An Optimized Mouse Embryonic Stem Cell Based Reverse Poly-Transfection Technique for Rapid Exploration of Nucleic Acid Ratios

Summary

The present protocol describes a method for reverse poly-transfection of mouse embryonic stem cells during culture with 2i and LIF media. This method yields higher viability and efficiency than traditional forward transfection protocols, while also enabling one-pot optimization of plasmid ratios.

Abstract

Due to its relative simplicity and ease of use, transient transfection of mammalian cell lines with nucleic acids has become a mainstay in biomedical research. While most widely used cell lines have robust protocols for transfection in adherent two-dimensional culture, these protocols often do not translate well to less-studied lines or those with atypical, hard-to-transfect morphologies. Using mouse pluripotent stem cells grown in 2i/LIF media, a widely used culture model for regenerative medicine, this method outlines an optimized, rapid reverse transfection protocol capable of achieving higher transfection efficiency. Leveraging this protocol, a three-plasmid poly-transfection is performed, taking advantage of the higher-than-normal efficiency in plasmid delivery to study an expanded range of plasmid stoichiometry. This reverse poly-transfection protocol allows for a one-pot experimental method, enabling users to optimize plasmid ratios in a single well, rather than across several co-transfections. By facilitating the rapid exploration of the effect of DNA stoichiometry on the overall function of delivered genetic circuits, this protocol minimizes the time and cost of embryonic stem cell transfection.

Introduction

Delivery of DNA and RNA into mammalian cells serves as a core pillar of biomedical research¹. A common method for introducing exogenous nucleic acids (NA) into mammalian cells is through transient transfection^2,3. This technique relies on mixing NA with commercially available transfection reagents capable of delivering them into the recipient cells. Typically, NA is delivered via forward transfection, where cells adhering to a two-dimensional surface receive the transfection complex. While forward transfection for the most common established cell lines is robust and protocols are well-published, more niche cell types with non-monolayer morphologies do not transfect easily, limiting the amount of NA that can be delivered and the number of cells that receive it.

Pluripotent stem cells (PSCs) serve as an attractive model for understanding development and as a tool for regenerative medicine, given their ability to divide indefinitely and produce any bodily cell type. For mouse PSCs (mPSCs), routine in vitro culture conditions with 2 inhibitors and LIF (2i/LIF) maintain a dome-like colony morphology, directly limiting the number of cells exposed to a forward transfection^4,5,6. To address this, a reverse transfection can be performed: cells are added to a dish containing media and transfection reagent, rather than adding transfection reagent to adherent cells⁷. While this increases the number of cells exposed to the reagent, it also requires the cells to be passaged and transfected concurrently.

Moving beyond simple single-NA transfections, researchers often aim to deliver several NA constructs into a population of cells in vitro. This is typically achieved through a co-transfection, where the NAs are mixed at a given ratio (1:1, 9:1, etc.) and are then combined with the chosen transfection reagent⁸. This yields a mix of NAs and reagent that preserves the original ratio of NAs to one another - while cells in the treatment may receive different amounts of this mix, they all receive the same ratio⁹. While this is advantageous when the desired ratio of parts is known, determining this ratio ahead of time can be labor-intensive, with each ratio constituting a different condition. One alternative is to perform a "poly-transfection," where individual NAs are mixed with the transfection reagent independently from one another⁹. By combining transfection complexes containing individual NAs (rather than combining NAs before creating the complexes), researchers can explore a wide array of NA stoichiometries in a single transfection experiment⁹. This is particularly valuable in cases where the products of several NAs are expected to interact with one another, such as with inducible transcription systems or systems with feedback built in^1,10,11. However, to do so effectively, a high transfection efficiency is needed. Indeed, as the number of unique transfected NAs increases, the probability of a given cell receiving all of the desired NAs decreases exponentially^{9, 12}.

The following report describes a reverse transfection protocol for mPSCs using a cationic lipid-based transfection reagent, in which cells are exposed to the reagent-NA mix for a maximum of 5 min to maximize viability and minimize the time outside of typical culture conditions. Comparing this protocol to the standard forward transfection of these cells demonstrates a higher transfection efficiency and an increase in the total number of surviving transfected cells. By combining this reverse transfection with a three-plasmid poly-transfection involving simple fluorescent reporters, an expanded potential to screen NA ratios with high transfection efficiency is demonstrated.

Protocol

1. Preparation of reagents for mPSC culture

1. Prepare N2 supplement.
   1. In non-sterile conditions, prepare the following stock solutions (step 1.1.1.1) in a chemical safety fume hood. Add the solid powder of each chemical to a pre-weighed 50 mL conical tube. Weigh each tube after adding the powder and add an appropriate amount of solvent to achieve the concentrations below. For one batch of media, prepare the minimum amount listed.
      NOTE: The following reagents are hazardous and should be handled in accordance with local chemical safety guidelines. Ensure proper PPE when handling, and only work with the solid powder forms of these chemicals in a chemical fume hood to prevent inhalation.
      1. Minimum of 0.05 mL of sodium selenite in water at 0.518 mg/mL, 0.5 mL of putrescine in water at 160 mg/mL, 0.165 mL of progesterone in 100% ethanol at 0.6 mg/mL (see Table of Materials).
      2. Store solutions at -20 °C for up to 2 years.
   2. In a biosafety cabinet (BSC), add the following to 58.035 mL of DMEM-F12.
      1. 0.05 mL of a 0.518 mg/mL sodium selenite solution, 0.5 mL of a 160 mg/mL putrescine solution, 0.165 mL of a 0.6 mg/mL progesterone solution.
      2. Filter sterilize the above mixture with a 0.22 µm filter.
   3. Still in the BSC, prepare a 100 mg/mL solution of apotransferrin.
      1. Add 5 mL of DMEM-F12 to 500 mg of apotransferrin (see Table of Materials). Slowly resuspend and wash the container, avoiding bubbles.
      2. After resuspending and removing as much as possible with 5 mL, add it to the selenite/putrescine/progesterone solution. Take more of the previous solution and wash the apotransferrin bottle, getting as much of it out as possible.
   4. Add 5 mL of 7.5% BSA fraction V (see Table of Materials) to the solution.
   5. Mix and aliquot 6.875 mL per tube. Store aliquots at -80 °C for up to 1 year.
   6. When using the above N2 aliquots to make N2B27 media, thaw the desired aliquot and add 3.125 mL of a 4 mg/mL insulin solution (see Table of Materials) to the 6.875 N2 for 10 mL of 100x N2.
2. Prepare N2B27 basal media.
   1. Thaw N2 and B27 supplements in a 4 °C fridge for a few hours or overnight.
   2. In a BSC, mix 484.5 mL of DMEM-F12 and 484.5 mL of Neurobasal media (see Table of Materials) in a sterile 1 L bottle.
   3. Add 10 mL of B27 and 10 mL of N2 supplement to the media.
   4. Add 1 mL of 55 mM beta-mercaptoethanol (see Table of Materials). Add 10 mL of 100x glutamax. Mix vigorously by swirling the bottle.
   5. Aliquot the desired volume per aliquot (typically 100 mL) and store at -80 °C for up to 1 year. Store in use after thawing at 4 °C for up to 3 weeks.
3. Prepare NBiL media.
   1. Thaw a 100 mL aliquot of N2B27 basal media from -80 °C in the 4 °C fridge.
   2. In a BSC, add LIF (see Table of Materials) to a final concentration of 10 µg/L.
   3. Add CHIR99021 (3 µM final) and PD0325901 (1 µM final) (see Table of Materials). Mix well with a 50 mL serological pipette. Store at 4 °C for up to 2 weeks.
4. Prepare 0.2% gelatin solution.
   1. Transfer 500 mL of double-distilled H₂O into a 1 L glass bottle.
   2. Measure out 1 g of gelatin (see Table of Materials) and add it to the water. Mix by shaking the bottle until mostly dissolved.
   3. Autoclave the gelatin and water mixture at 15 psi, 121 °C for 35 min. Once cooled, filter sterilize it with a 0.22 µm filter in a BSC. Store at 4 °C.
5. Prepare wash media.
   1. In a BSC, mix 160 µL of 7.5% BSA per 10 mL of DMEM-F12.
   2. Scale the above and prepare in large quantities for ease of future use (e.g. 8 mL of 7.5% BSA to 500 mL of DMEM-F12). Store at 4 °C.

2. Preparation of reagents for mPSC poly-transfection

NOTE: The following values are provided for a single well of a 24-well plate. Values can be scaled accordingly. The sequences of all the DNA/plasmids are detailed in Supplementary File 1.

1. Calculate the volume of DNA solution needed per treatment.
   1. Prepare 500 ng of DNA per well/condition.
      1. If transfecting a single plasmid with a DNA solution of 100 ng/µL, prepare one tube with 5 µL of DNA.
      2. If transfecting two plasmids at 100 ng/µL each, prepare two tubes with 2.5 µL in each, one for each plasmid.
2. In a BSC, perform the following: For each 500 ng transfection treatment (see Table of Materials), aliquot 25 µL of OptiMEM into a 1.5 mL tube.
   1. Scale this accordingly - for the example above, prepare two tubes, each with 250 ng of DNA (2.5 µL) and 12.5 µL of OptiMEM.
3. Add the required volume of DNA for that treatment to the OptiMEM in the tube.
4. For each tube with 500 ng DNA and OptiMEM, create a matching tube with another 25 µL of OptiMEM and 1 µL of cationic lipid-based transfection reagent.
   1. Again, these values must be scaled accordingly. For the above example, prepare two tubes, each with 12.5 µL of OptiMEM and 0.5 µL of transfection reagent.
      ​NOTE: When scaling values, use the mass of DNA added, not the volume required for that amount of DNA. Reactions with transfection reagent and DNA can be scaled (e.g., if 5 wells are to be transfected with the same DNA). Keep the ratios of reagents the same, but scale accordingly (e.g., 1000 ng DNA: 50 µL OptiMEM: 2 µL transfection reagent: 50 µL OptiMEM).

3. Preparation of mPSCs for transfection

NOTE: For reverse transfection, prepare the culture vessel and passage the mPSCs directly before adding the transfection reagents. For forward transfection, passage and plate the cells 12-18 h prior to transfection to allow the cells to adhere to the plate.

1. Prepare a 0.2% gelatin-coated cell culture vessel.
   1. Plan the number of wells needed for the transfection (one well of a 24-well plate per treatment/group).
   2. Add 200 µL of the 0.2% gelatin solution to each of the desired wells in the 24-well plate.
   3. Gently shake the plate to evenly spread the solution, ensuring it covers the entire bottom of the well.
   4. Allow the wells to coat for a minimum of 15 min at room temperature.
   5. Using a vacuum aspirator, carefully remove any leftover gelatin from the wells (tilt the plate to ensure removal of all liquid without scraping off the gelatin layer).
   6. Add 0.5 mL of NBiL media (step 1.3) to each well and leave the plate in a tissue culture incubator to prewarm.
2. Passage mPSCs.
   1. Aliquot 30 mL of wash media (step 1.5) into a 50 mL conical tube.
   2. Aspirate the old media from the tissue culture dish of mESCs.
      ​NOTE: Several aspiration techniques are permissible. Whatever technique is chosen, ensure that cells do not remain dry for extended periods (approximately 30 s maximum).
   3. Add the required amount of commercially available cell detatchment medium (1.5 mL for a 10 cm dish, scale accordingly, see Table of Materials).
   4. Wait for 3-5 min before pipetting up and down 15-20 times with a P1000 pipette to obtain a single-cell suspension. View cells under a microscope to ensure a single-cell suspension.
   5. Transfer the cells in the detatchment medium into the 50 mL tube containing wash media.
   6. Centrifuge at 300 x g for 5 min at room temperature. Aspirate wash media, ensuring no residual liquid remains in the tube.
   7. Resuspend the pellet in a small volume of wash media (~300 µL). Count the cell suspension using a hemocytometer to obtain the cell density.

4. Reverse transfection of mPSCs

1. Prepare poly-transfection reagents according to step 2.
2. Prepare the cell culture vessel and passage mPSCs according to step 3.
3. Aliquot 200 µL of NBiL media to 1.5 mL tubes, one for each treatment.
4. Add 26 µL of the OptiMEM:transfection reagent mixture to the DNA:OptiMEM mixture. Mix the reagents quickly and vigorously by pipetting approximately 10 times. Allow the mixture to incubate at room temperature for 5 min.
5. Based on cell density, transfer the required volume of cells from the 300 µL cell suspension to the 200 µL NBiL tubes to obtain 25,000 cells per tube.
6. After 5 min of incubation of the Lipofectamine 2000 (L2K, transfection reagent) and DNA mixture, add it to the 1.5 mL tube of cells and mix well by pipetting. Allow the cells to incubate with the reagent for 5 min at room temperature.
7. Centrifuge at 300 x g for 5 min at room temperature. Pipette out the liquid, leaving approximately 50 µL.
8. Resuspend the cell pellet with 100 µL of NBiL media. Transfer the cells to the plate and place the plate in the incubator. Change the media 24 h later with fresh NBiL media.

5. Forward transfection of mPSCs

1. 12-18 h prior to transfection, prepare the cell culture vessel and passage mPSCs according to step 3.
2. Based on cell density, transfer the required volume of cells from the 300 µL cell mixture to seed 25,000 cells per well. Place the plate in the incubator.
3. 1 h prior to transfection, aspirate the media from all wells and replace it with 0.5 mL of NBiL media. Place the plate in the incubator.
4. At the time of transfection, prepare mPSC poly-transfection reagents according to step 2.
5. Add 26 µL of the OptiMEM:transfection reagent mixture to the DNA:OptiMEM mixture. Mix the reagents quickly and vigorously by pipetting approximately 10 times. Allow the combined mixture to incubate at room temperature for 5 min.
6. Add the combined mixture to the wells dropwise. Place the plate in the incubator. Change the media 24 h later.

6. Flow cytometry

1. Aspirate the media from the transfected 24-well plate 48 h after transfection.
2. Add 200 µL of trypsin to each well, swirl, and incubate for 30 s at 37 °C. Tap the sides of the plate and add 200 µL of ice-cold FACS buffer (2% FBS in PBS).
3. Open and label a V-bottom 96-well plate, starting from A1. Mix the contents of each well on the transfected plate to dislodge cells and make single-cell solutions with a P200 pipette. Transfer 200 µL from each well to the appropriate well on the 96-well plate.
4. Add 15 mL of FACS buffer to a 50 mL reagent reservoir. Centrifuge the plate at 300 x g for 5 min at room temperature. Discard the supernatant by inverting the 96-well plate into the sink with a fast and smooth movement.
5. Use a multichannel pipette to resuspend the pellets on the 96-well plate in 150 µL of FACS buffer from the reagent reservoir. Repeat steps 6.4-6.5 twice. Place the 96-well plate in an ice-filled box protected from light.
6. Run the samples through a flow cytometer to obtain the population distributions of the fluorescent reporters.
   1. Ensure the cytometer is appropriate in terms of laser and filter combinations for the experiment to be performed (e.g., do not use an infrared reporter if it is not possible to excite or detect in the far-red spectrum).
   2. Include additional samples for standardization beads to allow for MEFL conversion of data during analysis. Ensure that enough cells are collected per sample for appropriate statistical analysis⁹.
7. Convert fluorescent units from the raw cytometer files and analyze the data using any of several methods, such as Python- or MATLAB-based pipelines or commercially available software^9,13.

Results

Both forward and reverse transfections rely on the interaction between the cell membrane and incoming transfection reagent-DNA complexes, allowing the delivery of NA to the recipient cells. Where these techniques differ is the state of the cell upon delivery - while DNA is typically delivered to adherent cell monolayers in traditional forward transfection, reverse transfection instead relies on having the reagent-DNA complex meet the cells while in a single-cell suspension. This difference can be particularly crucial in ...

Discussion

A key reason for the widespread adoption of transfection protocols is their reproducibility and accessibility; however, these protocols do require optimization across experimental contexts. Not mentioned above is the standard testing required when attempting to transfect a new cell line for the first time. First, the choice of transfection reagent is key, as commercially available reagents are not one-size-fits-all and will vary in the efficiency of NA delivery viability across cell types. Additionally, finding the ideal...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Accutase	MilliporeSigma	SCR005
Apotransferrin	MilliporeSigma	T1147-500MG
B27 supplement	ThermoFisher Scientific	17504044
Beta-mercaptoethanol	ThermoFisher Scientific	21985023
BSA fraction V (7.5%)	Gibco	15260-037
CHIR99021	MilliporeSigma	SML1046-25MG
DMEM-F12	MilliporeSigma	D6421-24X500ML
Flow cytometry standardization beads	Spherotech	URCP-38-2K
Gelatin	MilliporeSigma	G1890
GlutaMAX supplement	ThermoFisher Scientific	35050061
Insulin	Gibco	12585-0014
Lipofectamine 2000	Invitrogen	11668-019	Transfection reagent
Neurobasal media	ThermoFisher Scientific	21103049
OptiMEM	Invitrogen	31985-070
PD0325901	MilliporeSigma	PZ0162-25MG
Progesterone	MilliporeSigma	P8783	Chemical hazard - consult local safety guidelines, ensure proper PPE is worn, and work with the solid powder form only in a chemical fume hood
Putrescine	MilliporeSigma	P6780	Chemical hazard - consult local safety guidelines, ensure proper PPE is worn, and work with the solid powder form only in a chemical fume hood
Recombinant mLIF	BioTechne	8878-LF-500/CF
Sodium selenite	MilliporeSigma	S5261-25G	Chemical hazard - consult local safety guidelines, ensure proper PPE is worn, and work with the solid powder form only in a chemical fume hood
Trypsin-EDTA	ThermoFisher Scientific	25200056