| 摘要: | Background: Fine particulate matter (PM 2.5) is deemed a carcinogenic factor and one of the most dangerous health causes with persistent high ambient concentrations, also, producing free radicals related to oxidative stress has been linked with PM2.5 in living cells [1]. Researchers are continuing to explore the toxicity mechanism and also solutions to regulate the effect of harmful components of PM2.5. Exposure to PM2.5 can cause toxicity to animals and humans or induce cellular activities, including oxidative stress, cell death/apoptosis pathway, and inflammation that involves mitochondria with their own essential functions [2].
Thus, mitochondria influence the host that exposure to PM2.5 upon infection but how is yet known clearly. Besides, mitochondria can be transferred into the new cells by two methods: centrifuge, and endocytosis, respectively [3], [4], [5]. Therefore, centrifuge could help enhance the pervade capacity of isolated mitochondria in other cells.
Mitochondria are referred to as the powerhouse of cells that enable cells to function. Severe mitochondrial dysfunction is linked to several critical human genetic diseases, including cancer [6]. Nowadays, treatment of mitochondria-related disease still is incompletely efficient. So, the transplantation of mitochondria opens up a potential for mitochondrial therapy despite its still unclear mechanism. However, cure results have been recorded depending on the donor’s mitochondria. Thus, extraction and preservation of mitochondria of healthy donor cells is an effective therapeutic therapy in clinical [7]. Isolation techniques have been searched and developed to maintain the intact and function of mitochondria [8]. Although differential centrifugation (DC), density gradient high speed/ultracentrifugation (UC), or magnetic and protein affinity cooperation have been continuing to be used as the most popular methods. They have some limitations in evaluating yield and high purity, the target mitochondrial sub-populations or taking the time-consuming procedures and complex operation process, and need to optimize with
different cell types of different mechanical properties [9], [10].
To explore the strong microfluidic from the lab-on-chip devices that reduce human intervention through automation capabilities [11]. However, because of the complex operation process and low yield, widely using the microfluidic device as mitochondria
extraction tool has been limited [12]. Thus, we develop a simple and effective method for extracting functional mitochondria based on a novel microfluidic device with a centrifuge used in general laboratories. The purpose was to extract an equal amount of mitochondria to a commercial kit and keep mitochondrial function from damage in the extraction process.
Methods: We used human myocardial cells (AC16) and human alveolar basal epithelial cells (A549) exposed to PM2.5 and evaluated the effects on proliferation, apoptosis, reactive oxygen species (ROS) production, and mitochondrial membrane potential. After delivery and co-culturing with PM2.5 administrated AC16 cells, we will analyze the functional mitochondria.
We design and fabricate the microfluidic chip with the proposed geometry via traditional methods of PDMS micro-fabrication. Then, the microfluidic chip will be put into a centrifugal device that is like a container with a cap to hold and allow the component of cell to fall down to the centrifuge tube. And mitochondria will be extracted by using this device and a commercial kit as the control group. The extraction process operates to general centrifuge at 200xg in 3 minutes for the first step to lysing cell membrane. After extraction mitochondria, we transfer it into recipient cells by two different methods that are centrifuge at 2.000xg for 5 minutes, and endocytosis as control group. We will analyze the mitochondrial morphology and function such as proliferation, mitochondria membrane potential, oxygen consumption rate.
Results: Firstly, we found that PM2.5 have affected to viability at concentrations 47.16 g/ml on AC16 cells, but no statistically significant differences on A549 cells. These followed results showed that PM2.5 may be relevant oxidative stress in cell mitochondria on both cell lines. Real-time detection demonstrated an increase in ROS production in cells after exposure to PM2.5. JC1 assay, which indicated a loss of mitochondrial membrane potential (ΔΨm) in cells exposed to PM2.5. The disruption of mitochondrial membrane potential further supports the detrimental effects of PM2.5 on cells. Additionally, apoptosis analysis showed a higher percentage of early apoptosis in PM2.5-exposed cells compared to the control group.Then, mitochondria were isolated from healthy donors and transported without needing an additional source with only one step within 5 minutes at a normal centrifugal rate (2000xg) into PM2.5 administrated AC16 cells. To discovery our goals, the confocal images and flow cytometry results showed an efficiency of replacing and removing damaged mitochondria that were affected by PM2.5. On the other, ATP production- related OCR was investigated for metabolic activity of recipient cells that recovered by transferred mitochondria. Through the transfer directly healthy mitochondria from donors into PM2.5-exposured AC16 cells by general centrifugation in the laboratory, we established a simple and efficient approach to the transfer and treatment that may offer a platform for mitochondria therapy in the future.
The device can lysis the cell membrane and isolate mitochondria with three centrifuge steps within below 30 minutes at low speed (300xg for AC16 and 400xg for HL60) to disrupting cell and efficiency of extraction is high as like using commercial kit. Moreover, mitochondria membrane still was preserved integrity and function. Indeed, extracted mitochondria can be uptake by host cells and can enhance the proliferation and mitochondrial function of recipient cells.
Conclusion: We develop a microfluidic device that is able to monitor cell biology under continuous PM2.5 treatment based on the effects of PM2.5 on human cell lines and reveal mitochondria as a therapy to prevent and destroy fine particle exposure.
We establish a centrifuge protocol that applies to extract mitochondria from any cell with a shorter time, under 30 minutes with only three simple centrifuge steps, to co-culture extracted mitochondria. Extracted mitochondria is healthy and maintain functions.
Background: Fine particulate matter (PM 2.5) is deemed a carcinogenic factor and one of the most dangerous health causes with persistent high ambient concentrations, also, producing free radicals related to oxidative stress has been linked with PM2.5 in living cells [1]. Researchers are continuing to explore the toxicity mechanism and also solutions to regulate the effect of harmful components of PM2.5. Exposure to PM2.5 can cause toxicity to animals and humans or induce cellular activities, including oxidative stress, cell death/apoptosis pathway, and inflammation that involves mitochondria with their own essential functions [2].
Thus, mitochondria influence the host that exposure to PM2.5 upon infection but how is yet known clearly. Besides, mitochondria can be transferred into the new cells by two methods: centrifuge, and endocytosis, respectively [3], [4], [5]. Therefore, centrifuge could help enhance the pervade capacity of isolated mitochondria in other cells.
Mitochondria are referred to as the powerhouse of cells that enable cells to function. Severe mitochondrial dysfunction is linked to several critical human genetic diseases, including cancer [6]. Nowadays, treatment of mitochondria-related disease still is incompletely efficient. So, the transplantation of mitochondria opens up a potential for mitochondrial therapy despite its still unclear mechanism. However, cure results have been recorded depending on the donor’s mitochondria. Thus, extraction and preservation of mitochondria of healthy donor cells is an effective therapeutic therapy in clinical [7]. Isolation techniques have been searched and developed to maintain the intact and function of mitochondria [8]. Although differential centrifugation (DC), density gradient high speed/ultracentrifugation (UC), or magnetic and protein affinity cooperation have been continuing to be used as the most popular methods. They have some limitations in evaluating yield and high purity, the target mitochondrial sub-populations or taking the time-consuming procedures and complex operation process, and need to optimize with
different cell types of different mechanical properties [9], [10].
To explore the strong microfluidic from the lab-on-chip devices that reduce human intervention through automation capabilities [11]. However, because of the complex operation process and low yield, widely using the microfluidic device as mitochondria
extraction tool has been limited [12]. Thus, we develop a simple and effective method for extracting functional mitochondria based on a novel microfluidic device with a centrifuge used in general laboratories. The purpose was to extract an equal amount of mitochondria to a commercial kit and keep mitochondrial function from damage in the extraction process.
Methods: We used human myocardial cells (AC16) and human alveolar basal epithelial cells (A549) exposed to PM2.5 and evaluated the effects on proliferation, apoptosis, reactive oxygen species (ROS) production, and mitochondrial membrane potential. After delivery and co-culturing with PM2.5 administrated AC16 cells, we will analyze the functional mitochondria.
We design and fabricate the microfluidic chip with the proposed geometry via traditional methods of PDMS micro-fabrication. Then, the microfluidic chip will be put into a centrifugal device that is like a container with a cap to hold and allow the component of cell to fall down to the centrifuge tube. And mitochondria will be extracted by using this device and a commercial kit as the control group. The extraction process operates to general centrifuge at 200xg in 3 minutes for the first step to lysing cell membrane. After extraction mitochondria, we transfer it into recipient cells by two different methods that are centrifuge at 2.000xg for 5 minutes, and endocytosis as control group. We will analyze the mitochondrial morphology and function such as proliferation, mitochondria membrane potential, oxygen consumption rate.
Results: Firstly, we found that PM2.5 have affected to viability at concentrations 47.16 g/ml on AC16 cells, but no statistically significant differences on A549 cells. These followed results showed that PM2.5 may be relevant oxidative stress in cell mitochondria on both cell lines. Real-time detection demonstrated an increase in ROS production in cells after exposure to PM2.5. JC1 assay, which indicated a loss of mitochondrial membrane potential (ΔΨm) in cells exposed to PM2.5. The disruption of mitochondrial membrane potential further supports the detrimental effects of PM2.5 on cells. Additionally, apoptosis analysis showed a higher percentage of early apoptosis in PM2.5-exposed cells compared to the control group.Then, mitochondria were isolated from healthy donors and transported without needing an additional source with only one step within 5 minutes at a normal centrifugal rate (2000xg) into PM2.5 administrated AC16 cells. To discovery our goals, the confocal images and flow cytometry results showed an efficiency of replacing and removing damaged mitochondria that were affected by PM2.5. On the other, ATP production- related OCR was investigated for metabolic activity of recipient cells that recovered by transferred mitochondria. Through the transfer directly healthy mitochondria from donors into PM2.5-exposured AC16 cells by general centrifugation in the laboratory, we established a simple and efficient approach to the transfer and treatment that may offer a platform for mitochondria therapy in the future.
The device can lysis the cell membrane and isolate mitochondria with three centrifuge steps within below 30 minutes at low speed (300xg for AC16 and 400xg for HL60) to disrupting cell and efficiency of extraction is high as like using commercial kit. Moreover, mitochondria membrane still was preserved integrity and function. Indeed, extracted mitochondria can be uptake by host cells and can enhance the proliferation and mitochondrial function of recipient cells.
Conclusion: We develop a microfluidic device that is able to monitor cell biology under continuous PM2.5 treatment based on the effects of PM2.5 on human cell lines and reveal mitochondria as a therapy to prevent and destroy fine particle exposure.
We establish a centrifuge protocol that applies to extract mitochondria from any cell with a shorter time, under 30 minutes with only three simple centrifuge steps, to co-culture extracted mitochondria. Extracted mitochondria is healthy and maintain functions. |
