A method and apparatus for cell processing based on sinusoidal alternating current stimulation

Abstract

The application discloses a cell treatment method based on sinusoidal alternating current stimulation, and the method comprises the following steps: applying sinusoidal alternating current stimulation to target stem cells, and the mitochondrial membrane potential, ATP synthesis and aerobic respiration level of the stem cells treated by the electric stimulation are improved. The application further discloses a cell stimulation device based on sinusoidal alternating current stimulation, and the device comprises a cell culture container and a planar interdigital electrode substrate placed in the cell culture container, wherein the planar interdigital electrode substrate comprises an optically transparent substrate and metal planar interdigital electrodes on the substrate. The application discloses the application of the stem cells treated by the above method or the stem cells treated by the device in the preparation of a medicine for mitochondrial transplantation treatment. The application improves the mitochondrial membrane potential, ATP synthesis capacity and aerobic respiration level of the cells by applying sinusoidal alternating current stimulation to the target stem cells.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, and specifically relates to a cell processing method and apparatus based on sinusoidal alternating current stimulation. Background Technology

[0002] In cell biology research and the development of cell-based therapeutic strategies, applying precise and controllable physical stimulation to adherent cells cultured in vitro has become a key technical approach for regulating cell fate and enhancing cell function. Among these methods, electrical stimulation has attracted considerable attention due to its ability to mimic the in vivo bioelectrical environment and directly intervene in cell metabolism and signal transduction. However, the depth and breadth of this technology's application have long been limited by the performance of the stimulation equipment. Currently, the mainstream method of applying electrical stimulation via immersion electrodes struggles to create a uniform field strength on the two-dimensional plane of adherent cell growth, resulting in cells in different regions of the culture system receiving vastly different stimulation intensities. This leads to large fluctuations and low reproducibility in experimental results, severely hindering the establishment of accurate dose-response models. Furthermore, the opacity of traditional electrodes and the significant thermal effects they introduce cause physical obstruction and additional biological interference for real-time live-cell observation.

[0003] Mitochondria are vital organelles in eukaryotic cells, and their dysfunction is closely linked to a variety of diseases. Mitochondrial damage and loss of function are significant causes of functional cell injury and the development of many diseases. Mitochondrial transplantation, as an emerging therapeutic strategy, restores cellular function by transplanting functional mitochondria into damaged tissues or cells to replace or repair damaged mitochondria. The number of mitochondria delivered to the site of injury and their activity are key factors influencing this therapeutic strategy.

[0004] Mitochondrial transplantation can be achieved through direct delivery of free mitochondria, intercellular mitochondrial transfer, and delivery of natural vesicles containing mitochondria. However, these methods suffer from drawbacks such as loss of activity of free mitochondria during systemic administration or low efficiency of natural intercellular mitochondrial transfer. Furthermore, the delivery of low-quality or damaged mitochondria carries the potential risk of exacerbating recipient cell damage. Therefore, developing a novel method for efficiently delivering high-quality, active mitochondria to damaged cells has become a critical technological bottleneck in mitochondrial transplantation therapy.

[0005] Stem cells, such as mesenchymal stem cells, can serve as excellent donors of mitochondria. However, stem cells cultured under conventional conditions exhibit low mitochondrial activity and primarily rely on glycolysis for respiration, limiting their therapeutic potential as "living pharmaceutical factories." Previous studies have shown that moderate physical stimulation, such as electrical stimulation, can promote cell metabolism and function, and thus could potentially be used to regulate mitochondrial respiration patterns and enhance mitochondrial activity. However, there are currently no reports on using electrical stimulation to enhance mitochondrial activity in stem cells, thereby addressing the insufficient mitochondrial activity in existing mitochondrial transplantation therapies. Summary of the Invention

[0006] This invention discloses a cell treatment method and device based on sinusoidal alternating current stimulation, which is used to improve the mitochondrial membrane potential, ATP synthesis capacity and aerobic respiration level of target stem cells by applying sinusoidal alternating current stimulation.

[0007] A method for cellular electrical stimulation includes applying sinusoidal alternating current to target stem cells, thereby increasing the mitochondrial membrane potential, ATP synthesis, and aerobic respiration levels of the treated stem cells. The stem cells may be embryonic stem cells, adult stem cells, or induced pluripotent stem cells (iPSCs).

[0008] Furthermore, the electrical stimulation is sinusoidal alternating current stimulation, the frequency of the electrical stimulation is 2.5~3.5kHz, the voltage of the electrical stimulation is 5V, the duration of a single electrical stimulation is 10 minutes, and the frequency of the electrical stimulation is twice a day for three consecutive days.

[0009] This invention provides a specific electrical stimulation method that can significantly enhance the mitochondrial activity of stem cells. After electrical stimulation, the key indicators such as mitochondrial membrane potential and ATP synthesis capacity of the cells are significantly improved. Furthermore, after co-culturing with damaged cells, the apoptosis of the damaged cells is improved, which fully demonstrates the great potential of this platform in the engineering of adherent cells.

[0010] After electrical stimulation using the method provided in this invention, the mitochondrial membrane potential of the target stem cells significantly increased, and their ATP synthesis capacity was enhanced, indicating improved mitochondrial activity. These "functionally enhanced" stem cells can serve as delivery carriers for highly active mitochondria, providing a novel and efficient solution to the problem of mitochondrial quality regulation during mitochondrial transplantation.

[0011] To achieve the above method, this invention provides a cell electrical stimulation device based on a planar interdigital electrode design. This device can provide uniform, stable, precisely quantifiable, and real-time observation-compatible electrical stimulation for in vitro cultured cells, achieving reliable, efficient, and targeted enhancement of cell function.

[0012] A cell electrical stimulation device includes a cell culture container and a planar interdigitated electrode substrate placed in the cell culture container, the planar interdigitated electrode substrate including an optically transparent substrate and metal planar interdigitated electrodes located on the substrate.

[0013] The device generates sinusoidal alternating current stimulation at a frequency of 2.5–3.5 kHz and a voltage of 5 V. Each stimulation session lasts 10 minutes. The stimulation is applied twice daily for three consecutive days.

[0014] The cell electrostimulation device provided by this invention is a device that can provide uniform, stable, precisely quantifiable, and real-time observation-compatible electrostimulation for cells cultured in vitro. This cell electrostimulation device provides a standardized and highly controllable physical stimulation platform for cell therapy, tissue engineering, and basic life science research, achieving reliable, efficient, and targeted enhancement of cell function. It overcomes the technical defects of existing technologies, such as uneven electric field distribution, poor correlation between stimulation parameters and biological effects, and the inability to perform non-destructive live cell imaging during stimulation.

[0015] Furthermore, the device includes an electrical stimulation generator connected to a metal planar interdigital electrode.

[0016] This invention provides a cell electrostimulation device based on a planar interdigital electrode design. The device utilizes micro-nano fabrication techniques such as photolithography to construct precise interdigital metal circuits on an optically transparent substrate. When connected to an electrostimulation generator with controllable output parameters, the electrode generates a highly uniform electric field with precisely adjustable intensity on the horizontal plane where the cell adherent layer is located. This design ensures that all cells in the culture area are in a consistent electrical microenvironment, thus providing unprecedented reliability and reproducibility for scientific research and process development.

[0017] The superior characteristics of this device make it a universal platform for regulating cell function. Its exceptional optical transparency allows for non-destructive, real-time imaging of dynamic cellular responses using a high-resolution microscope while electrical stimulation is being applied. Furthermore, thanks to the micrometer-scale electrode spacing, the device requires only a relatively low operating voltage to achieve an effective stimulation field strength. This not only reduces reliance on dedicated high-voltage equipment but also fundamentally avoids cellular thermal stress damage caused by Joule heating, ensuring that the observed biological effects originate purely from the electrical stimulation itself.

[0018] Furthermore, the planar interdigitated electrode substrate comprises an optically transparent substrate on which planar interdigitated electrodes of metallic material are fabricated using micro-nano fabrication techniques. The fabrication method of the planar interdigitated electrode substrate includes: constructing a polyimide layer substrate on a glass slide, patterning it by photolithography, and then sequentially depositing chromium / gold thin films through vacuum thermal evaporation to form a metallic planar interdigitated electrode structure. After removing the adhesive, a planar interdigitated electrode with good conductivity and structural stability is obtained, which can be used for applications such as electrical stimulation.

[0019] Furthermore, by aseptically assembling or directly placing the cell culture container into the planar interdigitated electrode substrate after sterilization, an electrical stimulation device is constructed that enables aseptic cell culture, precise and controllable electrical stimulation, and real-time dynamic observation.

[0020] The present invention also provides the application of stem cells cultured by the above method in the preparation of drugs for treating mitochondrial transplantation.

[0021] This invention also provides an application of the aforementioned "enhanced" stem cells in the protection of damaged cells. Using the electrical stimulation device provided by this invention, "enhanced" stem cells with highly active mitochondria are constructed. When co-cultured with damaged cells, these enhanced stem cells can transfer highly active healthy mitochondria to the damaged cells via the intercellular mitochondrial transfer pathway, supplementing or replacing the mitochondrial network of the damaged cells, thereby achieving the protection of the damaged cells.

[0022] The main advantages of this invention compared to existing technologies include: The cell electrostimulation method provided by this invention applies a series of precisely controllable electrical stimulation parameters in terms of intensity, frequency, waveform, and duration to adherent cells through a stimulation output unit, thereby achieving targeted regulation of cell state. The universality of this method lies in its independence from specific cell types or predetermined biological outcomes, instead providing a standardized operating procedure.

[0023] The interdigitated electrode structure of the cell stimulation device provided by this invention enables it to generate a highly uniform electric field with precisely calculable intensity on the plane where cells adhere to the substrate for growth. This characteristic fundamentally solves the problems of electric field edge effects and uneven stimulation caused by traditional immersion electrodes, ensuring that all cells in the culture system receive a consistent stimulation intensity, and laying a physical foundation for establishing a reliable dose-response relationship.

[0024] The outstanding advantages of the cell electrostimulation device provided by this invention lie in its excellent optical compatibility and extremely low thermal effect. The transparent electrodes and substrate allow researchers to perform real-time, dynamic live-cell imaging of key indicators such as cell morphology and organelle dynamics using a high-resolution microscope throughout the entire electrostimulation process, providing a unique window for revealing the immediate mechanisms of electrostimulation. Simultaneously, the micron-level electrode spacing enables the device to generate high field strength at low operating voltages, effectively avoiding the confounding effects of thermal stress on cells and ensuring the purity of the biological effects. Attached Figure Description

[0025] Figure 1 The fabrication process of the photolithography electrode in Example 1; Figure 2 This is an example of a design drawing for the interdigitated electrode in Example 1; Figure 3 This is a schematic diagram of the integration of the interdigitated electrode and the cell culture device in Example 1; Figure 4 This is a simulation example of the electric field of the interdigitated electrodes in Example 1; Figure 5 This refers to the matching cell culture container and electrical stimulation device in Example 2; Figure 6 The cell morphology observed under an inverted microscope in Example 2; Figure 7 The stimulation parameters in Example 2 were used to increase the ATP level of stem cells; Figure 8 The stimulation parameters in Example 2 were used to increase the mitochondrial membrane potential of stem cells; Figure 9 The stimulation parameters in Example 2 were used to increase the aerobic respiration level of stem cells; Figure 10 In the comparative study, the stimulation parameters failed to increase the ATP levels of stem cells. Figure 11 In Example 3, the co-culture of electrically stimulated "enhanced" stem cells with damaged cells improved the apoptosis rate of damaged cells. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Operating methods not specifically specified in the following embodiments are generally performed under conventional conditions or as recommended by the manufacturer.

[0027] Example 1: Fabrication of a planar interdigitated electrode made of metal The electrical stimulation electrodes are designed with an interdigitated electrode structure, with both the electrode finger width and the gap between them being 100 μm (electrode / gap). Figure 2As shown, a uniform and stable alternating electric field can be provided. The interdigitated electrode array is constructed on the surface of an adhered glass slide (25 mm × 75 mm), and its fabrication process is as follows (e.g. Figure 1 (as shown) The first step is cleaning the glass slide: the glass slide is placed in an ethanol solution for ultrasonic cleaning, then dried and plasma cleaned for 100 seconds to remove surface impurities and ensure the cleanliness of the glass substrate. The second step is the construction of the polyimide (PI) layer: a uniform polyimide coating is spin-coated onto the glass substrate using a spin coater and left to cure overnight at 200 °C. This treatment results in better adhesion between the subsequent metal electrodes and the glass surface. The third step is photoresist coating: a photoresist layer with a thickness of about 1-2 μm is spin-coated onto the surface of the glass substrate on which the PI layer has been formed to obtain a flat and uniform photoresist layer. The fourth step is ultraviolet lithography patterning: using lithography equipment and a mask with interdigitated electrode patterns (such as...). Figure 2 As shown, the surface of the glass slide is exposed to ultraviolet light and then developed with a developer to obtain the photoresist pattern of the desired electrode pattern. Step 5, metal thin film deposition: chromium and gold are selected as electrode materials. The patterned glass substrate is placed in a vacuum thermal evaporation apparatus. After evacuation, a chromium layer with a thickness of 50 Å is deposited first, followed by a gold layer with a thickness of 1000 Å, to form a chromium / gold bilayer metal thin film structure. Step 6, photoresist removal: Immerse the vapor-deposited glass slide in acetone for about 2 hours and perform ultrasonic photoresist removal to remove residual photoresist. After this step, the interdigitated electrode with the predetermined structure can be obtained.

[0028] After the prepared interdigitated electrodes are integrated with a cell culture device, they can be used for cell culture and subsequent electrical stimulation experiments (such as...). Figure 3 (As shown).

[0029] Electric field simulation was performed on the prepared planar interdigitated electrode, and the results are as follows: Figure 4 As shown in the diagram, the electric field distribution between the interdigital electrodes exhibits a periodic distribution, with the electric field mainly concentrated in the gap region between adjacent electrodes, while the electric field is weaker in areas far from the gap. A field concentration effect is formed at the edges of adjacent electrodes, with electric field lines originating from the tip of the positive electrode pointing towards the adjacent tip of the negative electrode, passing through the intermediate medium to form a high-gradient electric field. Overall, the electric field intensity reaches its peak along the electrode edge and gradually decreases with increasing distance from the electrode edge, thus forming a stable and concentrated electric field distribution in the target region. This electric field distribution characteristic is beneficial for achieving a uniform and controllable electrical stimulation environment.

[0030] Example 2: Preparation of an "enhanced" stem cell preparation with highly active mitochondria As an empirical demonstration of the effectiveness of the present invention, by using the device and method, and by pretreating human umbilical cord-derived mesenchymal stem cells (MSCs) with specific optimized parameters, “enhanced” stem cells (eMSCs) with significantly improved mitochondrial activity can be successfully prepared.

[0031] According to Figure 2 The prepared planar interdigitated electrodes were fixed on a glass slide of the same size and sterilized with ultraviolet light. Figure 5 The matching four-well cell culture chambers shown are aseptically assembled. Add 1 mL of 2×10⁻⁶ cells to each well. 4 A suspension of stem cells adhered to the culture vessel for 24 hours. Cell adhesion and growth could be observed under an inverted phase-contrast microscope. Figure 6 As shown. For three consecutive days, MSCs were given alternating current stimulation twice daily. The waveform of the stimulation was a sine wave, the voltage was 5 V, the frequency was 3 kHz, and the stimulation time was 10 min per session. MSCs stimulated with these parameters showed ATP levels (e.g., ... Figure 7 As shown), mitochondrial membrane potential (as shown) Figure 8 (as shown) and aerobic respiration level (e.g.) Figure 9 (as shown in the figure) a significant improvement.

[0032] Comparative Example: Controlled Experiment with Unoptimized Stimulus Parameters To verify the unique effects of the electrical stimulation parameters (sine wave, 5 V, 3 kHz, 10 min per session) described in this invention, a control experiment was set up. In this control example, except for the electrical stimulation parameters, all other experimental conditions (including apparatus, cell type, seeding density, culture time, and detection method) were kept consistent with Example 2, except that the stimulation parameters were replaced with a combination of parameters not protected by this invention (direct current, 1 V / mm, 20 min per session). The results showed that MSCs stimulated with these control parameters did not exhibit the significant increase in ATP levels described in Example 2 of this invention (e.g., ...). Figure 10 (As shown). This result, conversely, confirms that not all electrical stimulation parameters can achieve the "enhanced" stem cell effect achieved by this invention, thus highlighting the superiority and non-obviousness of the specific parameter combination selected in this invention in enhancing mitochondrial activity.

[0033] Example 3: The protective effect of "enhanced" stem cells on damaged cells Inoculate 2×10⁶ cells per well of the six-well plate. 5One lung epithelial cell line, TC-1 cells (transfected with GFP fluorescence), was incubated in complete medium containing 10 μg / mL bleomycin (BLM) after adherence to the culture vessel to construct BLM-damaged TC-1 cells (BLM-TC-1), ensuring that the number of damaged cells reached approximately 50% of the total cell count. Subsequently, the culture supernatant was discarded, and after washing with PBS, MSCs or "enhanced" stem cells (eMSCs) were co-cultured with the damaged TC-1 cells at a cell ratio of 3:1. After 24 h of co-culture, the apoptosis rate of the GFP-positive cell population was assessed. Figure 11 As shown, electrically stimulated "enhanced" stem cells significantly reduced the apoptosis rate of damaged TC-1 cells compared to unstimulated stem cells. This application example demonstrates the great potential of this invention in the precise and controllable implementation of cell electrical stimulation and the preparation of therapeutic stem cell formulations with highly active mitochondrial transfer capabilities.

Claims

1. A cell treatment method based on sinusoidal alternating current stimulation, characterized in that, The method includes: applying sinusoidal alternating current stimulation to target stem cells, thereby increasing the mitochondrial membrane potential, ATP synthesis, and aerobic respiration levels of the stem cells after the electrical stimulation treatment.

2. The cell treatment method based on sinusoidal alternating current stimulation according to claim 1, characterized in that, The electrical stimulation is sinusoidal alternating current stimulation, the frequency of the electrical stimulation is 2.5~3.5kHz, the voltage of the electrical stimulation is 5V, and the duration of a single electrical stimulation is 10 minutes.

3. The cell treatment method based on sinusoidal alternating current stimulation according to claim 2, characterized in that, The electrical stimulation was applied twice a day for three consecutive days.

4. The cell treatment method based on sinusoidal alternating current stimulation according to claim 1, characterized in that, The stem cells are selected from embryonic stem cells, adult stem cells, or induced pluripotent stem cells.

5. A cell stimulation device based on sinusoidal alternating current stimulation, characterized in that, The device includes a cell culture container and a planar interdigitated electrode substrate placed in the cell culture container. The planar interdigitated electrode substrate includes an optically transparent substrate and metal planar interdigitated electrodes located on the substrate.

6. The cell electrical stimulation device according to claim 5, characterized in that, The device includes an electrical stimulation generator connected to a metal planar interdigital electrode.

7. The cell electrical stimulation device according to claim 5, characterized in that, The method for preparing the planar interdigitated electrode substrate includes: constructing a polyimide layer substrate on a glass slide, and after photolithographic patterning, sequentially depositing chromium / gold thin films through vacuum thermal evaporation to form a metal planar interdigitated electrode structure.

8. The cell electrical stimulation device according to claim 5, characterized in that, The cell culture container is combined with the planar interdigitated electrode substrate by aseptic assembly or direct placement after sterilization.

9. The cell electrical stimulation device according to claim 5, characterized in that, The electrical stimulation generated by the device is sinusoidal alternating current stimulation, the frequency of the electrical stimulation is 2.5~3.5kHz, the voltage of the electrical stimulation is 5V, and the duration of a single electrical stimulation is 10 minutes.

10. Use of stem cells treated by the method of any one of claims 1-4 or stem cells treated by the apparatus of any one of claims 5-9 in the preparation of a medicament for mitochondrial transplantation therapy.

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