A portable high-throughput single-cell dissociator

Abstract

This invention presents a high-throughput, portable single-cell dissociation instrument specifically designed to meet the needs of large-scale tissue sample dissociation. By optimizing the number of channels and the heating mechanism, this invention not only improves dissociation efficiency but also significantly reduces costs and enhances the device's mobility and flexibility by eliminating the traditional bottom heating module and replacing it with existing laboratory facilities such as incubators for temperature control. Simplified components, carefully selected materials, and optimized motor connections ensure the dissociation instrument's lightweight, portability, and robustness. These improvements make this invention demonstrate greater application potential in laboratory settings and environments requiring on-site operation.

Description

Technical Field

[0001] This invention belongs to the field of biochemical testing instruments and medical devices, and in particular relates to a portable high-throughput single-cell dissociation instrument. Background Technology

[0002] A tissue homogenizer is a device used to dissociate tissue samples into single-cell suspensions or tissue homogenates. Its primary purpose is to prepare tissue homogenates or single-cell suspensions. Its working principle combines mechanical and enzymatic digestion methods, using disposable tissue homogenization tubes with a rotor / stator system to gently process tissue structures, thereby obtaining high-quality single-cell suspensions or subcellular substances. This instrument can process multiple different tissues and samples simultaneously, with each sample handled independently under closed, sterile conditions, protecting operator safety and preventing cross-contamination. Furthermore, the tissue homogenizer is equipped with an independent heating module, suitable for various tissue homogenization procedures, and can process both fresh and frozen tissues. The prepared tissue homogenates can be used for protein extraction and analysis, and can also meet the needs of subsequent applications such as mRNA extraction, cDNA synthesis, purification, and sorting. Using a tissue homogenizer can significantly improve laboratory efficiency, reduce the tedium of manual operations, and ensure cell viability and yield. It is an important experimental tool for researchers who need to perform various experiments such as single-cell sequencing, primary cell culture, magnetic cell sorting, and flow cytometry / sorting.

[0003] Miltenyi Biotec's gentleMACS is a widely used, high-efficiency tissue dissociation device, typically configured with 2 or 8 channels. It mechanically processes solid tissue, breaking it down into single-cell suspensions. This device is primarily used for pretreatment of biomedical and clinical samples, especially in single-cell genomics and cell function studies. Used with dissociation tubes, the gentleMACS offers multi-channel processing capabilities, improving experimental efficiency. However, its design is primarily geared towards processing small to medium-sized samples, particularly for genomics and transcriptomics research. The device features a programmed control system to adapt to different tissue types, ensuring cell integrity and viability. The dissociator is equipped with an integrated heating base, primarily designed to optimize temperature conditions during enzyme processing, thereby improving dissociation efficiency and cell viability. This design allows the device to independently perform temperature-sensitive dissociation processes without an external oven. While the gentleMACS dissociator performs exceptionally well in single-cell applications, its heating system design is primarily geared towards specific experimental setups and may have limitations for scenarios requiring rapid, large-scale processing of diverse samples. While its integrated heating module increases the convenience of experiments, its accuracy may be affected by the ambient temperature, and it also significantly increases procurement costs. Furthermore, the device's size and non-portable nature may limit its use in applications requiring rapid on-site processing.

[0004] Existing dissociation instruments have the following drawbacks: 1. Low throughput: The gentleMACS dissociation instrument, with a similar size and heavier weight, can only provide a maximum of 8 channels for dissociation. This limited number of channels restricts its ability to process large batches of samples, making it unsuitable for large-scale biological sample dissociation needs. Other dissociation instruments typically have a maximum of 9 channels. 2. Poor portability and heavy weight: Due to the integrated heating and cooling modules, the gentleMACS is large in size and weight, which limits its mobility in different experimental environments. Especially in situations requiring on-site sample processing, its portability and setup are unsatisfactory. 3. High cost: The integrated heating module design significantly increases the overall cost. High cost not only affects the purchasing willingness of laboratories but also limits wider market promotion and application. 4. High potential failure rate and high maintenance costs: During operation, due to the design of the heating and cooling modules, the heat dissipation efficiency may be poor in harsh environments. This not only affects the stable operation of the machine but may also lead to overheating after prolonged use, affecting its performance and lifespan. 5. The connection between the motor and the dissociation tube is not very stable. Therefore, in the process of tissue dissociation, there is an urgent need for a dissociation instrument that is high-throughput, portable, low-cost, and stable. Summary of the Invention

[0005] To address the shortcomings of current dissociation instruments, this invention proposes a high-throughput, portable single-cell dissociation instrument specifically designed to meet the needs of large-scale tissue sample dissociation. By optimizing the number of channels and the heating mechanism, this invention not only improves dissociation efficiency but also significantly reduces costs and enhances the device's mobility and flexibility by eliminating the traditional bottom heating module and replacing it with existing laboratory facilities such as a temperature control chamber. Simplified components, carefully selected materials, and optimized motor connections ensure the dissociation instrument's lightweight, portability, and robustness. These improvements make this invention demonstrate greater application potential in laboratory settings and environments requiring on-site operation.

[0006] This invention is achieved through the following technical solution: A portable high-throughput single-cell dissociation device comprises three parts: a lower layer, a middle layer, and an upper layer. The lower layer is a dissociation device base 1, the middle layer is a motor base 2, and the upper layer is a dissociation tube fixing bracket 3. The dissociation device base 1 is square, and each of its top four sides has a first insert plate 4 protruding upwards, forming a receiving space on the dissociation device base 1. The motor base 2 is square, and 24 motor mounting holes 8 are evenly arranged on the motor base 2. The top two sides of the motor base 2 are provided with upper baffles that protrude upwards, and slots 6 are formed on the inner side of the upper baffles. The bottom of the motor base 2 is provided with lower baffles that protrude downwards around the perimeter. The lower baffles around the perimeter form a second receiving space under the motor base 2. The first insert plate 4 protruding around the perimeter of the dissociator base 1 can be inserted into the second receiving space and fits against the lower baffles protruding around the perimeter of the bottom of the motor base 2, thereby realizing the engagement of the dissociator base 1 and the motor base 2. The first receiving space on the dissociator base 1 and the second receiving space under the motor base 2 together form a receiving cavity to accommodate the motor control circuit and circuit board. The dissociation tube fixing bracket 3 is also square, and 24 dissociation tube placement holes 7 are evenly arranged on the dissociation tube fixing bracket 3. A second insert plate 5 protrudes downwards from both sides of the bottom of the dissociation tube fixing bracket 3. The second insert plate 5 can be inserted into the slot 6, thereby achieving the engagement of the dissociation tube fixing bracket 3 and the motor base 2. A receiving cavity with hollow ends is formed between the dissociation tube fixing bracket 3 and the motor base 2 to accommodate the motor and the dissociation tube. The dissociation instrument base 1 is integrally formed with the first insert plate 4, the motor base 2 is integrally formed with the upper baffle and the lower baffle, and the dissociation tube fixing bracket 3 is integrally formed with the second insert plate 5. Two speed control knobs 9 are symmetrically arranged on both sides of the outer wall of the motor base 2, each speed control knob 9 corresponding to 12 motors. Two direction buttons 10 for controlling the motor direction are also provided on one side of the outer wall of the motor base 2. The speed control knobs 9 and the direction buttons 10 are connected to the motor control circuit and circuit board. Two power cord sockets 11 are also provided next to the direction buttons 10. When storing the dissociation device, the power cord can be placed inside the housing cavity. The dissociation device also includes a rotor connector 12, which is a hollow cylindrical shape with a motor rotor insertion hole 14 inside. Six prisms 13 protrude from the outer wall, and the width of the prisms 13 gradually decreases along the longitudinal direction of the rotor connector. Both the motor rotor insertion hole 14 and the motor rotor are semi-circular. In use, the narrower end of the prism 13 is inserted into the groove of the dissociation tube, the rotor connector 12 is sleeved on the motor, and the motor rotor is inserted into the motor rotor insertion hole 14, thereby achieving a stable connection between the motor and the dissociation tube.

[0007] Preferably, the dissociation device base 1, motor base 2, and dissociation tube fixing bracket 3 can also be other shapes besides square.

[0008] Preferably, there may be multiple speed control knobs 9, and each speed control knob 9 can be used to control one or more motors.

[0009] Preferably, the speed control knob 9 is provided with a speed scale around its periphery.

[0010] Preferably, the positions of the speed control knob 9, the direction button 10, and the power cord socket 11 can be adjusted according to the actual situation.

[0011] Preferably, there may be 24 or more motor mounting holes and dissociation tube placement holes.

[0012] Advantages and effects of the present invention: 1. Greatly improves throughput: The present invention designs a dissociation instrument that supports more channels and can process 24 samples at the same time, which significantly improves dissociation efficiency and throughput, making it suitable for high-throughput single-cell analysis needs, especially for applications in large-volume tissue flow cytometry analysis.

[0013] 2. Enhanced Portability and Weight Reduction: First, through optimized design, the dissociation device of this invention eliminates the built-in heating module, replacing it with an optional external constant temperature chamber for heating. Second, this invention significantly simplifies and streamlines existing dissociation devices, retaining only essential components. Finally, the dissociation device base, motor base, and dissociation tube fixing bracket in this invention are all made of ABS material. ABS is a high-strength, tough, and easily processed thermoplastic polymer material with high strength, corrosion resistance, and high-temperature resistance. Therefore, this invention greatly reduces the weight of the equipment, enhances its portability, and makes it more convenient for on-site use.

[0014] 3. Improved heat dissipation performance: Since it no longer relies on the built-in heating module, but uses external constant temperature control, the heat dissipation performance of the present invention is significantly improved, enhancing the stability and safety of the equipment.

[0015] 4. Reduced Costs: By eliminating the expensive heating module and complex heat dissipation system and simplifying the components, the cost of this invention is significantly reduced. The high cost of gentleMACS (approximately RMB 500,000 per unit) makes it unaffordable for most traditional laboratories, which typically purchase it as an instrument platform. In contrast, the manufacturing cost of this invention is approximately RMB 1,500, and its selling price makes it an affordable small instrument for individual laboratories, greatly expanding its market potential and application scope.

[0016] 5. Improved motor connection stability: The present invention adopts a uniquely designed motor rotor connector, which can be fitted onto the motor and securely connected to the motor rotor. At the same time, the six prisms on the outer wall of the connector can be securely engaged with the groove of the release tube, ensuring the stability of the motor connection.

[0017] Through these improvements, the present invention not only overcomes many shortcomings of the prior art, but also provides a more efficient dissociation scheme that is better suited to the needs of modern biotechnology. These advantages are expected to greatly promote its widespread use in biomedical research and clinical applications. Attached Figure Description

[0018] Figure 1 This is a side view of the dissociation apparatus of the present invention.

[0019] Figure 2 This is a side view of the dissociation device of the present invention from another angle.

[0020] Figure 3 This is a schematic diagram of the rotor connector of the present invention.

[0021] Figure 4 shows the main flow cytometry cluster analysis of the dissociation effects of three dissociation methods on mouse liver.

[0022] In the diagram: 1. Dissociator base; 2. Motor base; 3. Dissociation tube fixing bracket; 4. First insert plate; 5. Second insert plate; 6. Slot; 7. Dissociation tube placement hole; 8. Motor mounting hole; 9. Speed ​​control knob; 10. Direction button; 11. Power cord socket; 12. Rotor connector; 13. Prism; 14. Motor rotor socket. Detailed Implementation

[0023] I. A portable high-throughput single-cell dissociation device, comprising three parts: a lower layer, a middle layer, and an upper layer. The lower layer is a dissociation device base 1, the middle layer is a motor base 2, and the upper layer is a dissociation tube fixing bracket 3. The dissociation device base 1 is square, with first insert plates 4 protruding upwards on all four sides of its top, forming a receiving space on the dissociation device base 1. The motor base 2 is square, and 24 motor mounting holes 8 are evenly arranged on the motor base 2. The top two sides of the motor base 2 are provided with upper baffles that protrude upwards, and slots 6 are formed on the inner side of the upper baffles. The bottom of the motor base 2 is provided with lower baffles that protrude downwards around the perimeter. The lower baffles around the perimeter form a second receiving space under the motor base 2. The first insert plate 4 protruding around the perimeter of the dissociator base 1 can be inserted into the second receiving space and fits against the lower baffles protruding around the perimeter of the bottom of the motor base 2, thereby realizing the engagement of the dissociator base 1 and the motor base 2. The first receiving space on the dissociator base 1 and the second receiving space under the motor base 2 together form a receiving cavity to accommodate the motor control circuit and circuit board. The dissociation tube fixing bracket 3 is also square, and 24 dissociation tube placement holes 7 are evenly arranged on the dissociation tube fixing bracket 3. A second insert plate 5 protrudes downwards from both sides of the bottom of the dissociation tube fixing bracket 3. The second insert plate 5 can be inserted into the slot 6, thereby achieving the engagement of the dissociation tube fixing bracket 3 and the motor base 2. A receiving cavity with hollow ends is formed between the dissociation tube fixing bracket 3 and the motor base 2 to accommodate the motor and the dissociation tube. The dissociation instrument base 1 is integrally formed with the first insert plate 4, the motor base 2 is integrally formed with the upper baffle and the lower baffle, and the dissociation tube fixing bracket 3 is integrally formed with the second insert plate 5. Two speed control knobs 9 are symmetrically arranged on both sides of the outer wall of the motor base 2, each speed control knob 9 corresponding to 12 motors. Two direction buttons 10 for controlling the motor direction are also provided on one side of the outer wall of the motor base 2. The speed control knobs 9 and the direction buttons 10 are connected to the motor control circuit and circuit board. Two power cord sockets 11 are also provided next to the direction buttons 10. When storing the dissociation device, the power cord can be placed inside the housing cavity. The dissociation device also includes a rotor connector 12, which is a hollow cylindrical shape with a motor rotor insertion hole 14 inside. Six prisms 13 protrude from the outer wall, and the width of the prisms 13 gradually decreases along the longitudinal direction of the rotor connector. Both the motor rotor insertion hole 14 and the motor rotor are semi-circular. In use, the narrower end of the prism 13 is inserted into the groove of the dissociation tube, the rotor connector 12 is sleeved on the motor, and the motor rotor is inserted into the motor rotor insertion hole 14, thereby achieving a stable connection between the motor and the dissociation tube.

[0024] Preferably, the dissociation device base 1, motor base 2, and dissociation tube fixing bracket 3 can also be other shapes besides square.

[0025] Preferably, there may be multiple speed control knobs 9, and each speed control knob 9 can be used to control one or more motors.

[0026] Preferably, the speed control knob 9 is provided with a speed scale around its periphery.

[0027] Preferably, the positions of the speed control knob 9, the direction button 10, and the power cord socket 11 can be adjusted according to the actual situation.

[0028] Preferably, there may be 24 or more motor mounting holes and dissociation tube placement holes.

[0029] II. Dissociation Effect Verification Test 1. Experimental design: The livers of six C57BL / 6J mice were dissociated using the GentleMACS dissociation instrument, the traditional shaking digestion method, and the dissociation instrument of this invention, respectively, and flow cytometry analysis was performed to observe the distribution of the main population.

[0030] 2. Reagents and materials: 1× phosphate-buffered saline (PBS); enzymes: collagenase I, DNase I, 0.25% trypsin (EDTA-free); cell filter (70 µm); 50 ml centrifuge tubes; gentleMACS C tubes (dissociation tubes); dissociation termination medium: RPMI 1640 containing 2% FBS; erythrocyte lysis buffer.

[0031] 3. Preparation of digestive enzyme mixture: Collagenase I (Yeason Cat#40507ES60): 1 mg / ml (prepared as 100 mg / ml stock solution, 100×); Trypsin (Yuanpei Cat#S310KJ): 0.01% (prepared as 0.25% stock solution, 25×); DNase I (Yeason Cat#10608ES25): 50 U / ml (prepared as 25 mg / ml stock solution, 2000 U / mg, 1000×). Note: Collagenase is used to digest connective tissue, trypsin is used to digest cell junctions, and DNase I is used to degrade free DNA to prevent cell aggregation and apoptosis.

[0032] 4. Experimental steps: (1) Tissue collection: The mice were euthanized, and the livers were removed and placed in 12-well plates containing PBS. The whole liver was cut into 1-2 mm fragments, and half of the tissue was weighed and dissociated using a Miltenyi dissociator and the dissociator of this invention, respectively.

[0033] (2) Tissue dissociation: Transfer the tissue to a dissociation tube, add 5 ml of digestive enzyme mixture, and tighten the dissociation tube. Place the dissociation tube on the Miltenyi dissociation apparatus and the dissociation apparatus of this invention for dissociation. The parameters of the Miltenyi dissociation apparatus are based on the official liver dissociation procedure (catalog number 130-105-807). The dissociation apparatus of this invention uses a rotation speed of 20 rpm and is placed at 37 degrees Celsius for dissociation for 30 minutes. For the traditional method, the shredded tissue suspension is placed on a shaking incubator at 200 rpm for digestion for 1 hour.

[0034] (3) Cell filtration and erythrocyte lysis: Digested cells were filtered into a 50ml centrifuge tube with a 70μm cell filter, and 5ml of 2% FBS RMPI 1640 medium was added. The tube was centrifuged at 500g for 3 minutes. The supernatant was removed using a vacuum pump. 3ml of erythrocyte lysis buffer was added, and the tube was allowed to stand for 3 minutes. PBS was added to 30ml, and the tube was centrifuged at 500g for 3 minutes. The supernatant was removed using a vacuum pump, and the cells were resuspended in 500μl of 2% FBS RPMI 1640.

[0035] (4) Flow cytometry analysis: Filter the cells into a 1.5 ml centrifuge tube with a 40 μm filter. Take 100 μl of cells for flow cytometry analysis.

[0036] 5. Results Analysis: The results of flow cytometry are shown in the figure. The horizontal axis represents FSC, and the vertical axis represents SSC. The area within the box contains dissociated immune cells and hepatocytes, while the area outside the box represents cell debris.

[0037] The results showed that in the traditional shaking method, the dissociation effect of the livers of the six mice exhibited significant heterogeneity. The target cell populations inside and outside the frame were affected by different operators, resulting in large differences in dissociation. In contrast, the dissociation instrument of this invention, along with gentleMACS, produced uniform dissociation effects with minimal differences between samples, demonstrating a high degree of consistency and reproducibility of the technology.

[0038] The dissociation apparatus of this invention demonstrated similar high-efficiency dissociation performance to gentleMACS in experiments. In experiments dissociating C57BL / 6J mouse livers, both gentleMACS and the dissociation apparatus of this invention exhibited excellent dissociation uniformity and repeatability. In contrast, the conventional 37°C oscillating dissociation method showed greater heterogeneity and significant operator-to-operator variability.

[0039] These experimental data and evidence clearly demonstrate the significant advantages of this invention over existing technologies in several key aspects. These advantages enable this invention to not only provide performance comparable to high-end equipment on the market, but also to offer greater cost-effectiveness and ease of operation, and it is expected to find widespread application in biomedical research and clinical settings.

Claims

1. A portable, high-throughput single-cell dissociation instrument, characterized in that, The device comprises three parts: a lower layer, a middle layer, and an upper layer. The lower layer is the base of the dissociation device, the middle layer is the motor base, and the upper layer is the dissociation tube fixing bracket. The dissociation device base is square, with first insert plates protruding upwards on all four sides of its top, forming a receiving space 1 on the dissociation device base. The motor base is square, with 24 motor mounting holes evenly distributed on its surface. Upper baffles protrude upwards on both sides of the top of the motor base, with slots formed on their inner sides. Lower baffles protrude downwards on all four sides of the bottom of the motor base, forming a receiving space 2 below the motor base. The first insert plates protruding around the dissociation device base can be inserted into this receiving space 2 and fit against the lower baffles protruding around the bottom of the motor base, thereby achieving the dissociation device base... The base engages with the motor base, and the receiving space on the top of the dissociation device base and the receiving space below the motor base together form a receiving cavity to accommodate the motor control circuit and circuit board; the dissociation tube fixing bracket is also square, and 24 dissociation tube placement holes are evenly arranged on the dissociation tube fixing bracket. The bottom two sides of the dissociation tube fixing bracket are provided with a second insert plate that can be inserted into the slot, thereby realizing the engagement of the dissociation tube fixing bracket and the motor base. A receiving cavity with hollow ends is formed between the dissociation tube fixing bracket and the motor base to accommodate the motor and the dissociation tube; the dissociation device base is integrally formed with the first insert plate, the motor base is integrally formed with the upper baffle and the lower baffle, and the dissociation tube fixing bracket is integrally formed with the second insert plate. Two speed control knobs are symmetrically arranged on both sides of the outer wall of the motor base. Each speed control knob corresponds to one of the 12 motor mounting holes. Two steering buttons for controlling the motor direction are also provided on one side of the outer wall of the motor base. Both the speed control knobs and steering buttons are connected to the motor control circuit and circuit board. Two power cord sockets are also provided next to the steering buttons. When storing the dissociation device, the power cord can be stored in the receiving cavity. The dissociation device also includes a rotor connector. The rotor connector is a hollow cylindrical shape with a motor rotor insertion hole inside. Six prisms protrude from the outer wall. The width of the prisms gradually decreases along the longitudinal direction of the rotor connector. The motor rotor insertion hole and the motor rotor are both semi-circular. In use, the narrower end of the prism is inserted into the groove of the dissociation tube. The rotor connector is sleeved on the motor, and the motor rotor is inserted into the motor rotor insertion hole, thereby achieving a stable connection between the motor and the dissociation tube.

2. The portable high-throughput single-cell dissociation instrument according to claim 1, characterized in that: The base of the dissociator, the motor base, and the dissociation tube fixing bracket can also be in shapes other than square.

3. The portable high-throughput single-cell dissociation instrument according to claim 1, characterized in that: There may be multiple speed control knobs, and each speed control knob can be used to control one or more motors.

4. The portable high-throughput single-cell dissociation instrument according to claim 1, characterized in that: The speed control knob is surrounded by a speed scale.

5. A portable high-throughput single-cell dissociation instrument according to claim 1, characterized in that: The positions of the speed control knob, directional button, and power cord socket on the outer wall of the motor base can be adjusted according to actual conditions.

6. A portable high-throughput single-cell dissociation instrument according to claim 1, characterized in that: The number of motor mounting holes and the number of dissociation tube placement holes can also be 24 or more.

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