Patent Application
20230407336
The present invention relates to mitochondrial transfer based on a droplet microfluidics system, and more particularly, to high-efficiency quantitative mitochondrial transfer based on a droplet microfluidics system.
Heteroplasmy, which is the ratio of mutant to wild-type mitochondria DNA (mtDNA), determines the severity of mitochondria-related disorders. In muscle tissues, when heteroplasmy exceeds a certain level or the mitochondria becomes dysfunctional, less ATP and excessive levels of reactive oxygen species (ROS) are produced, which can trigger muscle atrophy, weakness, and loss of endurance. Previous clinical and preclinical animal studies demonstrated links between increased mitochondrial damage and poor skeletal muscle health. Since 1990s, cell therapy, especially myoblast transplantation, has been proposed to improve the regeneration of injured skeletal muscles. However, failure of early clinical trials with myoblast transplantation has been observed due to host immune cells such as CD8+ T lymphocytes causing massive cell death. The accumulation of immune cells produces sarcolemma damage, induces activation of caspase 3 in myofibers, and further induces apoptosis in muscle fibers. Therefore, new approaches, such as macrophages regulation and chemical induction of stem cells, need to be developed to treat skeletal muscle disorders. Restoring or improving mitochondrial functions to facilitate muscle regeneration is an attractive approach.
Apart from energy production for cells, the mitochondria can influence cell proliferation, aging, apoptosis, innate immunity, calcium homeostasis, and even stem cell differentiation potential. Mutations of mtDNA impair the functions of cells and tissues. It is observed that spontaneous transfer of mitochondria can occur in nature between healthy and damaged cells via different mechanisms, which is believed to protect the damaged cells and restore their cellular function. Mitochondrial transfer is a technique to alter mtDNA in cells, and has attracted more and more attention since it was first reported by Clark and Shay. Mitochondrial transfer has already been used in cell therapy for mtDNA-related diseases. It is easier to perform and practically more efficient than other techniques modifying the mitochondrial genome, such as mitoZFNs and mitoTALENs. Transferring exogenous mitochondria into recipient cells could also reduce the ratio of mutant to wild-type mtDNA, and promote the restoration or improvement of cell and tissue functions. Previous studies showed that exogenous isolated mitochondria could be delivered into cells via co-culture or microinjection. In the method of co-culture with isolated mitochondria, the isolated mitochondria are engulfed by the recipient cells via endocytosis, which is a kind of cellular activity to uptake objects ranging from nanometers to several micrometers from the surrounding environment. The isolated mitochondria randomly move around the recipient cell and have chances to be engulfed by the cell when contacting it; this phenomenon could be regarded as a random and sporadic process. The transfer efficiency of co-culture method is influenced by the quantity of extracellular isolated mitochondria. Though it could reach the highest at 28% in a previous study, the number of transferred mitochondria is considerably heterogeneous (1-60 mitochondria per recipient cells) even when subjected to an equal amount of isolated mitochondria. Despite being a rather simple process, the success of the co-culture method is dependent on many uncontrollable factors that underlie the unsatisfactory cell metabolism recovery rate of recipient cells in previous work (˜0.2%). An automated optical tweezers-based manipulation system (OT-based manipulation system) was used for qualitative and quantitative mitochondrial transfer to reduce influences from uncontrollable factors. The OT-based system could precisely pick up the healthy mitochondria and transport them to the target recipient cell. However, this method suffers from the limitation of low throughput, which makes it difficult to be used for clinical applications.
Unlike co-culture methods, microinjection injects the isolated mitochondria preloaded in a microneedle directly into the recipient cells; thus, it may cause damage to the recipient cells owing to the cell membrane opening during the delivery process. In addition, the throughput of the microinjection technique is low.
All the methods mentioned above provide useful tools for studying the mechanism of cell functions' restoration or improvement upon mitochondrial transfer. However, they still could not fulfill the demand of a large quantity of mitochondria-transferred recipient cells in the cell therapy industry. The co-culture technique shows considerable advantage due to being harmless, but its low efficiency and heterogeneity are still a major bottleneck.
Droplet microfluidics is a technology that disperses continuous flow carrying chemical reagents, cells, or other biomaterials into discrete volumes at micrometer scale, called droplets. These droplets are the basic unit for further chemical reactions, cell life activities, target detections, and material synthesis. Droplet microfluidics provides a much smaller and constrained environment than the bulk volume method, thus allowing more rapid reaction and detection of molecules/particles and interactions with encapsulated cells. Previous works have demonstrated that the droplet generation rates could be as high as thousands of droplets per second, making droplet microfluidics a high-throughput technique intrinsically. An important application of droplet microfluidics is single-cell analysis, in which a single cell is encapsulated in one droplet for analyzing cell life activities or cell modification, such as antibody analysis or gene editing.
U.S. Patent Application Publication No. 2017/159017A1 entitled: “Method for introducing exogenous mitochondria into a mammalian cell” and US patent U.S. Patent Application Publication No. 2013/149778A1 entitled: “Method and Applications of Peptide-Mediated Mitochondrial Delivery System” applies natural cell membrane engulfing processes also known as endocytosis to transfer isolated mitochondria.
U.S. Patent Application Publication No. 2019/276852A1 entitled: “Method for delivering exogenous mitochondria into cells” and Europe Patent Application Publication No. EP3169338A1 entitled: “Methods for the intercellular transfer of isolated mitochondria in recipient cells” apply the centrifugation method to transfer mitochondria. In these methods, isolated mitochondria and cells were centrifuged together, which improved the mitochondria transfer efficiency by forcing mitochondria into the cells.
U.S. Pat. No. 10,760,040B1 entitled: “Mechanical transfection devices and methods” applies mechanical forces induced by the fluid to open the cell membrane and deliver isolated mitochondria into the cells.
The traditional microinjection method causes physical harm to recipient cells. The co-culture technology is able to generate the number of cells required for cell therapy. However, the co-culture technology cannot control the number of mitochondria transferred into each recipient cell, so some cells that do not have enough mitochondria transferred may not be able to perform their full therapeutic function.
Therefore, a mitochondrial transfer based on droplet microfluidics system is provided as a high-efficiency quantitative mitochondrial transfer system. In the present invention, droplet microfluidics-based mitochondrial transfer method causes only slight/minor physical harm to recipient cells, which is unavoidable in microinjection, and shows high-throughput advantage. Compared with the traditional co-culture method, the proposed method could also control the number of mitochondria transferred to the recipient cells at the single-cell level, and achieve high efficiency and high throughput.
The present invention provides a high-efficiency quantitative mitochondrial transfer based on a droplet microfluidics system. A droplet generation module configured to generate droplets containing isolated mitochondria and a single cell; a droplet observation module configured for observation of the generated droplets under a microscope; and a droplet collection module configured to collect the generated droplets.
In another embodiment, the droplet generation and observation modules are connected by a conduit.
In other embodiments, the system is disposed on a chip.
In yet another embodiment, the chip is smaller than 8 cm in length.
In a further embodiment, the droplet generation module comprises three inlets.
In another embodiment, the droplet generation module further comprises mitochondrial recipient cell suspension, isolated mitochondria suspension, and surfactant-added fluorinated oil.
In other embodiments, the mitochondrial recipient cell is recipient C2C12 cell.
In a further embodiment, the droplet generation module comprises a flow-focusing structure configured to separate the mitochondrial recipient cell suspension and the isolated mitochondria suspension into droplets.
In yet another embodiment, the droplet generation module comprises a wave-like structure which is configured to focus randomly distributed cells from the inlet to a line.
In other embodiment, the wave-like structure is configured to improve the single cell encapsulation ratio more than 47%.
In a further embodiment, the wave-like structure is configured to suppress the multiple cell encapsulation ratio less than 6%. In other embodiments, the droplet comprises isolated mitochondria and a single
cell has a diameter of 40 μm.
In another embodiment, an efficiency of mitochondrial transfer is at least 75%.
In other embodiments, the system is configured to yield at least 2×106 recipient cells in the droplets for mitochondria transfer in 30 minutes.
The present invention also provides a method for quantitative control of mitochondrial transfer based on droplet microfluidics, comprising the steps of preparing a first suspension, a second suspension, and an oil fluid; co-flowing of the first suspension, the second suspension, and the oil fluid to a system for quantitative control of mitochondrial transfer based on droplet microfluidics; co-encapsulating the first and the second suspensions in droplets; collecting the droplets; and co-culturing the first and the second suspensions in droplets.
In a further embodiment, the first suspension is a mitochondrial recipient cell suspension and the isolated mitochondria suspension.
In yet another embodiment, the second suspension is an isolated mitochondria suspension.
In other embodiments, the droplets have a diameter of 40 μm.
In other embodiments, the system comprises a wave-like structure.
In a further embodiment, the droplets comprise mitochondria, mitochondrial recipient cell, and mitochondrial recipient cell.
Turning to the drawings in detail,
The designed chip was fabricated using soft-lithography. Prior to experiments, the chip channels were coated with a surface modification agent to make them hydrophobic for stable water-in-oil droplet generation and transporting.
C2C12 myoblasts were cultured in Dulbecco modified Eagle medium (DMEM) with high glucose (Gibco™, 11965084) supplemented with 10% fetal bovine serum (Gibco™, 12800058) and 1% Antibiotic-Antimycotic (Gibco™, 15240096) at 37° C. in 5% CO2
The mitochondria used in this work were freshly isolated from C2C12 myoblasts cells by following the protocol of mitochondria isolation kit (Beyotime, C3601) before each mitochondrial transfer experiment. First, the mitochondria of donor C2C12 cells were stained with MitoTracker™ Green FM (Invitrogen™, M7514). Second, the stained cells were washed three times with PBS, detached from the culture flask with Trypsin/EDTA Solution (Gibco™, R001100), and centrifuged at 500 g for 5 minutes. Third, the supernatant was removed, and the collected cells were resuspended with 1 ml of cell lysis reagent (Beyotime, C3601-1) and placed in ice bath for 15 minutes. Fourth, the lysed cells were homogenized with a glass homogenizer for 30 cycles. Fifth, the homogenized cells were centrifuged at 1,000 g for 10 minutes at 4° C. Then, the supernatant was resuspended with 1 ml of cell lysis reagent (Beyotime, C3601-1) and centrifuged at 1,000 g for 10 minutes at 4° C. again for more purity. Finally, the supernatant was collected and centrifuged at 3,500 g for 10 minutes at 4° C. The pellet collected was the isolated mitochondria. Mitochondria storage reagent (Beyotime, C3601-3) was used to suspend the isolated mitochondria at the required concentration for further experiments. The mitochondria isolated from 1×106 cells and suspended in 10 μL of mitochondria storing reagent was set as one unit of mitochondrial suspension.
After co-culturing the recipient cells and exogenous isolated mitochondria in droplets for 2 hours, the droplets were loaded into the observation module, as shown in
The collected droplets floated on the top the fluorinated oil. Before the droplets broke, the extra oil on the bottom of the tube was removed. Then, 1 ml of 50% 1H,1H,2H,2H-Perfluorooctanol (PFO, Thermo Scientific™, AAB2015609) was added to the tube containing the collected droplets and gently vortexed for 1 minute for the droplets to merge into a bulk solution. Afterwards, the upper bulk solution was carefully moved to a new tube and centrifuged at 300 g for 3 minutes to collect the cells.
For evaluating C2C12 differentiation, 5,000 cell/cm2 were seeded in a six-well plate and cultured in growth media until reaching 80% confluence. The media were then replaced with DMEM (Gibco™, 11965084) supplemented with 2% horse serum (Gibco™, 16050130). The cells were kept in differentiation medium until the end of the assay, typically between day 5 to day 7. Myotube formation was monitored every two days. The time-points were days 0, 3, and 7.
The cell proliferation rate of each C2C12 cell group was determined by MTT assay. In brief, C2C12 cells were plated at a density of 5,000 cell/cm2 in 96-well plates and incubated for 24 h. After incubation, the cells were treated with 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, 100 μL, 0.5 mg/mL) for 3 hours at 37° C. The produced dark blue formazan crystals were solubilized by 100 μL DMSO. The absorbance at 570 nm was measured with a microplate reader.
In use, the developed droplet-based mitochondria transfer system of
As shown in
For the observation and 3D rebuilding under a confocal fluorescence microscope (LEICA SP8LIA++ TRUE Confocal Laser Scanning Microscope), MitoTracker™ Green FM (Invitrogen™, M7514) at a concentration of 2 μM is used to stain the mitochondria before isolation from donor cells.
As presented in
As mentioned above, the cell encapsulation efficiency is improved beyond the Poisson distribution by using the wave-like structure.
The closed microenvironment of droplets limits the travelling distance of isolated mitochondria and increases the probability of the isolated mitochondria to contact with the cell, thereby making mitochondria taken up by the cell easily, and improving the mitochondria transfer efficiency. Moreover, due to the smaller size of isolated mitochondria than that of the droplets (1-40 μm in diameter), the isolated mitochondria are evenly encapsulated in each droplet. The number of isolated mitochondria encapsulated in droplets can be controlled by adjusting the concentration of isolated mitochondria suspension.
In one embodiment, three different concentrations of isolated mitochondrial suspension (0.25, 0.5, and 1.0 units of concentration) are used to verify the transfer efficiency of the present invention.
Myogenesis assay is performed to test the differentiation ability of C2C12 myoblasts after mitochondrial transfer with the proposed droplet microfluidics-based method. After 7 days of induction,
The present invention provides a mitochondrial transfer based on droplet microfluidics system is provided as a high-efficiency quantitative mitochondrial transfer system due to the following advantages:
The number of mitochondria needed to be transferred into recipient cells is an import issue in precise medicine. The development of the presented invention, which can achieve a precise quantity-control on mitochondrial transfer at the single cell level, can help us to determine the mitochondria number needed to make a significant function improvement on the recipient cells before conducting the cell therapy for mtDNA-related diseases.
Compared to existing mitochondrial transfer methods, the invented method can produce massive quantitative mitochondria transferred cells for cell therapy purpose. Although the co-culture with isolated mitochondria method can also produce the number of cells needed for cell therapy, it cannot control the number of mitochondria transferred into each recipient cell, thus, some cells without enough mitochondria transferred may cannot fully play their therapeutic functions. However, using the invented system, the mitochondria transferred into each recipient cell is controlled, thus, the cells used for cell therapy can fully play their therapeutic functions.
While the present disclosure has been described and illustrated with reference to specific embodiments thereof, these descriptions and illustrations are not limiting. It should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present disclosure as defined by the appended claims. The illustrations may not necessarily be drawn to scale. There may be distinctions between the artistic renditions in the present disclosure and the actual apparatus due to manufacturing processes and tolerances. There may be other embodiments of the present disclosure which are not specifically illustrated. The specification and the drawings are to be regarded as illustrative rather than restrictive. Modifications may be made to adapt a particular situation, material, composition of matter, method, or process to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the claims appended hereto. While the methods disclosed herein have been described with reference to particular operations performed in a particular order, it will be understood that these operations may be combined, sub-divided, or re-ordered to form an equivalent method without departing from the teachings of the present disclosure. Accordingly, unless specifically indicated herein, the order and grouping of the operations are not limitations.
