Application Cases

Experiment Name: Rapid Magnetic Labeling of Ex Vivo H-22 Tumor Cells Using Ultrasound

Research Direction: Biomedicine

Objective:
The labeling agents used for cell magnetic labeling are mostly superparamagnetic iron oxide (SPIO) nanoparticles. Since both the cell membrane surface and SPIO surfaces carry negative charges, they repel each other, making it difficult for cells to naturally uptake iron oxide particles. To improve cell labeling efficiency and achieve magnetic labeling of cells, surface modification of particles using carriers or transfection agents is typically employed. This promotes cellular uptake through processes such as phagocytosis or liquid-phase pinocytosis. In recent years, magnetic particles have also been conjugated with antibodies or receptors to bind to corresponding receptors on the cell surface, enabling particle entry into cells. Alternatively, magnetic iron oxide particles can be formulated into specific labeling agents that enter cells via nonspecific membrane surface absorption in mammalian cells. When labeling target cells with SPIO, the SPIO is complexed with transfection reagents in a specific manner and then co-cultured with the target cells in a culture medium, typically requiring extended incubation times (24–48 hours). SPIO particles enter cells through nonspecific membrane surface absorption.

Dextran-coated ultrasmall superparamagnetic nanoparticles are currently the most widely used contrast agents for magnetic resonance imaging (MRI) and were among the earliest agents used for in vitro cell magnetic labeling experiments. However, compared to standard-sized (50–150 nm) or micron-sized iron oxide particles, their contrast enhancement effect is relatively weaker. When using larger dextran-coated SPIO particles for cell labeling, more Fe particles accumulate in the cytoplasm, resulting in better labeling efficiency. Additionally, using larger particles, such as micron-sized iron oxide particles, enables magnetic labeling of individual cells and subsequent single-cell MRI. This represents a novel research direction. However, due to the protective role of the cell membrane, loading these larger iron oxide particles into the cytoplasm remains an unresolved challenge.

Testing Equipment:
ATA-8202 RF power amplifier, signal generator, ultrasound probe, water tank, distilled water, etc.

Experimental Procedure:
Dextran-coated superparamagnetic iron oxide nanoparticles were synthesized in-house using the chemical co-precipitation method to produce a water-based magnetic suspension. The core particle diameter ranged from 10 to 20 nm, and the concentration could be adjusted as needed. Previously, experiments were conducted using a safe mass fraction of 25 μg/mL and an output electrical power of 1 W. It was observed that ultrasound exposure for up to 2 minutes at this power had minimal impact on cell viability. However, due to the low concentration of SPIO particles in the cell suspension, the loading efficiency was unsatisfactory. Therefore, this experiment employed higher SPIO concentrations, divided into five groups (A, B, C, D, E) based on their final concentrations in the cell suspension. The mass fractions were 22.5, 90, 410, 1500, and 2250 μg/mL, respectively, with an output electrical power of 2 W for ultrasound loading experiments. The experimental setup is shown in Figure 1.

Figure 1: Experimental Setup

Experimental Results:
This experiment investigated the labeling efficiency and cell viability under the following conditions: probe excitation frequency of 1.43 MHz, generator output electrical power of 2 W, SPIO mass fractions in the cell suspension ranging from 22.5 to 2250 μg/mL, and ultrasound exposure times ranging from 10 to 120 seconds.

1. Cell Labeling Results
   Cells showing Prussian blue positivity were considered labeled. Under conditions where the SPIO mass fraction was 410 μg/mL, the ultrasound generator output power was 2 W, the probe center frequency was 1.43 MHz, and the ultrasound exposure time was 10 seconds, the labeling efficiency of H-22 cells was approximately 82%. In contrast, cell suspensions without ultrasound treatment were incubated with nano-superparamagnetic iron oxide water-based magnetic suspension at 37°C for 30 minutes, followed by Prussian blue staining. Microscopic observation revealed almost no labeled cells, indicating that without ultrasound treatment, short-term incubation alone could not achieve cell labeling. In other words, without ultrasound involvement, SPIO particles could not enter the cells within a short time. This demonstrates that ultrasound, as an external force, can indeed facilitate the entry of large particles—nanoscale iron oxide particles—into cells, enabling magnetic labeling.

   To quantify the relationship between cell labeling efficiency and ultrasound exposure time or SPIO concentration, under acoustic conditions of 1.43 MHz and an output electrical power of 2 W, ten randomly selected microscopic fields were counted to determine the cell labeling efficiency for different ultrasound exposure times and SPIO concentrations. The statistical results showing the relationship between cell labeling efficiency (%) and ultrasound exposure time (t in seconds) for different SPIO concentrations are presented in Figure 2.

   
   

Figure 2: Relationship Between Ultrasound Exposure Time and Cell Labeling Efficiency at Different SPIO Concentrations

Relationship between labeling efficiency and ultrasound exposure time at specific SPIO concentrations:
When the SPIO mass fraction was 22.5 μg/mL, the cell labeling efficiency increased approximately linearly with longer ultrasound exposure times. However, overall labeling efficiency remained low, with less than 25% labeling even after 120 seconds of ultrasound exposure. For SPIO mass fractions ranging from 90 to 2250 μg/mL, the relationship between labeling efficiency and ultrasound exposure time exhibited an oscillatory pattern. Except for 410 μg/mL, labeling efficiency decreased with longer exposure times below 30 seconds. Between 30 and 60 seconds, labeling efficiency increased with longer exposure times. Beyond 60 seconds, labeling efficiency decreased again.

Relationship between labeling efficiency and SPIO concentration at fixed ultrasound exposure times:
Overall, when ultrasound exposure times were within 60 seconds, cell labeling efficiency was relatively high. Beyond 60 seconds, labeling efficiency generally declined. At an ultrasound exposure time of 30 seconds and an SPIO mass fraction of 410 μg/mL (Group C), cell labeling efficiency exceeded 90%. When SPIO mass fractions were too high (e.g., 1500 μg/mL in Group D and 2250 μg/mL in Group E) or too low (e.g., 22.5 μg/mL in Group A), cell labeling efficiency was less than 20%. This indicates that there is an optimal concentration of SPIO for labeling H-22 cells, and deviations from this concentration result in poorer labeling efficiency.

The precise relationship between labeling efficiency, SPIO mass fraction, and ultrasound exposure time requires further investigation.

Aigtek ATA-8000 Series RF Power Amplifier:

Figure: Specifications of the ATA-8000 Series RF Power Amplifier