Tailoring transfection for cardiomyocyte cell lines: balancing efficiency and toxicity in lipid versus polymer-based transfection methods in H9c2 and HL-1 cells

Abstract

Cardiovascular research relies heavily on the veracity of in vitro cardiomyocyte models, with H9c2 and HL-1 cell lines at the forefront due to their cardiomyocyte-like properties. However, the variability stemming from nonstandardized culturing and transfection methods poses a significant challenge to data uniformity and reliability. In this study, we introduce meticulously crafted protocols to enhance the culture and transfection of H9c2 and HL-1 cells, emphasizing the reduction of cytotoxic effects while improving transfection efficiency. Through the examination of polymer-based and lipid-based transfection methods, we offer a comparative analysis that underscores the heightened efficiency and reduced toxicity of these approaches. Our research provides an extensive array of step-by-step procedures designed to foster robust cell cultures and outlines troubleshooting practices to rectify issues of low transfection rates. We discuss the merits and drawbacks of both transfection techniques, equipping researchers with the knowledge to choose the most fitting method for their experimental goals. By offering a definitive guide to these cell lines’ culturing and transfection, our work seeks to set a new standard in procedural consistency, ensuring that the cardiovascular research community can achieve more dependable and reproducible results, thereby pushing the boundaries of current methodologies toward impactful clinical applications.

NEW & NOTEWORTHY We have developed standardized protocols that significantly reduce cytotoxicity and enhance transfection efficiency in H9c2 and HL-1 cardiomyocyte cell lines. Our detailed comparative analysis of polymer-based and lipid-based transfection methods has identified optimized approaches with superior performance. Accompanying these protocols are comprehensive troubleshooting strategies to address common issues related to low transfection rates. Implementing these protocols is expected to yield more consistent and reproducible results, driving the field of cardiovascular research toward impactful clinical breakthroughs.

INTRODUCTION

Cardiomyocyte functionality is central to the cardiac contraction-relaxation cycle, with their compromise leading to a spectrum of cardiomyopathies that culminate in heart failure (1). As adult cardiomyocytes exhibit limited proliferative and regenerative capabilities, delineating the mechanisms of pathological remodeling is paramount to mitigating cardiomyocyte attrition and improving cardiac health (2–4). In vitro models, particularly cardiomyocyte cell lines, offer a controlled environment to dissect the molecular intricacies underlying cardiomyocyte pathology (5). The H9c2 and HL-1 cell lines, representing murine and rat models, respectively, are instrumental in such studies; however, the absence of optimized transfection protocols hampers the reliability and reproducibility of outcomes in cardiovascular research.

Our investigation tackles this gap by developing refined methods to optimize transfection in H9c2 and HL-1 cardiomyocyte cell lines. This initiative is not merely an enhancement of laboratory techniques but a crucial step in ensuring the fidelity of experimental data that drives the field forward. The challenge of transfection lies in the dichotomy between efficacy and toxicity, a balance yet to be mastered, especially for those new to the domain (6).

The purpose of this study is to systematically evaluate and optimize the transfection methods for H9c2 and HL-1 cell lines, with a focus on maximizing efficiency while minimizing cytotoxic effects. We aim to provide a robust protocol that delineates the optimal conditions for both polymer and lipid-based transfection methods. By doing so, we intend to bridge the efficiency-toxicity gap, presenting a set of best practices that enhance experimental accuracy and reliability. Moreover, this study seeks to offer a comprehensive understanding of the unique characteristics of H9c2 and HL-1 cell lines derived from mouse atrial tissue and rat ventricular tissue, respectively. By exploring their differential response to transfection, we aspire to establish standardized practices that can be adopted by the cardiovascular research community, thus fostering data uniformity and propelling translational advancements in the field.

In summary, the study’s purpose is twofold: to refine transfection protocols that are critical for in vitro cardiomyocyte research and to ensure these protocols are accessible and effective for both seasoned researchers and those at the inception of their scientific careers. The ultimate goal is to enable the pursuit of precise molecular interventions that could retard or reverse the pathological remodeling of cardiomyocytes, offering hope for the treatment of heart disease.

MATERIALS AND METHODS

Cell Lines Description

The H9c2 and HL-1 cell lines, derived from distinct origins, exhibit unique cardiomyocyte characteristics detailed in Table 1. H9c2 cells are myoblasts, which can adopt a cardiac-like phenotype upon differentiation (7, 8). Specifically, under reduced serum conditions and in the presence of all-trans-retinoic acid (RA), H9c2 cells can differentiate into multinucleated cells with a cardiac phenotype, albeit with a diminished capacity for proliferation (9). In contrast, HL-1 cells, which originate from adult mouse atrial tissue, contract and maintain phenotypic features characteristic of adult cardiomyocytes (10, 11). H9c2 cardiomyoblasts exhibit energy metabolism characteristics more closely resembling those of primary cardiomyocytes compared with atrial HL-1 cells, making them a more suitable in vitro model for simulating cardiac pathogenesis (12).

Table 1.

Comparative characteristics of HL-1 and H9c2 cardiomyocyte cell lines

Characteristic	HL-1 Cells	H9c2 Cells
Origin	Derived from mouse atrial cardiomyocytes	Derived from rat ventricular cardiomyocytes
Age	Adult cardiomyocyte cell line	Embryonic cardiomyocyte cell line
SV40 transfection	SV40 large T antigen transfected to achieve immortalization	Not transfected with SV40
Differentiation state	Semidifferentiated, express markers of mature cardiomyocytes.	Less differentiated than HL-1 cells
Proliferation	Can multiply continuously due to SV40 transfection	Limited proliferative capacity, have a potential to differentiate at late passages.
Growth rate	Rapid growth rate, doubling time of ∼20 h	Slower growth rate, doubling time of ∼40 h
Cell morphology	Spindle-shaped cells that form irregularly shaped clusters	Elongated cells that grow in a parallel orientation
Contractile activity	Contractile cell line, display spontaneous contractions at early passages.	Do not show spontaneous contractions
Gene expression	Express markers for atrial cardiomyocytes such as ANP and Nppa	Express markers for ventricular cardiomyocytes such as Myh6 and Myh7
Optimal culture conditions	Claycomb medium with 10% FBS, 0.1 mM norepinephrine, and 4 mM l-glutamine at 37 °C with 5% CO2	DMEM with 10% FBS at 37 °C with 5% CO2
Freezing media	90% Culture medium (plain Claycomb medium), 10% DMSO	90% Culture medium (plain DMEM), 10% DMSO

ANP, atrial natriuretic peptide; Nppa, natriuretic peptide A; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; DMSO, dimethyl sulfoxide.

Cell Culture Components and Preparation

For the establishment and maintenance of cell cultures, the following components are meticulously selected and prepared:

• 1) 
  Culture media:

  • i) 
    Claycomb medium (Millipore-Sigma, 51800C): specifically formulated for cardiac cells, providing a nutrient-rich environment. The formulation of the original Claycomb medium has been published (11).
  • ii) 
    Dulbecco’s modified Eagle’s medium (DMEM; Sigma, D5796-500ML): a versatile medium supporting a wide range of cell types.
  • iii) 
    Opti-MEM (Thermo Fisher Scientific, 31985070): reduced serum medium optimized for transfection efficiency.
  • iv) 
    Fetal bovine serum (FBS; Millipore-Sigma, F2442): fetal bovine serum to supplement the growth media with essential nutrients.
  • v) 
    Norepinephrine: added to Claycomb medium to stimulate cardiac muscle contraction.
  • vi) 
    Glutamine: an amino acid to support cell metabolism and growth.
  • vii) 
    Antibiotics: to prevent bacterial contamination in the culture.
• 2) Coating solution for HL-1 cells: a mixture of fibronectin and gelatin to promote cell adhesion and growth.
• 3) Trypsin (Sigma-Aldrich, T4049): an enzyme used to detach cells during passaging.
• 4) Phosphate-buffered saline (PBS; Lonza, 51226): a balanced salt solution used for washing cells.
• 5) Plasmid DNA: for genetic manipulation and studies in transfection.
• 6) Medium filter (Thermo Scientific, 5660020): to sterilize the culture medium by removing potential contaminants.
• 7) Lactate dehydrogenase (LDH; Thermo Scientific, 88953): an enzyme assay to assess cell viability.
• 8) Lipofectamine (Thermo Fisher Scientific, 11668019): a lipid-based reagent for efficient DNA transfection.
• 9) PolyJet (SignaGen, SL100688): a polymer-based transfection reagent offering an alternative method for gene delivery.

Each component is crucial for the specific requirements of cardiac cell culture and transfection protocols. All media and solutions need to be prepared under sterile conditions to maintain the integrity and cleanliness of cell cultures.

Plasmid DNA Vector

To standardize transfection procedures in HL-1 and H9c2 cell lines, we used a 6-kb plasmid DNA vector, pcDNA3.1 + egfp+P2A (GenScript Biotech, SC2092). The plasmid was carefully diluted and transformed into Escherichia coli DH5α, and incubated overnight on LB agar plates with 1% ampicillin at 37°C. Following a 16- to 18-h incubation, a single colony was selected and propagated in 300 mL of LB medium supplemented with 1% ampicillin. A maxi-prep was performed according to the Qiagen manufacturer’s instructions. The DNA’s quantity and purity were assessed using a NanoDrop spectrophotometer, with an expected yield of ∼900 µg from the initial elution.

Critical step.

Ensuring DNA purity with a 260-to-280 ratio of around 2.00 is imperative, as contaminated, or impure DNA can significantly diminish transfection efficiency and lead to increased cell mortality.

Cell Culture Revival Protocol

The successful revival of frozen cell lines is a critical step in establishing a viable cell culture. The reagents and flasks for cell culture are included in Table 2. The standard procedure for cell line revival is as follows:

Table 2.

Consumables and reagents for cell culture and transfection

Name	Company	Catalog No.
15-mL conical sterile polypropylene centrifuge tubes	ThermoFisher	339650
96-well plate	ThermoFisher	171734
Dark-well 96-well plate	ThermoFisher	152036
24-well plate	ThermoFisher	930186
6-well plate	ThermoFisher	130184
T25 flask	ThermoFisher	156367
T75 flask	ThermoFisher	156499
Lipofectamine 2000	ThermoFisher	11668019
PolyJet	SignaGen	SL100688

• 1) 
  Begin by warming the culture medium for 20 to 30 min. Remove the frozen vial of cells from the liquid nitrogen storage and promptly thaw it in a 37°C water bath that has been preheated, aiming for a rapid thaw of less than 2 min to minimize cell damage.

  • i) 
    Alternatively, the vial may be placed in the cell culture incubator for 2 min for a partial thaw.
• 2) Following partial thawing, disinfect the vial’s exterior with 70% ethanol and proceed inside a sterile hood. Gently mix the cells with prewarmed medium and transfer the contents to a conical tube.
• 3) 
  Centrifuge the tube at a low speed (e.g., 300 g) for 5 min to pellet the cells.

  • i) 
    Concurrently, disinfect the flask’s plastic bag with 70% ethanol and label the flask with the date and passage number.
• 4) Discard the supernatant and resuspend the cell pellet in 1 mL of prewarmed medium, pipetting up and down to ensure a uniform suspension.
• 5) Transfer the cell suspension into the appropriate volume of medium for the culture vessel (e.g., 10 mL for a T75 flask).
• 6) Place the culture vessel in an incubator at 37°C with 5% CO₂ to promote overnight recovery.

Adhering to this protocol will facilitate the effective revival of cells for subsequent culture and experimentation. It is crucial to monitor cell morphology, medium color, and confluency by the end of the initial day and the following day. For H9c2 cells, passage at 70–80% confluency is recommended, and for HL-1 cells, passage at 90–95% confluency (Fig. 1). The precise revival conditions may vary based on the cell type and specific growth requirements; thus, it is essential to consult the manufacturer’s guidelines or relevant literature for detailed instructions. Refer to Fig. 1 for a visual guide to the process.

Figure 1.

Overview of cell culture revival and seeding protocol. A: depiction of the process for reviving frozen cells: Cells are initially resuspended in 1 mL of complete medium, then transferred to a 15-mL conical tube where an additional 5 mL of complete medium is added. This dilution step is followed by cell counting to determine the appropriate seeding density for various plate formats. A guide for adjusting cell confluency specific to HL-1 and H9c2 cell lines for 6-well, 48-well, and 96-well plates is included. The cell suspension is thoroughly mixed to ensure even cell dissociation and consistent distribution across the culture wells. After overnight incubation, cultures are assessed for adherence and confluency; medium color changes and dead cells floating indicate the need for medium replacement. B: illustration of the expected homogeneous distribution of cells within a culture well postseeding.

Cell Passaging Protocol

Effective passaging is crucial for maintaining cell line integrity, as higher passage numbers can lead to differentiation and significant molecular and morphological changes.

The H9c2 cardiomyocyte cell line retains characteristic cardiomyocyte features within the initial five passages, making it a dependable model for early-stage studies (13). In contrast, HL-1 cells, commercially available through Millipore (catalog no. SCC065), display consistent cardiomyocyte markers and functionality up to the 10th passage. However, the original stock of HL-1 can maintain the ability to contract and retain characteristic cardiomyocyte features through at least passage 240 (10). These specific passage ranges are optimal for conducting transfection studies, ensuring both the integrity of cardiomyocyte characteristics and the reliability of experimental outcomes.

• 1) Gently aspirate the spent media from the culture vessel and wash the cell layer with 2 mL of plain medium to remove residual serum that may inhibit trypsin action.
• 2) Dispense 2 mL of trypsin into the vessel and return it to the incubator for 2–4 min to allow cells to detach. The exact time may vary depending on the cell type and confluency.
• 3) After incubation, gently agitate the flask to dislodge any remaining adherent cells. Confirm cell detachment under a microscope. Immediately neutralize the trypsin by adding an equal volume of complete medium and pipette the mixture to dislodge and suspend the cells completely.
• 4) Transfer the cell suspension to a 15-mL conical tube and centrifuge at 300 g for 5 min at room temperature to pellet the cells.
• 5) Carefully aspirate the supernatant containing trypsin and resuspend the cell pellet in 1 mL of complete medium promptly to minimize cell stress.
• 6) Count the cells if necessary and seed an appropriate number of cells into a new culture vessel with the desired volume of medium, ensuring proper distribution for uniform growth.

Transfection Techniques for Cardiomyocyte Cell Lines

Transfection is a pivotal procedure in molecular biology, instrumental for delving into gene function, analyzing protein roles and structures, and administering gene therapy. With technological progress, a plethora of transfection reagents and techniques have surfaced, falling into three broad categories: chemical, physical, and biological (14, 15). The distinction between transient and stable transfection is fundamental; the former offers temporary gene expression, while the latter ensures permanent integration into the host genome, allowing for continuous expression and passage to progeny (15). The choice of transfection type is inherently tied to the objectives of the research and the cell lines in question. In this study, we have employed both lipid-based and polymer-based transfection reagents to transiently transfect H9c2 and HL-1 cardiomyocyte cell lines, allowing for a comparative analysis of these two methodologies in terms of efficiency and cellular viability.

The arena of transfection reagents is diverse, with lipid-based and polymer-based reagents being prominent due to their cost-effectiveness and simplified methodology. These reagents are favored for their high transfection efficacy. A detailed comparative analysis of these two reagents is presented in Table 3, highlighting their advantages and limitations.

Table 3.

Comparative efficacy and cytotoxicity profile of lipofectamine and PolyJet transfection reagents in cardiomyocyte cell lines

	Lipofectamine	PolyJet
Transfection method	Lipid-based transfection	Polymer-based transfection
Efficiency	High	High
Toxicity (e.g., cell death)	Moderate-high	Low
Transfection medium	Optimum medium	Serum-free culture medium (e.g., Claycomb or DMEM)
Working concentration	1:2, 1:3 (DNA: Lipofectamine 2000)	1:2, 1:3 (DNA: PolyJet)
Price	More expensive compared with other transfection reagents	Cost-effective

DMEM, Dulbecco’s modified Eagle’s medium.

Lipid-based reagents, also known as liposomes, form complexes with DNA through the interaction of their cationic components with the negatively charged phosphate groups of the DNA. These complexes then bind to cell membranes and enter cells via endocytosis. Despite their effectiveness, lipid-based reagents can be expensive, which may limit their use in large-scale studies. Moreover, optimizing transfection conditions for various cell types can be challenging, with excessive use of reagents like Lipofectamine potentially leading to cytotoxic effects and increased cell mortality.

Polymer-based reagents offer an alternative, forming complexes with DNA in a manner akin to liposomes. They are particularly advantageous for cell lines resistant to lipid-based methods and are associated with a lower immune response posttransfection, translating into reduced cytotoxicity. While generally less expensive than lipid-based options, polymer-based reagents might exhibit lower transfection efficiencies in some contexts.

Optimal Culturing of H9c2 Cardiomyoblasts

The H9c2 cell line, consisting of embryonic rat cardiomyoblasts, is procured from the American Type Culture Collection and plays a vital role in cardiac research. For H9c2 cell culturing, DMEM with high glucose levels is used, enriched with 10% FBS to foster growth and 1% penicillin-streptomycin to provide prophylactic antibacterial protection.

A specific number of cells, tailored to the experiment’s scale, are plated and incubated for a predetermined number of hours, allowing for adhesion and initial growth. Vigilant monitoring is essential to ensure that cells are subcultured before surpassing 60–70% confluence to maintain an optimal growth rate and cellular function. The cells were maintained in an incubator set to 37°C with a 5% CO₂ atmosphere to replicate physiological conditions conducive to cell growth.

Critical alert.

It is imperative to avoid allowing H9c2 cells to reach 100% confluence. Full confluency can trigger contact inhibition, negatively impacting cell health and viability, and potentially confounding experimental results. To preserve cell vigor and ensure reproducible data, it is recommended to keep H9c2 cells below 80% confluency.

When cells approach the 70% confluence threshold, they are passaged at a 1:2 split ratio into fresh flasks, with each receiving equal parts of the cell suspension. This practice is considered the start of a new passage. The culture medium is diligently checked every day and refreshed every 24 to 48 h to maintain an optimal environment for the cells.

By adhering to these meticulous culturing protocols, H9c2 cells can be maintained in a state conducive to robust growth and viability, facilitating their use in a wide array of cardiac-related experimental assays.

Optimized Culture and Seeding of HL-1 Cardiomyocyte Cell Line

HL-1 cells, a line of continuously proliferating cardiomyocyte cell line, exhibit characteristic cardiac phenotypes under specific culture conditions (10). These cells thrive in flasks coated with a fibronectin-gelatin matrix, which mimics the extracellular matrix of cardiac tissue and supports cellular adhesion and proliferation. Our culture protocol involves maintaining HL-1 cells in Claycomb medium, supplemented with 10% FBS to provide essential growth factors, 100 µg/mL of penicillin and streptomycin to prevent microbial contamination, 2 mM l-glutamine for cellular metabolism support, and 0.1 mM norepinephrine, a crucial additive that promotes cardiomyocyte contractility. The cells were cultured in a controlled environment within an incubator calibrated to a temperature of 37°C and an atmosphere containing 5% CO₂ to simulate the in vivo conditions essential for optimal cell proliferation and function.

For coating, we use a fibronectin concentration of 50 µg/mL combined with 0.02% gelatin in a sufficient volume to cover the growth surface area of each flask, typically 5 mL for a T75 flask. This coating substrate is applied at least 1 h before cell seeding to ensure adequate surface preparation, although overnight incubation is preferred to achieve optimal cell attachment.

It is important to note that fibronectin-gelatin coating can be reused two to three times, reducing waste and costs without compromising cell health.

Once cells reach 90–95% confluency, they are passaged at a ratio of 1:3, ensuring continued growth and viability. Daily monitoring of the culture medium is imperative to assess cell morphology, detect any potential infections, and confirm the maintenance of contractility.

It is imperative to note that overconfluence in cell cultures can precipitate a decline in cell viability and centrality, resulting in altered morphology and heightened cell mortality. Consequently, to harness the peak vitality and contractility of HL-1 cells, transfection procedures are recommended to be conducted between passages 4 and 8. Earlier passages have been observed to enhance transfection efficiency significantly.

In terms of cell seeding for transfection, we have determined that strategic seeding and distribution techniques are essential for achieving uniform cell coverage across culture plates. The initial placement of cells, followed by gentle tilting and swirling of the plate, ensures an even distribution. For the transfection procedure itself, we advocate adding the transfection mixture in incremental volumes, using a pipette set to deliver small droplets across the well to enhance distribution and uptake efficiency. For example, a 300-µL transfection volume should be administered in 50-µL increments using a P200 pipette.

Finally, due to the inherent size difference between HL-1 and H9c2 cells, it is crucial to adjust the seeding density accordingly. For our experiments, we reduced the H9c2 seeding quantity by 55% to match the confluency levels of HL-1, facilitating a standardized comparison of transfection outcomes between the two cell lines.

Mycoplasma Infection Assay Protocols

To safeguard the integrity of our cell culture experiments, a rigorous mycoplasma infection screening was implemented. Every 20 passages, both HL-1 and H9c2 cell lines were meticulously tested for the presence of mycoplasma, a prevalent and often undetected contaminant in cell cultures. The assay was conducted using sensitive PCR-based methods capable of detecting even low levels of mycoplasma contamination (16). Postassay, all tested cultures were confirmed to be mycoplasma-free, ensuring that the experimental data obtained were not compromised by these common cell culture pathogens.

In addition to the periodic mycoplasma testing, routine microscopic examinations were performed to monitor for bacterial contamination and to assess the general health of the cell cultures. Observations were made at both 20× and 40× magnification, which allowed for the detailed inspection of cell morphology and the detection of any signs of contamination or cellular distress. This dual approach of targeted mycoplasma testing and continuous microscopic vigilance provided a robust quality control mechanism to maintain the highest standards of cell culture health and experimental validity.

Optimized Seeding Technique for Uniform Cell Distribution

Uniform cell distribution within culture plates is a critical determinant for the reproducibility and success of cellular transfections. To achieve this, we have refined our seeding technique, focusing on the precise placement of cells within the wells and employing strategic swirling motions immediately after seeding to distribute cells evenly. These steps are essential in avoiding the formation of cell clusters and ensuring homogenous cell growth.

In addition to physical techniques, we have developed a methodical approach to the addition of transfection reagents. By introducing the transfection mixture in gradual, drop-wise increments, we have significantly enhanced the evenness of coverage across the cell layer, which is crucial for achieving consistent transfection rates.

Cell density adjustments are also pivotal. Given the inherent size disparity between HL-1 and H9c2 cells, we meticulously calculate the number of cells required to attain similar confluence levels, ensuring that each cell type reaches its optimal density for transfection without overgrowth or underuse of space (Fig. 1A). This careful calibration of cell numbers is instrumental in standardizing transfection conditions and outcomes across different cell lines within our experiments.

Comprehensive Transfection Protocol for Cardiomyocyte Cell Lines

Sample and solutions preparation.

• 1) 
  Before transfection, cell-specific medium is warmed to physiological temperature (37°C) using either a water bath or an incubator.

  • i) 
    Note: for Lipofectamine-mediated transfection, Opti-MEM is the recommended medium for optimal transfection efficiency.
  • ii) 
    An hour before transfection, switch the cell culture medium to Opti-MEM.
  • iii) 
    Prepare a complete medium without antibiotics for posttransfection recovery; the use of plain medium is discouraged as it may impact transfection efficiency and cell proliferation.
• 2) Both the transfection reagent and plasmid DNA should be kept on ice until ready for use to preserve stability.
• 3) Use of autoclaved tubes within the sterile field of a biosafety cabinet is crucial to prevent contamination.
• 4) Medium change 1 h before transfection can enhance transfection efficiency by providing fresh nutrients and removing potential inhibitors of transfection.
• 5) Assess cell health and confluency under the microscope to ensure cells are in optimal condition for transfection.

Medium Preparation

For DMEM, a complete medium typically contains 5–10% FBS and a standard percentage of antibiotics, although antibiotics should be omitted in medium used for Lipofectamine transfections because of potential adverse interactions.

PolyJet Transfection Protocol (for a 6-Well Plate)

• 1) Prepare precoated 6-well plates with a gelatin/fibronectin mixture to facilitate cell adhesion.
• 2) 
  Cell seeding:

  • i) 
    Seed HL-1 cells at a density of 700,000 cells per well in 2 mL of Claycomb medium supplemented with FBS, norepinephrine, glutamine, penicillin, and streptomycin.
  • ii) 
    Seed H9c2 cells at a density of 400,000 cells per well in 2 mL of DMEM with 10% FBS and antibiotics.
• 3) Incubate the plates at 37°C with 5% CO₂.
• 4) Perform transfection the following day, targeting 60–70% confluency for HL-1 and 50–60% for H9c2.
• 5) Remove the medium and add 1 mL of plain medium to each well.
• 6) Prepare the transfection complex with PolyJet and plasmid DNA according to the manufacturer’s instructions. Incubate at room temperature to allow complex formation.
• 7) Add the transfection complex to each well dropwise, using gentle swirling to mix.
• 8) Incubate the plates under standard conditions for 5 h.
• 9) 
  Aspirate the transfection medium and replace with fresh complete medium. Observe for transfection efficiency after 24–48 h.

  • i) 
    Optionally, replace the medium after 24 h with fresh complete medium to promote cell recovery and growth.

Lipofectamine 2000 Transfection

Follow similar initial steps for cell seeding and media preparation as described for PolyJet when using Lipofectamine 2000:

• 1) Prepare the Lipofectamine 2000/DNA complexes as per the manufacturer’s guidelines.
• 2) Carefully add the complexes to the cells, minimizing disruption to the cells.
• 3) Allow for a 6-h incubation period before changing the medium to complete medium without antibiotics.

In both protocols, it is essential to handle the cells gently during the transfection process to maintain cell integrity and viability. Posttransfection care is critical to support cell recovery and expression of the transfected genes. Regular monitoring and media changes ensure optimal cell health and experimental conditions.

Troubleshooting Cardiomyocyte Cell Line Transfection

Transfecting cardiomyocyte cell lines such as HL-1 and H9c2 can be challenging, with the potential for low transfection efficiency, high cell death rates, and nonreproducible results, even when adhering closely to established protocols. To address these issues, we have compiled Table 4, which outlines common problems encountered during transfection and provides potential causes along with suggested solutions. By systematically addressing each potential issue as outlined in Table 4, researchers can improve the efficiency and reliability of transfection experiments in cardiomyocyte cell lines. It is also advisable to keep detailed records of all experimental parameters to aid in troubleshooting and ensure reproducibility.

Table 4.

Troubleshooting H9c2 and HL-1 cardiomyocyte cell lines transfection

Transfection Problem/Potential Causes	Suggested Solutions
Low transfection efficiency
Incorrect transfection reagent used	Select an appropriate transfection reagent for cardiomyocytes H9c2 and HL-1.
Formation issues with DNA-reagent complexes	Avoid serum during complex formation. Use Opti-MEM medium for lipofectamine. Use fresh prewarmed media. Ensure purity and plasmid integrity (e.g., endotoxins, residual RNA, proteins, salts, ethanol residues, and genomic DNA can significantly reduce transfection efficiency). Ensure proper storage of the transfection reagent. Ensure thorough mixing of the DNA-reagent complex before incubation to achieve uniformity.
Presence of inhibitors during complex formation	Use serum-free DMEM, avoiding high-concentration phosphate and sulfated proteoglycans during complex formation. Avoid using antibiotics posttransfection.
Improper storage temperature	Ensure the transfection reagents are not frozen. Store at +4°C to maintain their effectiveness as recommended by the manufacturer. Avoid leaving the reagent at room temperature for too long.
Cell density	Aim for 70% confluency for DNA and cotransfection.
Plasmid length	Additional optimization might be needed for longer plasmids (e.g., >12 kb).
Transfection assay issues	Incorporate a positive control (e.g., scramble plasmid with similar length) in the transfection.
Unrecognized promoter-enhancer sequences	Ensure the compatibility of the transfected plasmid with cardiomyocytes H9c2 and HL-1, as both are originated from different species.
Passage number	Ensure the cells are in healthy conditions (e.g., observing cells for their characteristic shape, size, and uniform growth; ensuring the medium is clear and pH-balanced; preventing over confluency; monitoring for microbial contamination; maintaining high cell viability; and ensuring cells adhere properly)
Transfection complex left at room temperature too long	Form the transfection complex for 10–30 min at room temperature, depending on the manufacture protocol.
Cell density	Aim for 70% confluency for DNA and cotransfection.
High cell death (toxicity)
Cell stress	Ensure media are freshly prepared, prewarmed to 37°C, and pH balanced. Avoid keeping the cells for too long outside the incubator.
Excessive incubation time	It has been observed that changing the media 5 h posttransfection yields high transfection efficiency. Extending the incubation period beyond this may lead to increased cytotoxicity and cell death.
Cell disturbance during transfection	Minimize disturbances to cells during transfection. When adding the transfection complex, avoid excessive swirling and pipetting to ensure cells remain undisturbed for optimal uptake. Carefully avoid disturbing the cells with the Pasteur pipette while changing the medium.
Excessive DNA or transfection reagent	Conduct dose-response experiments to find the optimal DNA and reagent amounts (e.g., 1:1, 2:1, 3:1, 4:1, reagent to DNA)
Transfection not reproducible
Variable confluency or cell condition changes	Maintain consistent cell confluency and use cells of low passage number or fresh cultures for reliability. Regularly check for the plasmid integrity.

DMEM, dulbecco’s modified eagle’s medium; MEM, minimum essential medium.

RESULTS

In the quest to enhance the transfection efficiency while mitigating cytotoxic effects, we systematically evaluated cell viability posttransfection using lactate dehydrogenase (LDH) assays in both HL-1 and H9c2 cell lines. This was conducted against a backdrop of varying concentrations of the transfection reagents PolyJet and Lipofectamine. Subsequently, the effectiveness of gene delivery was ascertained to establish an optimal balance between high transfection rates and minimal cytotoxicity. The outcomes of these experiments allowed us to refine the transfection protocols for HL-1 and H9c2 cells, using both PolyJet and Lipofectamine to achieve the dual objectives of efficient transfection and low cell mortality.

Transfection Dosage Optimization to Curtail Cytotoxicity

Lactate dehydrogenase (LDH) release assay was employed to quantify cell membrane integrity, wherein LDH release is indicative of cellular lysis. Baseline LDH levels from intact cells were established as a negative control. To identify the transfecting reagent dosage that incurs the least cytotoxicity, we exposed cells to a gradient of transfecting reagent concentrations, subsequently inducing cell lysis to measure released LDH as an index of cytotoxicity.

Morphological assessments of both intact and lysed cells are depicted in Fig. 2A. For HL-1 cells (Fig. 2B) and H9c2 cells (Fig. 2C), the dosages of PolyJet and Lipofectamine were titrated from 0.2 to 2.0 µL. Notably, we observed cell-type-specific variations in cytotoxicity levels. The comparative analysis of cytotoxic effects revealed that at lower doses, PolyJet exhibited reduced toxicity relative to Lipofectamine in HL-1 cells (Fig. 2B). In contrast, PolyJet’s cytotoxicity was markedly lower than that of Lipofectamine across all tested doses in H9c2 cells (Fig. 2C). Furthermore, the cytotoxic response to PolyJet was less pronounced in H9c2 cells when compared with HL-1 cells (Fig. 2D). Conversely, Lipofectamine induced greater cytotoxicity in HL-1 cells as opposed to H9c2 cells (Fig. 2E). These patterns highlight both cell-type specific and transfecting reagent dose-specific effects on the cytotoxicity profiles of HL-1 and H9c2 cells.

Figure 2.

Assessment of transfection reagent cytotoxicity using lactate dehydrogenase (LDH) assay. A: representative phase-contrast micrographs illustrating H9c2 cardiomyocytes cultured in a 96-well format; intact cells serve as the negative control, and lysed cells provide a positive reference for the LDH assay. Scale bar = 400 µm. B: quantification of cytotoxicity induced by lipid-based (Lipofectamine) and polymer-based (PolyJet) transfection reagents in HL-1 cells, presented as a percent increase relative to control, across a gradient of reagent concentrations. C: percent cytotoxicity in H9c2 cells following application of increasing concentrations of Lipofectamine and PolyJet, demonstrating dose-dependent cellular response. D: dose-response comparison of the cytotoxic effects of PolyJet in HL-1 vs. H9c2 cells, highlighting differences in cell line susceptibility. E: dose-dependent cytotoxicity comparison between HL-1 and H9c2 cells upon treatment with varying concentrations of Lipofectamine, illustrating the relative tolerance of each cell line to the transfection reagent.

Refined PolyJet Transfection Protocols for Cardiomyocyte Cell Lines

Achieving high transfection efficiency while maintaining cellular integrity is critical for the success of gene expression studies. In our study, we focused on fine-tuning the dosage of PolyJet, a widely used transfecting reagent to optimize gene delivery to HL-1 and H9c2 cardiomyocyte cell lines with the least impact on cell viability.

An extensive series of experiments was conducted to explore the effects of various PolyJet-to-DNA ratios. Through these systematic investigations, we sought to identify the optimal conditions that would enable the highest transfection efficiency without inducing significant cytotoxicity. The effectiveness of PolyJet in transfecting HL-1 cells is illustrated in Fig. 3A, where a range of dosages was assessed for their ability to mediate gene transfer. Similarly, for H9c2 cells, the transfection efficiencies across a spectrum of PolyJet concentrations were evaluated and are presented in Fig. 3B.

Figure 3.

Calibration of PolyJet transfection dose for enhanced efficiency in HL-1 and H9c2 cardiomyocytes. A: assessing transfection efficiency in HL-1 cells using varied PolyJet-to-DNA ratios. Image features a series of green fluorescent protein (GFP) fluorescence images capturing the efficacy of transfection across a range of PolyJet-to-DNA ratios in HL-1 cells. Intensity and distribution of GFP signal provide a visual quantification of transfection success. Each image is standardized with a 400-µm scale bar, allowing for direct comparison of transfection extent and consistency, as well as cell morphology. B: refinement of PolyJet-to-plasmid DNA ratios for enhanced transfection in H9c2 cells (96-well plate). Image displays a series of phase contrast and fluorescence microscopy images that document the optimization process of transfection efficiencies in H9c2 cells using varying PolyJet-to-plasmid DNA ratios. Plasmid DNA encodes for GFP, serving as a reporter for successful transfection. Illustrated are distinct cellular morphologies as captured under phase-contrast microscopy alongside the expression levels of GFP as observed in fluorescence microscopy. These comparative visuals serve to determine the most effective PolyJet-to-DNA ratio by correlating the GFP expression with the associated ratio. A 400-mm scale bar is included to provide a reference for cellular dimensions. C: optimized transfection protocol for HL-1 and H9c2 Cells using PolyJet (6-well plate). Image presents detailed schematic outlining the standardized transfection regimen that has been optimized to achieve efficient gene delivery with minimal cytotoxicity in both HL-1 and H9c2 cell lines. Schematic details the specific doses of PolyJet and DNA that have been identified as optimal through systematic testing. For ensuring rigorous adherence to the established methodology and to facilitate accurate replication of the transfection procedure, the manufacturer’s protocol is included alongside the schematic. The visual schematics have been developed using a licensed version of BioRender.com.

In conjunction with these experiments, we adhered to the manufacturer’s recommended protocols to ensure the reproducibility and validity of our results. A standardized transfection protocol was established for both HL-1 and H9c2 cells, which is detailed in Fig. 3C. This protocol delineates the precise amounts of PolyJet and DNA that yielded the most effective transfection outcomes.

Through careful calibration of PolyJet dosages, we established a regimen that provides robust gene delivery while mitigating the cytotoxic effects often associated with transfection procedures [Supplemental Fig. S1; see https://doi.org/10.6084/m9.figshare.25481890.v1; and Supplemental Fig. S2; see https://doi.org/10.6084/m9.figshare.25481896.v1]. These findings contribute to the field by offering a reliable method for gene delivery into cardiomyocyte cell lines, an essential step for further cellular and molecular cardiac research.

Optimized Transfection Protocol for Lipofectamine in HL-1 and H9c2 Cells

Central to our study was the establishment of an effective transfection protocol that maximizes efficacy while minimizing cytotoxicity for HL-1 and H9c2 cardiomyocyte cell lines. To achieve this, we employed various ratios of Lipofectamine to DNA to ascertain the optimal conditions for gene delivery.

In HL-1 cells, the transfection efficiency was quantitatively analyzed through GFP fluorescence intensity, as shown in Fig. 4A. This approach allowed us to visually assess the level of gene expression, correlating it with the respective transfection mixture used. Similarly, in H9c2 cells, we used a combination of GFP fluorescence and phase-contrast imaging to evaluate both the transfection efficacy and the maintenance of cellular integrity, detailed in Fig. 4B.

Figure 4.

Dose optimization for Lipofectamine-mediated transfection in HL-1 and H9c2 cells. A: quantitative assessment of Lipofectamine-mediated transfection: representative green fluorescent protein (GFP) fluorescence images that detail the transfection efficiency across a spectrum of Lipofectamine-to-plasmid DNA ratios in HL-1 cells. The variability in GFP expression correlates with the Lipofectamine/DNA combinations, providing insight into the potency of each ratio. The images are meticulously annotated with the specific ratios used, offering a clear reference for comparison. A universal scale bar of 400 µm across all images ensures a consistent benchmark for assessing the extent of transfection and cellular detail. B1–B9: tuning Lipofectamine-to-plasmid DNA ratios for optimal transfection in H9c2 Cells (96-well plate). Displayed are phase contrast and fluorescence microscopy images that detail the optimization of transfection conditions in H9c2 cells. The experiment explores various Lipofectamine-to-plasmid DNA ratios, where the plasmid encodes for GFP. Through the phase contrast images, the cell morphology is revealed, while the fluorescence images depict GFP expression, thereby quantifying transfection efficiency. This comparative display allows for a visual determination of the most effective Lipofectamine-to-DNA ratio by correlating GFP fluorescence intensity to each specific ratio. A scale bar of 400 µm is included to provide a reference for size across the different conditions. C: optimized transfection protocols for HL-1 and H9c2 cardiomyocyte cell lines (6-well plate). A detailed schematic outlining the standardized methodology that has been developed to achieve efficient transfection in both HL-1 and H9c2 cell lines is provided. It encapsulates the finely tuned doses of the transfecting reagents identified through rigorous experimentation, to ensure maximum gene delivery with minimal cytotoxicity. The manufacturer’s guidelines are incorporated within this schematic as a fundamental reference point, ensuring the fidelity and uniformity of the transfection process across varying experimental conditions. These protocols are designed to facilitate reproducibility and maintain consistency in transfection outcomes, serving as a vital resource for morphological changes that may relate to cell health.

A series of experiments, guided by the manufacturer’s recommended protocol, enabled us to fine-tune the Lipofectamine-to-DNA ratios. Through this systematic process, we identified the dosages that facilitated the most efficient gene delivery with the least cytotoxic effect. The culmination of this effort is encapsulated in Fig. 4C, which presents the optimal transfection conditions for both cell lines.

Our results demonstrate a clear relationship between the Lipofectamine-to-DNA ratio and the transfection outcomes. By meticulously calibrating this ratio, we have developed a Lipofectamine transfection protocol that ensures high efficiency and viability in two key cardiomyocyte cell lines, providing a robust methodology for future cellular and molecular biology research.

Delineating the Influence of Seeding Density on Cell Viability and Transfection Reagent Efficiency on Gene Delivery

Our investigation into the effect of cell seeding density on HL-1 cell death revealed critical insights for optimizing cell culture conditions. We systematically cultured cells at varying densities, ranging from 5,000 to 40,000 cells per well within a 96-well plate. The morphological observations, including cell distribution and health, were documented and are illustrated in Fig. 5A. After the culture period, we quantified cell death employing an LDH assay. The results depicted in Fig. 5B indicated an escalation in cytotoxicity proportionate to the increase in cell density. Notably, cell death remained relatively low at seeding densities between 5,000 and 10,000 cells per well. However, a significant increase in cell death was observed as densities ranged from 20,000 to 40,000 cells per well. Thus our data suggest that optimal seeding density to minimize cell death falls below the threshold of 20,000 cells per well, where cytotoxicity is maintained at a minimal level of ∼1%.

Figure 5.

Influence of seeding density and transfection reagents on cell viability and transfection efficacy. This figure offers an evaluation of the roles that seeding density and transfection reagents play in modulating both the viability and the transfection efficiency of HL-1 cells. A: morphological analysis at different seeding densities. Displayed are phase-contrast images of HL-1 cells cultured within a 96-well plate at varying seeding densities. These images, complete with a 400-μm scale bar, allow for a visual assessment of cell confluence and morphological changes that may relate to cell health. B: lactate dehydrogenase (LDH) assay for cytotoxicity due to seeding density. The LDH assay results, depicted here, quantify cell death as influenced by seeding density. By systematically increasing the number of cells per well, we establish the correlation between confluence and cytotoxicity within the confines of a 96-well culture system. C: transfection efficiency vs. cytotoxicity with polyjet and lipofectamine. This part contrasts the transfection efficiency and resultant cytotoxicity when using PolyJet and Lipofectamine. Green fluorescent protein fluorescence images provide a quantitative measure of transfection success, while phase-contrast images offer insights into the associated cell viability. A 400-µm scale bar is applied throughout to ensure accuracy in evaluating the distribution of transfection and the subtleties of cell condition.

In parallel, we examined the influence of different transfection reagents on the efficiency of gene delivery, holding cell density constant. Our comparative analysis revealed that, with equivalent seeding densities, HL-1 cells exhibited varied levels of transfection efficiency when treated with PolyJet versus Lipofectamine. PolyJet consistently outperformed Lipofectamine in terms of transfection efficiency, as shown in Fig. 5C. This differential efficacy underscores the importance of selecting an appropriate transfection reagent tailored to the specific requirements of the cell type and experimental objectives.

Gene-Specific Transfection in HL-1 Cardiomyocyte Cell Line Validated Using Targeted shRNA and Protein Overexpression

In our experiments, a control green fluorescent protein (GFP) reporter plasmid was employed to eliminate gene-mediated impacts on transfection efficiency and cytotoxicity. This method was further advanced by incorporating gene-specific reporters for targeted genes, allowing for the visual confirmation and quantification of transfection efficacy to be conducted. shRNA sequences specifically targeting two crucial metabolic enzymes, 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) and succinyl-CoA:3-oxoacid CoA transferase (SCOT), were transfected into the HL-1 cardiomyocyte cell line using doses that had been optimally determined. The HMGCS2 shRNA was tagged with red fluorescent protein (RFP), SCOT shRNA was tagged with GFP, and an HMGCS2 overexpression plasmid was also tagged with GFP to facilitate the assessment of protein overexpression.

To provide a comprehensive overview of the cell culture conditions, phase-contrast imaging was used alongside fluorescence observations, effectively illustrating cell density and morphology. This combined imaging approach was crucial for simultaneously evaluating transfection efficiency, cell viability, and morphology, which are vital for the success of transfection studies.

As demonstrated in Fig. 6, the effective delivery and expression of both HMGCS2 and SCOT shRNAs, as well as the HMGCS2 protein overexpression, were confirmed by the distinct red and green fluorescence signals observed in the HL-1 cardiomyocyte cell line. These results not only affirm the reliability of our transfection protocol but also underscore the importance of using gene-specific reporters to accurately assess gene silencing and overexpression outcomes in the field of cardiomyocyte research.

Figure 6.

Demonstration of transfection efficacy in HL-1 cardiomyocyte cell line with gene-specific reporters. Image highlights the transfection of HL-1 cardiomyocytes using 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) shRNA tagged with red fluorescent protein (RFP), succinyl-CoA:3-oxoacid CoA transferase (SCOT) shRNA tagged with green fluorescent protein (GFP), and HMGCS2 plasmid also tagged with GFP showcasing optimal dosage effectiveness. Phase-contrast images are included to illustrate cell density and morphology. Scale bar = 400 µm.

DISCUSSION

Our investigation has established optimized protocols for PolyJet and Lipofectamine transfection in H9c2 and HL-1 cardiomyocyte cell lines, with the overarching goal of maximizing transfection efficiency while minimizing cellular toxicity. Using lactate dehydrogenase (LDH) assays, we were able to quantify cell viability posttransfection and adjust transfection reagent dosages accordingly.

Key findings from our study indicated that PolyJet, at certain dosages, was superior in maintaining cell viability in H9c2 cells when compared with Lipofectamine, which exhibited higher cytotoxicity levels, particularly in HL-1 cells. This underscores the importance of tailoring transfection protocols to specific cell types. Our results, depicted in Figs. 3C and 4C, detail these tailored protocols, providing a blueprint for effective gene delivery in cardiac cell models.

The study further highlighted the critical role of seeding density in cell viability. We identified an optimal seeding density range that preserves cell viability, with a marked increase in cell death above 20,000 cells per well in a 96-well plate. This threshold has important implications for designing transfection experiments, as overly dense cultures may lead to higher cell mortality, potentially confounding experimental results.

Moreover, we provided practical guidance for optimal seeding densities across various well-plate formats, which is critical for standardizing experimental conditions in cardiac cell research. The optimized seeding conditions for H9c2 and HL-1 cells, as presented in Fig. 1, are anticipated to enhance experimental accuracy and reproducibility across studies.

The novel aspect of our work is the empirical determination of transfection protocols that balance gene delivery and cell health, a crucial consideration for transfection experiments. The study’s significance extends beyond protocol optimization; it offers a refined approach to cardiomyocyte research, where the choice of transfection reagent and cell seeding density can have profound effects on the success of gene delivery and subsequent cellular responses.

The validation of gene-specific transfection, facilitated using GFP and RFP-tagged shRNAs and GFP-tagged plasmids, underscores the effectiveness of our refined transfection protocol within cardiomyocyte research. This methodology ensures precise delivery of shRNAs, significantly improving the accuracy of gene silencing. Furthermore, the addition of plasmids and shRNAs provides additional support for the versatility of this transfection protocol, allowing for both overexpression and inhibition of specific genes in cardiomyocyte cell lines. The employment of fluorescent tags enables effective observation and quantification of transfection efficacy, establishing a dependable approach for investigating cardiomyocyte molecular dynamics. This breakthrough in transfection technology marks a critical advancement in the field of cardiac biology and disease research, providing a valuable tool for future investigations into the complex mechanisms underlying cardiac function and pathology.

The transition to human-derived cardiomyocyte cell lines like AC16 and iPSCs marks a significant evolution in cardiovascular research, moving beyond the limitations of rodent models (17, 18). These human models offer a clearer window into heart disease mechanisms and treatment efficacy, addressing the translational challenges of cross-species differences (19). Our study not only contributes to this shift by developing refined transfection protocols and optimizing cell seeding densities but also establishes a comprehensive approach for future investigations into cardiac functionality and pathology. This work lays the groundwork for advancing our understanding of heart disease and fostering the development of innovative therapeutic strategies.

The use of commercial Claycomb medium in HL-1 cell culture highlights the challenges faced in scientific research because of undisclosed medium formulations. While the exact components of these media are not openly shared by manufacturers, their effectiveness in maintaining HL-1 cell contractility and overall health is established. Despite the potential unknown effects when introducing additional compounds due to undisclosed components, the detailed formulation by White et al. (11) provides an optimized nutrient mix for cardiomyocyte cultures. Addressing these challenges requires meticulous experimental design and stringent controls. This ensures the reliability of results, especially when new compounds are introduced into cultures. By maintaining a rigorous approach, research into cardiac function and disease mechanisms can progress, even within the constraints of using commercial culture media. The careful application of commercial media in cardiomyocyte research could maintain scientific rigor while managing the practical aspects of cell culture.

In conclusion, our tailored transfection protocols and insights into seeding density effects provide an advanced framework for cardiomyocyte research with potential applications in understanding cardiac function and disease, and in the development of novel therapeutic strategies.

For future research directions, it is imperative to extend these findings by exploring other cardiomyocyte lines, as well as primary cardiomyocytes, to generalize the applicability of the optimized transfection protocols. Additionally, further investigation into the molecular pathways impacted by the transfection process could provide deeper insights into minimizing cellular stress and enhancing transfection efficiency. The goal would be to refine these techniques to the point where they can be standardized across multiple forms of cardiomyocyte research, thereby facilitating advancements in cardiac biology and therapeutic development.

SUPPLEMENTAL DATA

Supplemental Fig. S1: https://doi.org/10.6084/m9.figshare.25481890.v1.

Supplemental Fig. S2: https://doi.org/10.6084/m9.figshare.25481896.v1.

GRANTS

This research was supported in part by National Institutes of Health Grants R56HL156806 and P50AA030407 (to P.K.M.), the University of Nebraska Collaboration Initiative (to P.K.M.), and the University of Nebraska Medical Center Presidential Graduate Fellowship (to F.I.G.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

F.I.G. and P.K.M. conceived and designed research; F.I.G. and H.R.S. performed experiments; F.I.G. and H.R.S. analyzed data; F.I.G. and H.R.S. interpreted results of experiments; F.I.G. and H.R.S. prepared figures; P.K.M. drafted manuscript; F.I.G., H.R.S., and P.K.M. edited and revised manuscript; F.I.G., H.R.S., and P.K.M. approved final version of manuscript.