Protocol for in vitro transfection of mRNA-encapsulated lipid nanoparticles

Summary

Lipid nanoparticle (LNP) platforms are promising for mRNA therapeutics; however, while highly effective in vivo, commercial mRNA-LNPs show reduced transfection efficiency in vitro under serum-starved conditions, limiting mechanistic studies and cell engineering applications. Here, we present a new standard for in vitro transfection of mRNA-LNPs, including cell culture preparation, mRNA-LNP treatment in complete media, and mRNA expression level quantification. This protocol improves efficiency and reproducibility in in vitro screening and ensures consistency in the assessment of mRNA-LNPs.

Graphical abstract

Highlights

• •Instruction for transfection of lipid nanoparticles (LNPs) to cells
• •Steps for cell/LNP preparation, transfection in complete media, and data quantification
• •Guidance on standardized procedures across multiple cell lines

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Lipid nanoparticle (LNP) platforms are promising for mRNA therapeutics; however, while highly effective in vivo, commercial mRNA-LNPs show reduced transfection efficiency in vitro under serum-starved conditions, limiting mechanistic studies and cell engineering applications. Here, we present a new standard for in vitro transfection of mRNA-LNPs, including cell culture preparation, mRNA-LNP treatment in complete media, and mRNA expression level quantification. This protocol improves efficiency and reproducibility in in vitro screening and ensures consistency in the assessment of mRNA-LNPs.

Before you begin

We provide the protocol for the transfection of mRNA-encapsulated lipid nanoparticles (mRNA-LNPs) into nine commonly used cell lines: HEK293, Huh-7, HeLa, HepG2, MCF-7, U-87 MG, HT22, raw264.7, and SH-SY5Y. Depending on cell type, the appropriate media can be selected for adaptation. The mRNA-LNPs in this protocol were prepared based on previously reported methods.^1,2,3 All procedures were performed under standard biosafety level 1 conditions with appropriate personal protective equipment, and waste was disposed of according to institutional guidelines. The overall experimental workflow is illustrated in the Graphical Abstract.

Innovation

While mRNA-LNPs perform excellently in vivo, they often exhibit drastically reduced transfection efficiency in vitro when using the well-established serum-starved method.⁴ Thereby, reducing the correlation between in vitro and in vivo data, compromising the reliability of LNP screening and mechanistic studies. Although several studies have reported using complete media for mRNA-LNP transfection, detailed protocols are rarely provided, making reproducibility difficult. In many cases, optimization is only demonstrated in hepatic cell lines, where mRNA-LNP uptake is inherently more efficient. This protocol introduces a simple and reproducible innovation. The protocol eliminates the need for serum-starvation and simplifies the workflow. Compared with serum-starved methods, this approach demonstrates 4- to 26-fold higher transfection efficiency across multiple cell types. This robust and reproducible protocol may support mechanistic investigation, design and optimization of LNP, and gene-editing applications by ensuring greater consistency and better in vitro–in vivo relevance in mRNA-LNP assessment.

Cell line maintenance

Timing: 1–2 weeks

• 1.Frozen cell stocks were purchased from the Korea Cell Line Bank (https://cellbank.snu.ac.kr) and Ubigene (https://www.ubigene.us/product/). Table 1 presents the detailed culture conditions for each cell line.
• 2.
  Thawing and initial culture of frozen cells.

  • a.
    Warm one cryovial of frozen cells in a water bath at 37°C until thawed.
  • b.
    Centrifuge the thawed cell suspension at 1,000 rpm (170 × g) for 2 min at room temperature.
  • c.
    Gently aspirate and discard cell supernatant.
  • d.
    Resuspend the cell pellet in fresh complete medium.
  • e.
    Transfer cell suspension to a 100-mm cell culture dish and incubate cells for 2–3 days under standard incubation conditions (37°C, 5% CO₂).
  • f.
    The cell-specific culture conditions used in this study are summarized in Table S1.
• 3.
  Cell subculture.

  • a.
    Gently aspirate the culture medium.
  • b.
    Wash the cells twice with sterile 1× DPBS (5 mL).
  • c.
    Add trypsin-EDTA (2 mL) to cover the cell layer.
  • d.
    Incubate them at 37°C for 2 min.
  • e.
    Observe under the microscope to confirm cell detachment from the dish.

    CRITICAL: All cells should be detached before proceeding to the next step.

    Note: HepG2 cells require extended trypsin-EDTA incubation (up to 5 min) owing to poor detachment.

  • f.
    Seed the detached cells into a 100-mm cell culture dish at an appropriate density. Adjust the total volume to 10 mL using complete medium, then incubate under standard culture conditions (37°C, 5% CO₂).

    Note: See Table 1 for a recommended cell density for each cell line.

Table 1.

Cell culture conditions for each cell line

Cell line	Seeding density per 100-mm dish (cells)	Subculture interval (days)	Growth medium	Passage number ranges
HEK293	1.0–2.0 × 106	4–5	DMEM	<50
Huh-7	4.0–7.0 × 105	4–5	RPMI-1640	<50
HT22	0.5–1.0 × 105	3–4	DMEM	20–40
U-87 MG	3.0–6.0 × 105	3–4	DMEM	20–40
HeLa	0.7–1.4 × 106	3–4	DMEM	20–40
HepG2	2.0–3.0 × 106	3–4	DMEM	20–40
MCF-7	0.7–1.5 × 106	3–4	RPMI-1640	20–40
Raw264.7	2.0–3.0 × 106	2–3	DMEM	10–25
SH-SY5Y	1.5–2.0 × 106	3–4	DMEM	10–25

All media were supplemented with 10% FBS and 1% penicillin-streptomycin.

Preparation for mRNA-LNPs

Timing: 1 day

• 4.
  Calculation of the amount of lipid components.

  • a.
    Calculate the required mRNA amount (ng).
  • b.
    Determine the N/P ratio (moles of positively charged nitrogens in ionizable lipids (N)/moles of negatively charged phosphates in mRNA (P)).

    Note: The N/P ratio of the commercial mRNA-LNP is 6.⁵

  • c.
    Convert mRNA mass to P value using the average molecular weight (337.45 g/mol) of ribonucleotides (rA, rU, rC, and rG; e.g., mRNA mass/337.45 = P).
  • d.
    Calculate the N value based on the desired N/P ratio (e.g., P × 6 = N).
  • e.
    Calculate moles of ionizable lipids (e.g., N = nmoles of SM-102).
  • f.
    Calculate the required moles for each lipid based on the moles of ionizable lipids. In this protocol, we used commercial mRNA-LNP (SM-102:1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC): Cholesterol: 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) = 50:10:38.5:1.5).⁵
• 5.
  Preparation of lipid stock solutions.

  • a.
    Weigh lipids in an Eppendorf tube using a precision balance.

    Note: Use at least 1.1× the calculated amount.

    Note: Use a pipette for viscous liquids such as SM-102.

  • b.
    Dissolve in methanol-chloroform mixed solvent (1:1, v/v) and vortex.

    Note: Prepare only the volume required; do not store long-term.

    Note: DMG-PEG2000 can be dissolved in EtOH.

    CRITICAL: Ensure the final stock concentrations can be pipetted.

• 6.
  Lipid mixture film formation.

  • a.
    Combine 1.1× volumes of each lipid into a 2-mL glass vial.

    Note: Use pipetting against the wall to transfer small volumes.

  • b.
    Evaporate solvent using a rotary evaporator at around 40°C for about 5 min.

    Note: If you do not use the film immediately, store it sealed at −20°C.

• 7.
  LNP formation via thermo-shaker.

  • a.
    Redissolve the lipid film in 55 μL of EtOH.

    Note: Ensure full redissolution. If the lipid film does not dissolve clearly, sonicate the solution and vortex the vial again.

  • b.
    Prepare mRNA solution: dilute the mRNA stock in citrate buffer (pH 4.0) to 153 μL and gently mix with a pipette (Do not vortex).

    Note: Handle mRNA in an RNase-free environment and avoid repeated freeze-thaw cycles. Use freshly thawed mRNA solutions promptly to minimize the risk of degradation.

    Note: EGFP-encoding mRNA is used to evaluate transfection efficiency via flow cytometry and fluorescence microscope.^4,6,7 Alternatively, firefly luciferase-encoding mRNAs can be used to evaluate transfection efficiency by measuring bioluminescence using a microplate reader.^{4,8,9,10,11,12,13}

  • c.
    Add 50 μL of lipid solution to a clean Eppendorf tube.
  • d.
    Place on a Thermo-shaker at 25°C, 1400 rpm.
  • e.
    Quickly add 152 μL of the mRNA solution, then shake for 15 s.

    Note: Do not depress the micropipette fully to expel the final drop. Instead, quickly dispense up to the first resistance point to ensure rapid and consistent injection.

    Note: This method is suitable for small-scale application (≤ 20 μg of RNAs) for in vitro transfection.¹⁴ At larger scales, the particle size and polydispersity index (PDI) may increase beyond the acceptable range.

• 8.
  Solvent exchange.

  • a.
    Wash an Amicon Ultra Centrifugal Filter with DPBS.
  • b.
    Transfer an mRNA-LNP solution into a centrifugal filter and fill DPBS until the boundary.
  • c.
    Centrifuge at 12,190 rpm (14,000 × g) for 10 min at 4°C, as per the manufacturer’s instructions.
  • d.
    Add 150 μL DPBS and resuspend concentrated mRNA-LNP in the filter using a pipette.
  • e.
    Centrifuge at 12,190 rpm (14,000 × g) for 10 min at 4°C.
  • f.
    Add 100 μL of DPBS and resuspend concentrated mRNA-LNP in the filter using a pipette.
  • g.
    Centrifuge at 12,190 rpm for 10 min at 4°C.

Note: If the remaining mRNA-LNP volume is > 50 μL, repeat the centrifugation.

• 9.
  mRNA-LNP recovery and adjustment of the concentration.

  • a.
    Resuspend the concentrated mRNA-LNP in the filter using a pipette.
  • b.
    Perform a reverse spin (mini centrifuge, 1340 rpm (170 × g), 10 s) into a clean matching collection tube, as modified from the manufacturer’s instructions.
  • c.
    Transfer to a 1.5 mL tube and measure the final volume.

    Note: Wash the filter with DPBS several times and combine them in the final tube.

  • d.
    Adjust to the final mRNA concentration in mRNA-LNP stock solution (e.g., 50 ng/μL) using DPBS.

    Note: Ensure that the mRNA-LNPs are of appropriate size (70–150 nm),¹⁵ PDI (< 0.2),¹⁶ and encapsulation efficiency (< 10% free RNAs by gel electrophoresis) before use.

    Note: Store the final mRNA-LNPs in a refrigerator and use them with a week.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Chemicals, peptides, and recombinant proteins
SM-102	Hanmi Fine Chemical Co., Ltd.	N/A; Lot#JKH-RNA18-003
1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)	Avanti Polar Lipids, Inc.	Cat#850365P
Cholesterol	Sigma-Aldrich (Merck)	Cat#C3045
1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000)	Avanti Polar Lipids, Inc.	Cat#880151P
Methanol (MeOH)	Sigma-Aldrich (Merck)	Cat#34860
Chloroform	Sigma-Aldrich (Merck)	Cat#366927
Lipofectamine 3000	Invitrogen (Thermo Fisher Scientific)	Cat#L300008
Ethanol (EtOH)	Sigma-Aldrich (Merck)	Cat#459836
Citric acid	Sigma-Aldrich (Merck)	Cat#C2404
Sodium chloride	Daejung Chemicals & Metals	Cat#7548-1100
Sodium hydroxide (aq.) (10 M)	Sigma-Aldrich (Merck)	Cat#72068
Critical commercial assays
Quant-it™ RiboGreen reagent and RNA assay kit	Invitrogen (Thermo Fisher Scientific)	Cat#R11490
Experimental models: Cell lines
HEK293	Korean Cell Line Bank	Cat#21573; RRID: CVCL_0045
MCF-7	Korean Cell Line Bank	Cat#30022; RRID: CVCL_0031
Huh-7	Korean Cell Line Bank	Cat#60104; RRID: CVCL_0336
HeLa	Korean Cell Line Bank	Cat#10002; RRID: CVCL_0030
HepG2	Korean Cell Line Bank	Cat#88065; RRID: CVCL_0027
U-87 MG	Korean Cell Line Bank	Cat#30014; RRID: CVCL_0022
HT22	Ubigene	Cat#YC-A004; RRID: CVCL_0321
Raw264.7	Korean Cell Line Bank	Cat#40071; RRID: CVCL_0493
SH-SY5Y	Korean Cell Line Bank	Cat#22266; RRID: CVCL_0019
Oligonucleotides
Enhanced green fluorescent protein (EGFP)-encoding mRNA (Cap1, 5-methoxyuridine [5-MOU])	TriLink BioTechnologies	Cat#L7201
EGFP-encoding mRNA (Cap1, 5-MOU)	GenScript Biotech Corporation	Cat#RP-A00008
Firefly luciferase-encoding mRNA (Cap1, 5-MOU)	TriLink BioTechnologies	Cat#L7202
Firefly luciferase-encoding mRNA (Cap1, 5-MOU)	GenScript Biotech Corporation	Cat#RP-A00022
Software and algorithms
CytExpert	Beckman Coulter	https://www.beckman.com/flow-cytometry/research-flow-cytometers/cytoflex/software
Gen5 Prime Software	BioTek Instruments (Agilent Technologies)	https://www.agilent.com/en/product/cell-analysis/cell-imaging-microscopy/cell-imaging-microscopy-software/biotek-gen5-software-for-imaging-microscopy-1623226
Other
Dulbecco’s modified Eagle’s medium (DMEM)	WELGENE	Cat#LM001-01
RPMI 1640 medium	WELGENE	Cat#LM011-03
Fatal bovine serum	WELGENE	Cat#S001-01
FBS	Gibco (Thermo Fisher Scientific)	Cat#26140079
Penicillin-streptomycin (100×)	WELGENE	Cat#LS202-02
DPBS (1×)	WELGENE	Cat#LB001-01
Trypsin-EDTA (0.05%, 1×)	WELGENE	Cat#LS015-01
Opti-MEM	Gibco (Thermo Fisher Scientific)	Cat#31985070
Cell culture lysis 5× reagent	Promega	Cat#E153A
Luciferase assay buffer	Promega	Cat#E152A
UltraPure DNase/RNase-free distilled water	Invitrogen (Thermo Fisher Scientific)	Cat#10977023
Amicon Ultra centrifugal filter	Millipore (Merck)	Cat#UFC5010
Cell culture microplate, 96 well, PS, F-bottom, μClear, Black, CELLSTAR, TC	Greiner	Cat#655090
CytoFLEX (flow cytometer)	Beckman Coulter	https://www.beckman.com/flow-cytometry/research-flow-cytometers/cytoflex
Cytation 5 cell imaging multimode reader	BioTek Instruments (Agilent Technologies)	https://www.agilent.com/en/product/cell-analysis/cell-imaging-microscopy/cell-imaging-multimode-readers/biotek-cytation-5-cell-imaging-multimode-reader-1623202

Materials and equipment

Complete medium

Reagent	Final concentration	Amount
RPMI-1640 or DMEM	N/A	445 mL
Fetal bovine serum	10% (v/v)	50 mL
Penicillin-streptomycin	1% (v/v)	5 ml
Total	N/A	500 mL

Store at 2°C to 8°C for up to 1 month.

Citrate buffer (pH 4.0)

Reagent	Final concentration	Amount
Citric acid (aq.) (1 M)	50 mM	25 mL
Sodium chloride (aq.) (1 M)	110 mM	55 mL
Sodium hydroxide (aq.) (10 M)	Adjust the pH to 4.0	4 ml
UltraPure DNase/RNase-Free Distilled Water	N/A	416 mL
Total	N/A	500 mL

Store at 2°C to 8°C for up to 6 months.

Step-by-step method details

Preparation of cells for seeding

Timing: 1 day

• 1.Count the cells in the culture dish using a hemocytometer to ensure accurate plating.

Note: Standardize cell numbers for consistency and confirm that cells are well-dispersed as single cells to prevent clumping, which can affect transfection efficiency and reproducibility.

• 2.Dilute the cells to the desired concentration in the appropriate growth media containing 10% fetal bovine serum and antibiotics (e.g., DMEM for HEK293 cells; Table 1).
• 3.Prepare a 24- or 96-well culture plate by adding calculated volume of cell suspension to each well.
• 4.Adjust the cell concentration to meet the experimental requirements.

Note: See Table 2 for recommended cell concentration for different plate formats.

Note: Maintain a single-layer distribution with ∼60% confluency to ensure uniform transfection conditions.

• 5.Incubate the culture plate in a humidified incubator at 37°C with 5% CO₂ overnight.

Table 2.

Concentration for mRNA-LNP transfection in the 24- and 96-well plates

Culture plate format	24-well	96-well
Well area (cm2)	1.9	0.32
Seeding density (cells/well)	8 × 104 cells	2 × 104 cells
Seeding volume (mL/well)	1.0	0.2
mRNA-LNP stock concentration (based on mRNA, ng/μL)	50	50
mRNA-LNP in medium preparation
mRNA-LNP stock volume (μL)	6	2.5
Conditioned media volume (μL)	594	122.5
Treated mRNA-LNP in medium per well (μL/well)	500	100
Treated mRNA amount per well (ng/well)	250	100
Final treated mRNA concentration (ng/μL)	0.5	1.0

Preparation of mRNA-LNP in medium

Timing: 10–30 min

• 6.Prepare the characterized mRNA-LNPs in DPBS at a 50 or 100-fold concentration relative to the final working concentration.

Note: See Table 2 for recommended mRNA-LNP concentrations for different plate formats.

Note: Before treatment, verify mRNA-LNP stability by checking for aggregation, excessive size (70–150 nm in general), or high PDI (< 0.2 in general). Encapsulation efficiency was also measured by measuring the proportion of unencapsulated RNA (< 10%) using gel electrophoresis or Ribogreen assay.

• 7.
  Dilute the mRNA-LNP stock solution with complete medium.

  CRITICAL: You can select conditioned (step 7-a) or fresh (step 7-b) or mixed (step 7-c) complete medium for dilution of mRNA-LNPs depending on the cell line. For hepatic cells, we recommend following step 7-a, whereas for non-hepatic cell lines, following step 7-b. However, if the preference is uncertain, perform a comparative test and select the approach with the highest transfection efficiency, or alternatively, follow step 7-c.

  Note: See Table 2 for recommended mRNA doses, mRNA-LNP concentrations, and mixing volumes for different plate formats.

  • a.
    Dilute the mRNA-LNP stock solution with conditioned complete medium.

    • i.
      Carefully transfer a portion of the media from each well to a separate tube for temporary storage and incubate the culture plate during the preparation of “mRNA-LNP in medium.”

      Note: Avoid disturbing the cell layer or causing detachment.

    • ii.
      Add the stored conditioned media (e.g., 594 μL per well for a 24-well culture plate) to the mRNA-LNP stock solution in DPBS (e.g., 6 μL per well for a 24-well culture plate) to achieve the desired final concentration (e.g., 0.5 ng/μL mRNA for a 24-well culture plate).

      Note: Adding mRNA-LNP stock solution directly to each well without removing conditioned medium is feasible; however, variations in residual medium volume remaining after overnight incubation following seeding can lead to inconsistencies in mRNA-LNP concentration across wells. To minimize such errors, it is preferable to use a separately prepared “mRNA-LNP in medium” solution.

  • b.
    Dilute the mRNA-LNP stock solution with fresh complete medium.

    • i.
      Add the fresh complete medium (e.g., 594 μL per well for a 24-well culture plate) to the mRNA-LNP solution in DPBS (e.g., 6 μL per well for a 24-well culture plate) to achieve the desired final concentration (e.g., 0.5 ng/μL RNA for a 24-well culture plate).
  • c.
    Dilute the mRNA-LNP stock solution with mixed complete medium.

    • i.
      Carefully transfer a portion of the media from each well to a separate tube for temporary storage and incubate the culture plate during the preparation of “mRNA-LNP in medium.”
    • ii.
      Mix the conditioned complete medium (e.g., 297 μL per well for a 24-well culture plate) and fresh complete medium (e.g., 297 μL per well for a 24-well culture plate) equally (1:1).
    • iii.
      Add the mixed complete medium (e.g., 594 μL per well for a 24-well culture plate) to the mRNA-LNP stock solution in DPBS (e.g., 6 μL per well for a 24-well culture plate) to achieve the desired final concentration (e.g., 0.5 ng/μL mRNA for a 24-well culture plate).
• 8.Perform gentle pipetting to ensure the thorough mixing of the media and mRNA-LNPs.

Note: Proceed directly to the transfection step after preparing the “mRNA-LNP in medium” without any additional incubation.

Transfection of mRNA-LNP to cells

Timing: 7–25 h

• 9.Carefully remove residual medium from the cells.
• 10.Without an additional washing step, add the prepared “mRNA-LNP in medium” to each well at the specified volume (e.g., 500 μL per well for a 24-well plate).

Note: See Table 2 for recommended mRNA-LNP in medium volumes for different plate formats.

• 11.Incubate the cells under standard conditions (37°C, 5% CO₂) for the recommended duration (e.g., 24 h for general mRNA transfection and 6 h for highly transfectable cells such as Huh-7 or HepG2).

Optional: After 6 h, replace the mRNA-LNP-containing medium with fresh growth medium if needed, and continue incubation.

Note: If LNP toxicity is minimal, omit this media change step.

Table 1 lists the culture conditions used for the maintenance of each cell line.

Table 2 summarizes the recommended mRNA amounts, mRNA-LNP stock concentrations, dilution volumes, and final working concentrations for both the 24- and 96-well plates.

Table 3 presents side-by-side comparisons of the transfection protocols. Details of transfection conditions of each protocol in 24- and 96-well plates are described in Table S1.

Table 3.

Side-by-side comparison of transfection protocols

Step	PC (Lipofectamin) (manufacturer’s protocol)	PC_LNP (serum-starved condition)	LNP in medium (this protocol)
1. Medium removal	Yes (Remove medium completely)	Yes (Remove medium completely)	Yes (before adding 1× mRNA-LNP in complete medium)
2. DPBS washing	Yes (1–2 times)	Yes (1–2 times)	No
3. Pre-incubation medium	Opti-MEM (20 min) 450 μL (24-well plates) 60 μL (96-well plates)	Opti-MEM (20 min) 450 μL (24-well plates) 60 μL (96-well plates)	No
4. Formulation	mRNA-Lipofectamine complex in Opti-MEM	mRNA-LNP diluted 10× in Opti-MEM	mRNA-LNP diluted 1× in complete mediaa
5. Formulation incubation	Yes (10–15 min at RT)	No	No
6. Transfection volume	50 μL (24-well plates) 40 μL (96-well plates)	50 μL (24-well plates) 40 μL (96-well plates)	500 μL (24-well plates) 100 μL (96-well plates)
7. Incubation	6–24 h	6–24 h	6–24 h
8. Media change	Yes (after 6 h)	Yes (after 6 h)	Optional (after 6 h)
9. Media used after media change	Complete growth medium	Complete growth medium	Complete growth medium

^aUse conditioned or fresh or mixed complete media depending on the cell line.

Transfection efficiency assessment using flow cytometric assay (method #1)

Timing: 1 h

For method #1, following steps 12–13.

This protocol describes the measurement of transfection efficiency using EGFP-encoding mRNA in a 24-well plate format.

• 12.
  Harvest transfected cells.

  • a.
    Remove media from the cells.
  • b.
    Add DPBS over the cells in each well and aspirate it three times.
  • c.
    Add 200 μL of 1× trypsin-EDTA per well.
  • d.
    Place the plate with trypsin-EDTA in the incubator for approximately 3 min.

    Note: Avoid excessive exposure to trypsin-EDTA to prevent damage to cell health and viability.

  • e.
    Deactivate trypsin-EDTA by adding 300 μL of complete media containing fatal bovine serum and antibiotics.
  • f.
    Move the cells in each well to 1.5 mL tube each.
  • g.
    Spin cells down through centrifugation (1000 rpm, 170 × g, 3 min).
  • h.
    Remove the media and wash the pellet with 1 mL DPBS.

    Note: Carefully preserve the cell pellet while removing the media to prevent loss of cells.

  • i.
    Repeat step g–h three times.
• 13.Conduct flow cytometry analysis.

Note: Place the harvested cells on ice to maintain their stability and viability.

Transfection efficiency assessment using luciferase assay (method #2)

Timing: 1 h

For method #2, following steps 14–16.

This protocol describes the measurement of transfection efficiency using firefly luciferase-encoding mRNA in a 96-well plate format.

• 14.
  Wash transfected cells.

  • a.
    Aspirate the culture medium from each well.
  • b.
    Add DPBS to each well and aspirate.
  • c.
    Repeat the wash step b twice.

Note: Avoid cell detachment during the washing step.

• 15.
  Cells lysis.

  • a.
    Add 30 μL of 1× lysis buffer to each well.
  • b.
    Incubate the plate at room temperature for 2–3 min.
• 16.
  Measure the luminescence.

  • a.
    Transfer 20 μL of cell lysate from each well to a white 96-well plates.
  • b.
    Add 100 μL of luciferase assay buffer to each well.
  • c.
    Measure luminescence using a microplate reader.

Note: Perform luminescence measurement within 30 min of adding the substrate solution.

Transfection efficiency assessment using live-cell imaging (method #3)

Timing: 1 h

For method #3, following steps 17–18.

This protocol describes the measurement of transfection efficiency using EGFP-encoding mRNA in a 96-well plate (F-BOTTOM, μCLEAR, BLACK) format.

• 17.
  Wash the transfected cells.

  Note: Use a clear-bottom, black-walled 96-well plate from the cell seeding step to ensure optimal conditions for live cell imaging.

  • a.
    Carefully aspirate the medium from each well.
  • b.
    Add DPBS (100 μL) to each well and aspirate.
  • c.
    Repeat the step b twice.
  • d.
    Add 100 μL of complete medium to each well.
  • e.
    Incubate the plate under standard culture conditions (37°C, 5% CO₂) to allow cell recovery.

    Note: Avoid cell detachment during the washing step.

• 18.
  Live cell imaging.

  • a.
    Remove the plate from the incubator.
  • b.
    Acquire fluorescence images for each well using an appropriate imaging system.

Expected outcomes

This protocol significantly enhances the in vitro transfection efficiency of mRNA-LNPs. Lipofectamine was used as a positive control using the conventional protocol (Table 3 and Supplementary Protocol 1). This protocol eliminates the starvation steps and enables direct mRNA-LNP treatment in serum containing complete media compared with the conventional method. Figure 1 illustrates a simplified workflow of the protocol. A detailed step-by-step comparison is presented in Table 3. mRNA-LNPs were prepared in-house using a 50:10:38.5:1.5 molar ratio (SM-102:DSPC:cholesterol:DMG-PEG2000)—a composition widely used in commercial mRNA vaccines.⁵ The in vivo efficacy of this commercial formulation has already been confirmed in previous studies,^1,3,17 making it a suitable benchmark for protocol validation.

Figure 1.

Simplified workflow of the protocol

A step-by-step schematic illustration of the experimental procedure for practical use.

Quantitative analysis of in vitro transfection efficiency can be achieved either by delivering EGFP mRNA and subsequently measuring EGFP fluorescence using flow cytometry or delivering firefly luciferase mRNA and quantifying bioluminescence from firefly luciferase with a microplate reader. Figure 2 presents the results obtained using both methods, comparing the transfection efficiency under the conditioned media protocol (LNP) and conventional serum-starved approach (PC_LNP). Using conventional transfection methods, which are commonly based on serum-starved conditions, the LNP-mediated transfection efficiency was significantly lower than that of Lipofectamine (PC). However, under the conditions defined in this protocol, flow cytometry analysis indicated that the transfection efficiency is comparable to or even superior to that of Lipofectamine. We observed a substantial improvement in transfection efficiency across multiple cell lines, including HEK293, Huh-7, HeLa, HepG2, MCF-7, U-87 MG, HT22, Raw264.7, and SH-SY5Y cells. While cell type-specific variation exists, the current protocol increased transfection efficiency by 1.4–25.6-fold based on EGFP fluorescence (Figures 2A and S1; Tables S1 and S3) and 31.8–133.8-fold based on bioluminescence from firefly luciferase relative to serum-starved groups (Figures 2B and S2; Tables S2 and S3).

Figure 2.

Quantitative assay for in vitro mRNA-LNP transfection by transfection method across cell lines

(A) Quantification of in vitro transfection efficiency using flow cytometric assay across multiple cell lines and transfection conditions. The mean fluorescence intensities (MFI) of EGFP were measured across multiple cell lines. Transfection conditions included non-transfected cells (NC, white bars), mRNA-only transfected cells (RO, diagonally striped bars), mRNA-Lipofectamine-transfected positive control (PC, light gray bars), mRNA-LNP-treated cells using this protocol (LNP, black bars), and mRNA-LNP-treated cells under serum-starved condition (PC_LNP, gray bars). Error bars indicate standard deviation (n = 3). Statistical significance was assessed by Tukey’s paired comparison test: n.s. (not significant), ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001. (B) Quantification of in vitro transfection efficiency using luciferase assay accross multiple cell lines and transfection conditions. The relative luminescence units (RLU) from firefly luciferase were measured across multiple cell lines. The transfection conditions and statistical analyses were the same as in (A).

Figure 3A presents histograms of EGFP fluorescence in individual cells, as measured by flow cytometry, demonstrating that the fluorescence intensity distribution in the mRNA-Lipofectamine-treated cells is often broad, suggesting heterogeneous intracellular delivery. In contrast, the mRNA-LNP-treated cells displayed narrower, more defined peaks, indicating more uniform transfection across the cell population. Although this method has not yet been tested in all cell lines, the observed improvements strongly suggest that it enhances the applicability of mRNA-LNPs for in vitro studies.

Figure 3.

Evaluation of in vitro EGFP expression of EGFP-encoding mRNA-LNP by transfection method

(A) Representative flow cytometry histograms showing EGFP fluorescence distribution across cell lines and transfection conditions. The cells were transfected under five different conditions: NC (Negative control, blue), RO (mRNA only, magenta), PC (positive control with mRNA-Lipofectamine, red), LNP (mRNA-LNP in medium, orange), and PC_LNP (mRNA-LNP transfection under serum-starved condition, light blue). (B) The live cell imaging across cell lines and transfection conditions. The cells were transfected under five different conditions as described in (A). Scale bars: 200 μm.

The fluorescence resulting from EGFP mRNA transfection can also be visualized by live-cell imaging (Figure 3B), which shows a pattern highly consistent with that quantified by flow cytometry. Although mRNA-Lipofectamine produced stronger fluorescence than that of the mRNA-LNPs, the fluorescence intensity is not uniform across cells. The images clearly demonstrate that mRNA–LNP transfection in complete media is superior to that under serum-starved conditions.

In vitro transfection of mRNA–LNPs using this protocol was conducted repeatedly under varying cell conditions, experimental dates, and experimenters. As shown in Figures 4A and S3–S5, the protocol yielded reliable transfections across HEK293, Huh-7, and HT22 cell lines. The protocol was evaluated by three independent experimenters A, B, and C on different days. All experiments were conducted in conditioned complete medium using EGFP mRNA, and the results were summarized as the mean fluorescence intensity (MFI) of EGFP quantified by flow cytometry. Despite these variations, reproducible and positive transfection efficiencies were consistently observed, with only minor differences. By contrast, reproducibility was comparatively lower in PC treated with mRNA-Lipofectamine.

Figure 4.

Evaluation of in vitro mRNA-LNP transfection efficiency under different experimental conditions

(A) Reproducibility of mRNA-LNPs transfection efficiency across different operators and experimental dates using either mRNA-Lipofectamine (PC, gray bars) or mRNA-LNP (LNP, black bars), including non-treated cells (NC, white bars). (B) Comparison of mRNA-LNP transfection efficiency in different medium conditions. Transfection conditions included non-treated cells (NC, white bars), cells transfected in fresh medium (Fresh, diagonally stripped bars), and cells transfected in conditioned medium (Conditioned, black bars). Error bars indicate standard deviation (n = 3). Statistical significance was assessed by Tukey’s paired comparison test: ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001.

In this protocol, complete medium was used for mRNA-LNP transfection, and the choice of serum supplier influences the outcomes. Additionally, the use of conditioned or fresh media may affect transfection efficiency. A direct comparison of media containing 10% FBS from two different manufacturers (Company W (Co. W) and Company G (Co. G)) revealed variations depending on whether the medium was conditioned or fresh, and these effects may differ depending on cell type (Figures 4B and S6). Thus, the optimal combination of supplier and medium conditions is important, but may vary case by case. Liver tropism has been previously reported for the commercial LNP formulation used in this study. Consistent with this finding, we observed that conditioned medium was preferable for hepatic cell lines, whereas fresh medium was advantageous for non-hepatic cell lines. This phenomenon can be interpreted in relation to protein corona formation.^18,19 In hepatic cells, conditioning facilitates the formation of an appropriate protein corona, thereby enhancing transfection efficiency. In contrast, in non-hepatic cells, the conditioned medium interfered with optimal protein corona formation, leading to reduced efficiency. Thus, the observed outcomes were a consequence of the liver tropism of the tested LNPs. Different LNP formulations with distinct organ tropisms may yield opposite results, underscoring the critical importance of selecting the appropriate medium conditions for a given mRNA–LNP system. When uncertainty exists regarding the optimal media, a 1:1 mixture of conditioned and fresh media may serve as a practical compromise; indeed, our recent report demonstrated transfection results obtained under such mixed medium conditions.²⁰

Compared to physical methods such as electroporation, the delivery of mRNA via LNPs is particularly important for maintaining cell viability. For instance, in the context of mRNA-based Chimeric Antigen Receptor T cell engineering, the use of mRNA-LNPs results in higher cell viability than electroporation.²¹ Our overall results demonstrated that mRNA-LNP transfection using this protocol exhibits a superior transfection profile compared to serum-starved conditions. Therefore, along with the growing interest in mRNA therapeutics, this protocol is expected to be useful for various applications, including gene-editing tool development, delivery and therapeutic mechanism studies, cell-based therapy, and other mRNA-based applications. To screen mRNA-LNPs when the target tissue is known, the use of matched cell lines may provide relevant in vitro screening models. However, high in vitro performance does not always predict selective in vivo delivery; thus, in vitro results should be interpreted in the context of in vivo complexity. In cases where the delivery target is not clearly defined, selecting cell lines align with the experimental goal is essential for obtaining meaningful data. Nevertheless, by enabling robust and reproducible transfection across diverse cell types, this protocol facilitates more reliable in vitro screening of mRNA-LNPs, mechanistic studies, and cell engineering strategies in contexts where serum-compatible high-efficiency delivery is critical.

Limitations

While conditioned media enhance mRNA-LNP transfection efficiency, several limitations must be considered. Batch-to-batch variability poses a major challenge, as secreted bioactive molecules vary depending on cell density, passage number, and incubation time, leading to inconsistent results. Additionally, uncontrolled factors within conditioned media, such as metabolic byproducts and degraded biomolecules, may introduce cytotoxic effects, further complicating standardization.

Conditioned media may alter cell physiology, potentially affecting transfection efficiency through mechanisms unrelated to nanoparticle uptake.

This protocol shows a consistent improvement in transfection efficiency across several cell lines, especially compared to that of serum-starved conditions, which often result in little transfection. However, similar to Lipofectamine and other transfection agents, commercial LNPs do not always function equally well in all cell types. This is a limitation of the intrinsic properties of the LNPs and how different cells take them up.

Finally, cell-type specificity remains uncertain, as different cell lines may respond variably, affecting viability and reproducibility.

Addressing these challenges may exhibit variable responses, impacting both cell viability and reproducibility through compositional analysis, controlled supplementation, and broader validation is essential for optimizing this approach in standardized experimental settings.

If the target organ or cell type for delivery is not clearly defined, it remains difficult to determine whether the transfection trends observed in one or two selected cell lines accurately reflect in vivo delivery pattern. However, for applications with specific delivery targets, such as cancer cells, or cases where in vitro research is essential, this protocol serves as a foundation for high-throughput in vitro screening to optimize nanoparticle formulations for targeted delivery.

This protocol is not guaranteed to be applicable to all cell lines, particularly primary and immune cells. Nevertheless, compared to the serum-starved conditions, we consistently observed the enhanced transfection efficiency of mRNA-LNPs in complete media with hundreds of mRNA-LNPs, even when they exhibit a different pattern of transfection efficiency. The manufacturer of the serum and medium, decision regarding the use of fresh or conditioned medium, number of cells seeded, and incubation time need to be further validated for each mRNA-LNP and cell line. In addition, variability between serum batches may influence reproducibility across experiments.

Finally, high-throughput in vitro screening may require substantial amounts of complete medium, increasing experimental costs.

Troubleshooting

Problem 1

The cells tested in this protocol are adherent, whereas suspension cells require modifications in the protocol.

Potential solution

For cells other than those specified, we recommend using the complete growth medium appropriate for that cell line. For suspension cells, the main difference is that the trypsinization step is unnecessary and therefore omitted. In addition, minor modifications of the transfection steps are recommended. For transfection in conditioned complete medium, add 50–100× mRNA-LNP stock solution to each well after overnight incubation of seeded cells. For transfection in fresh complete medium, add the 50–100× mRNA-LNP stock solution immediately after seeding. For transfection in mixed complete medium, seed cells at half the final medium volume, incubate overnight, and then fill the remaining half of the final volume with fresh complete medium, followed immediately by 50–100× mRNA-LNP stock solution. Compare this procedure with step 7 and steps 9–10 of this protocol.

Problem 2

The use of complete media is challenging for large-scale screening due to batch-to-batch variability and the logistical difficulty of maintaining consistent conditions across multiple experiments.

Potential solution

To enable scalability, a defined and optimized formulation of complete media should be developed. It is critical to identify the source of serum. This would allow for consistent application in high-throughput settings while preserving the transfection-enhancing properties. Additionally, automation and bioreactor-based media preparation could help ensure uniformity across experimental batches. Refer to the expected outcome section.

Problem 3

High doses of mRNA-LNPs may decrease cell viability. Although this protocol suggests an optimal treatment range of 0.5–1.0 ng/μL, the actual working concentration may vary depending on the specific LNP formulation and cell line used.

Potential solution

If reduced cell viability is observed using this protocol, we recommend decreasing the mRNA–LNP concentration or replacing the used medium with fresh growth medium 6 h post-transfection. As cell growth rate and conditions affect seeding density at transfection, seeding density should be adjusted. Refer to Step 11 of this protocol.

Problem 4

Excessive cell density can lead to reduced transfection efficiency. Even when seeding density is appropriately adjusted, rapid cell growth may result in high density at transfection, reducing efficiency.

Potential solution

We recommend testing this protocol at different seeding densities to determine the optimal conditions for the specific cell types used. We recommend to adjust the cell density to reach ∼60% confluency at the time of mRNA-LNP treatment. Refer to Step 4 of this protocol.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Eun-Kyoung Bang (eunkbang@kist.re.kr).

Technical contact

Technical questions on executing this protocol should be directed to and will be answered by the technical contact, Eun-Kyoung Bang (eunkbang@kist.re.kr).

Materials availability

This study did not generate new unique reagents.

Data and code availability

This study did not generate data sets.

Acknowledgments

This work was supported by Ministry of Science and ICT, South Korea (no. RS-2023-00229101); Ministry of Food and Drug Safety, South Korea (no. 22213MFDS421); and Korea Institute of Science and Technology Institutional Program (no. 2E33791).

Author contributions

K.Y. and S.Y. designed, set up, and standardized the protocol and wrote the manuscript. I.K.H., H.G., H.P., H.L., and S.O. validated the overall protocol. G.K. provided resources and contributed to funding acquisition. E.-K.B. supervised the study, secured funding, provided resources, and contributed to manuscript writing.

Declaration of interests

The authors declare no competing interests.