing 3 mL of StemFlex Medium to inactivate the TrypLE Select Enzyme.
6. Centrifuge the cell suspension at 200 x g for 4 minutes.
7. Aspirate the supernatant and resuspend the pellet to a single-cell suspension in 3 mL of StemFlex Medium with RevitaCell Supplement.
8. Perform a total viable cell count with the Invitrogen Countess II Automated Cell Counter or another method.
9. Dilute with additional StemFlex Medium with RevitaCell Supplement to a final concentration of 100,000 cells/mL.
10. Aspirate the Geltrex matrix or rhLaminin-521 from the wells of a precoated 24-well plate.
    Important: Proliferating PSC cultures need room to expand during transfection, so plate the recommended starting number of cells (step 11) to achieve 30% confluence on the day of transfection.
11. Add 0.5 mL of the PSC suspension in StemFlex Medium with RevitaCell Supplement to plate 50,000 cells/well in the precoated 24-well plate.
12. Return the plate to the incubator and culture the cells at 37°C with 5% CO₂, overnight.

Changing medium on the day of transfection

Prepare a solution of of Gibco Opti-MEM I medium with RevitaCell Supplement. Aspirate the StemFlex medium, and add 0.5 mL of the supplemented Opti-MEM I medium to each well just before transfection.

• Important: Transfect in Opti-MEM I Medium with RevitaCell Supplement, not in StemFlex Medium, which can inhibit transfection.

DNA transfection protocol

Perform the following steps, which have been optimized for using Invitrogen Lipofectamine Stem Transfection Reagent in StemFlex Medium:

Analysis of transfection efficiency

Observe PSCs transfected with a GFP reporter construct at 24 and 48 hours posttransfection by fluorescence microscopy or flow cytometry for endpoint analysis (Figure 1).

mRNA transfection protocol

Perform the following steps, which have been optimized for using Lipofectamine Stem Transfection Reagent in StemFlex Medium:

Analysis of transfection efficiency

Observe PSCs transfected with a fluorescent mRNA at 24 and 48 hours posttransfection by fluorescence microscopy or flow cytometry for endpoint analysis (Figure 2).

Figure 2. Posttransfection analysis of iPSCs. (A) Fluorescence image demonstrating 68% transfection efficiency, and (B) bright-field image. iPSCs (NCRM1) are shown 44 hours after transfection with 250 ng of GFP mRNA and 2 μL of Lipofectamine Stem reagent in StemFlex Medium on Geltrex matrix.

Ribonucleoprotein (RNP) transfection protocol

RNP complex components:

• Invitrogen GeneArt Platinum Cas9 Nuclease (Cat. No. B25641)
• gRNA (see “Designing and generating gRNA by in vitro transcription”)

On the day of transfection (1 day after plating PSCs in a single well of a 24-well plate) perform the following steps, which have been optimized for using Lipofectamine Stem reagent in StemFlex Medium:

Analysis of transfection efficiency

Observe PSCs transfected with a GFP reporter construct at 24 and 48 hours posttransfection by fluorescence microscopy or flow cytometry, and analyze double-stranded break (DSB) formation using the Invitrogen GeneArt Genomic Cleavage Detection Kit or a similar assay (Figure 3).

Designing and generating gRNA by in vitro transcription

In addition to RNP transfection efficiency, the efficiency of DSB/indel formation at a given locus can depend on gRNA design. Use the Invitrogen GeneArt CRISPR Search and Design Tool, available at thermofisher.com/crisprdesign, to search our database of >600,000 predesigned gRNA sequences specific to every gene in the human genome. These predesigned gRNAs are optimized for gene knockout and typically target the first 3 transcribed exons per gene.

Clone and generate your own gRNA using the Invitrogen GeneArt Precision gRNA Synthesis Kit (Cat. No. A29377). gRNA concentration can be quantified on the Invitrogen Qubit 3 Fluorometer (Cat. No. Q33216) coupled with the Invitrogen Qubit RNA BR Assay Kit (Cat. No. Q10210).