Automatic operation device for cell inoculation and cell inoculation method

Abstract

The invention relates to the technical field of biological experiment equipment, and provides an automatic cell inoculation operation device and a liquid separation method for cell inoculation, and the automatic cell inoculation operation device comprises a liquid separation module, an inoculation needle assembly, a vibration module and a positioning module. A liquid storage cavity, a flow dividing pipe, a buffering groove, a thin liquid cavity and a liquid dividing cavity are sequentially formed in the liquid dividing module from top to bottom. The inoculating needle assembly is located above the liquid storage cavity and used for injecting cell suspension into the liquid storage cavity. The inlet of the overflow channel is higher than the highest liquid level of the buffer tank, the tail end of the overflow channel is connected to the bottom of the liquid storage cavity, and the highest position of the overflow channel is located above the liquid level of the liquid storage cavity. A liquid outlet channel is arranged below the liquid separation cavity, the liquid outlet channel is in one-to-one correspondence with the culture holes of the culture dish, the vibration module comprises a vibration pad, and the control system is electrically connected with the inoculation needle assembly, the liquid separation module, the vibration module and the positioning module. Cells are prevented from being accumulated in the center of the hole bottom due to the inertia effect, and the cells are evenly distributed in the hole.

Description

Technical Field

[0001] This invention relates to the field of biotechnology experimental equipment technology, and in particular to an automated cell seeding operation device. This invention also relates to a liquid separation method for cell seeding using this automated cell seeding operation device. Background Technology

[0002] Cell seeding is a fundamental and core step in cell culture, biopharmaceuticals, and other fields. The quality of this procedure directly determines subsequent cell proliferation and differentiation, thus directly affecting the accuracy and reliability of experimental results. Currently, traditional cell seeding methods often employ manual dropping, which has several drawbacks.

[0003] First, during the liquid droppering process, liquid tends to adhere to the walls of the dropper or the edge of the culture dish, resulting in insufficient liquid entering the wells. This loss is particularly significant during micro-inoculation, directly leading to a lower-than-expected number of cells entering the wells. More seriously, the adhered liquid poses a risk of cross-contamination in subsequent operations. When the same dropper is used for different cell lines, residual cells may migrate through the dropper wall to other samples, causing confusion between experimental groups.

[0004] Secondly, when liquid is manually dripped, it enters the culture well in a free-fall manner. Its vertical falling speed is fast. When the liquid hits the bottom of the well, the cells form a high-density aggregation in the central area due to inertia. This results in the accumulation of cells in the center of the bottom of the culture dish well, while the cell density in the edge area is insufficient and cannot be evenly dispersed. This affects the cell growth space and nutrient absorption, and may even lead to cell death.

[0005] Furthermore, manual operation has poor repeatability, with significant differences in inoculation results between different operators or even within the same batch. Liquid volume errors are large, and cell number variations are high, seriously affecting the stability of experimental data. Summary of the Invention

[0006] In view of this, the present invention aims to provide an automated cell seeding device to avoid cells accumulating in the center of the well bottom due to inertia, thereby achieving the effect of uniform cell distribution within the well.

[0007] To achieve the above objectives, the technical solution of the present invention is implemented as follows: An automated cell seeding device includes a dispensing module, a seeding needle assembly, a vibration module, a positioning module, and a control system. The liquid separation module has, from top to bottom, a liquid storage chamber, a diversion pipe, a buffer tank, a fine liquid chamber, and a liquid separation chamber. The inoculation needle assembly is located above the reservoir and is used to inject cell suspension into the reservoir; The upper part of the buffer tank is provided with an overflow channel, the inlet of the overflow channel is higher than the highest liquid level of the buffer tank, the end of the overflow channel is connected to the bottom of the liquid storage cavity, and the highest part of the overflow channel is located above the liquid surface of the liquid storage cavity; A liquid outlet channel is arranged below the liquid distribution cavity and corresponds to the culture holes of the culture dish one by one, and the vibration module includes a vibration pad annularly arranged at the end of the liquid outlet channel. The positioning module is used for positioning the liquid distribution module above the culture dish, and the control system is electrically connected with the inoculation needle assembly, the liquid distribution module, the vibration module and the positioning module respectively.

[0008] Further, the liquid distribution module is made of PEEK material, the inner cavity of the liquid distribution module is coated with a polytetrafluoroethylene hydrophobic coating, the bottom of the liquid storage cavity is connected with a plurality of shunt pipes, and the shunt pipes, the buffer tank, the fine liquid cavity, the liquid distribution cavity and the liquid outlet channel are arranged one by one corresponding to the culture holes.

[0009] Further, the buffer tank is in a semispherical structure, a protrusion protruding upward is formed at the center of the bottom of the buffer tank, a plurality of annular through holes are arranged on the protrusion, and the through holes are used for the communication of the fine liquid cavity.

[0010] Further, the inoculation needle assembly includes an inoculation needle body, a micro-injection pump and a support, the micro-injection pump is in communication with the inoculation needle body, and is used for injecting cell suspension into the liquid storage cavity.

[0011] Further, annular mounting grooves are formed at intervals below the buffer tank, the vibration pad is arranged in the mounting groove, a lithium battery for driving the vibration pad to vibrate is also arranged in the mounting groove, a blocking plate is arranged at the end of the mounting groove, the blocking plate blocks the outlet of the mounting groove, and a liquid flow hole coaxially arranged with the liquid outlet channel is formed on the blocking plate.

[0012] Further, the control system includes a microcontroller, a relay, a time relay and a liquid level sensor. A liquid outlet valve is arranged at the lower part of the liquid distribution cavity, and the liquid outlet valve is used for limiting the flow of the outlet of the liquid distribution cavity. The liquid level sensor is arranged in the liquid storage cavity and each buffer tank, and the microcontroller is signal-connected with the relay, the time relay, the liquid level sensor, the micro-injection pump, the liquid outlet valve of the liquid distribution cavity and the positioning module respectively.

[0013] Further, the bottom end of the liquid outlet channel is provided with a 45° chamfer gradually expanding outward, and the spacing between the liquid outlet channel and the culture hole is adjustable, and the adjustment range is 2-3mm.

[0014] Further, the positioning module comprises a first driving part, a positioning cylinder connected to the power output end of the first driving part, and a positioning seat arranged on one side of the liquid distribution module, wherein the positioning cylinder is provided with a positioning column protruding upward, and the first driving part drives the positioning column to insert into or separate from the positioning hole of the positioning seat.

[0015] Further, the positioning module further comprises a locking assembly, wherein the locking assembly comprises a locking seat connected to the workbench, and the liquid distribution module is provided with an extension shaft extending outward at both ends, and a second driving part connected to the locking seat and a locking shaft connected to the power output end of the second driving part. The locking seat is provided with a support plate, and when the extension shaft is arranged above the support plate, the second driving part drives the locking shaft to extend outward to lock the extension shaft on the support plate.

[0016] Compared with the prior art, the present application has the following advantages: The cell inoculation automatic operation device provided by the present application realizes the improvement of the precision and reliability of the whole process of cell inoculation by driving the shunt of the liquid distribution module through air pressure and combining with the multi-stage flow stabilizing structure, adopts PEEK material as the intermediate layer to construct a closed fluid channel, the surface of which is smooth and has good biocompatibility, completely eliminates the liquid wall-hanging phenomenon, and ensures that the actual inoculation volume deviates from the target value within the allowable error range. At the same time, the multi-stage flow stabilizing structure makes the liquid flow rate decrease in a gradient, so that the cell suspension can enter the culture hole almost without impact, effectively avoids the accumulation of cells at the center of the hole bottom due to inertia, and realizes the uniform distribution of cells in the hole.

[0017] At the same time, the positioning module is arranged to position the intermediate layer and the culture dish to ensure that the upper and lower hole positions are aligned, which can improve the stability of the experiment of the operation device. The locking mechanism is also arranged to lock after positioning, so that the liquid outlet channel corresponds to the culture hole, and the positioning effect is further ensured.

[0018] Another object of the present application is to provide a cell inoculation liquid distribution method of the cell inoculation automatic operation device according to any one of the above, comprising the following steps: Step S1, device pretreatment: check the sealing property of the device, adapt the multi-specification channel according to the target container type, set the experimental parameters and calibrate each component; Step S2, sample preparation: load the cell suspension into the liquid storage pool of the micro-injection pump, and set the injection speed and volume; Step S3, accurate injection: the control system controls the micro-injection pump to start, and the inoculation needle injects the preset volume of cell suspension into the liquid storage cavity; S31, first-stage shunt flow stabilization: the cell suspension in the liquid storage cavity flows into each buffer groove through the shunt pipe, The excess liquid in each buffer groove is returned to the bottom of the liquid storage cavity through the corresponding independent overflow channel, so that the liquid level is stable. S32, secondary flow distribution homogenization: the cell suspension in the buffer groove flows into the distribution cavity through the fine liquid pipe, the fine liquid cavity accurately controls the speed, the distribution cavity temporarily stores the homogenate, and the cell content is uniform; Step S4, synchronous operation: the control system controls the opening of the liquid outlet valve, and simultaneously triggers the start of the vibration module, and the cell suspension is dropped into the target container through the liquid outlet channel; Step S5, operation cycle: the liquid outlet valve is closed, the time relay triggers the vibration module to stop, and batch inoculation is completed. Step S6, effect verification: detection is performed from four dimensions of distribution accuracy, cell distribution uniformity, cell survival rate and anti-overflow condition.

[0019] The cell inoculation and distribution method provided by the application eliminates faults through equipment pretreatment in the early stage of experiment, adapts to multi-channel specifications, and calibrates parameters, thereby laying a foundation for subsequent accurate operation; the sample preparation and injection link is combined with the quantitative control of the micro-injection pump, the double design of primary flow distribution and secondary flow distribution homogenization is matched, the volume and concentration of the cell suspension in each channel are ensured to be uniform, the inoculation accuracy is greatly improved; in the synchronous operation, the liquid outlet valve and the vibration module are started cooperatively, channel blockage and liquid residue are effectively avoided, cell uniform dropping is ensured, cell damage is reduced, and cell survival rate is improved; and one injection is adopted, automatic operation of multi-channel inoculation is realized, manual intervention is reduced, batch inoculation efficiency is improved, and experimental repeatability is ensured through a standardized process. BRIEF DESCRIPTION OF DRAWINGS

[0020] The accompanying drawings, which form a part of the present application, are used to provide further understanding of the present application, and the illustrative embodiments of the present application and their description are used to explain the present application, and do not constitute improper limitations on the present application. In the drawings: Figure 1 A schematic view of the cell inoculation automatic operation device described in the embodiments of the present application; Figure 2 A connection schematic view of the distribution module, the culture dish and the positioning module described in the embodiments of the present application; Figure 3 A Figure 2 A local enlarged view of I in FIG. 4; Figure 4 A Figure 2 A local enlarged view of II in FIG. 4; Figure 5 A perspective schematic view of the positioning module described in the embodiments of the present application; Figure 6 A perspective schematic view of the locking assembly described in the embodiments of the present application.

[0021] Explanation of reference signs: A liquid separation module; 2, a inoculation needle assembly; 3, a vibration module; 4, a positioning module; 5, a liquid outlet valve; 6, a culture dish; A liquid storage cavity; 102, a shunt pipe; 103, a buffer groove; 104, a fine liquid cavity; 105, a liquid separation cavity; 106, an overflow channel; 107, a liquid outlet channel; 108, a mounting groove; 109, a lithium battery; 110, a blocking plate; A vibration pad; A protrusion; 1032, a through hole; 401, a first driving part; 402, a positioning cylinder; 403, a positioning seat; 404, a positioning column; 405, a locking seat; 406, an extension shaft; 407, a locking shaft; 408, a support plate; 409, a second driving part. DETAILED DESCRIPTION

[0022] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0023] In the description of the present application, it should be noted that the orientation or position relationship indicated by the terms "upper", "lower", "inner", "back" and the like is based on the orientation or position relationship shown in the drawings, and is only for the convenience of describing the present application and simplifying the description, and does not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the present application. In addition, the terms "first" and "second" are only for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0024] In addition, in the description of the present application, unless otherwise explicitly limited, the terms "mounting", "connection", "connection", "connector" should be understood broadly. For example, it can be fixedly connected, or it can be detachably connected, or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium, or it can be the communication inside two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood in combination with the specific circumstances.

[0025] The present application will be described in detail below with reference to the accompanying drawings and in conjunction with embodiments.

[0026] The present embodiment relates to a cell inoculation automatic operation device, as a whole, like Figure 1As shown, the device comprises a liquid distribution module 1, an inoculation needle assembly 2, a vibration module 3, a positioning module 4 and a control system. The inside of the liquid distribution module 1 is sequentially shaped from top to bottom with a liquid storage cavity 101, a flow distribution pipe 102, a buffer groove 103, a fine liquid cavity 104 and a liquid distribution cavity 105. The inoculation needle assembly 2 is located above the liquid storage cavity 101 and is used to inject cell suspension into the liquid storage cavity 101. The upper part of the buffer groove 103 is provided with an overflow channel 106, the inlet of the overflow channel 106 is higher than the highest liquid level of the buffer groove 103, the end of the overflow channel 106 is connected to the bottom of the liquid storage cavity 101, and the highest part of the overflow channel 106 is located above the liquid surface of the liquid storage cavity 101. The lower part of the liquid distribution cavity 105 is provided with a liquid outlet channel 107, the liquid outlet channel 107 is arranged one-to-one corresponding to the culture holes of the culture dish 6, the vibration module 3 comprises a vibration pad 301 which is annularly surrounded at the end of the liquid outlet channel 107, the positioning module 4 is used to position the liquid distribution module 1 above the culture dish 6, and the control system is electrically connected with the inoculation needle assembly 2, the liquid distribution module 1, the vibration module and the positioning module 4 respectively.

[0027] According to the above design idea, the cell inoculation automatic operation device of the embodiment realizes the improvement of the precision and reliability of the whole process of cell inoculation by the setting of the air pressure driving and the flow distribution of the liquid distribution module 1, combined with the multi-stage flow stabilizing structure. PEEK material is used as the intermediate layer to construct a closed fluid channel, which has a smooth surface and good biocompatibility, completely eliminates the liquid wall hanging phenomenon, and ensures that the actual inoculation volume and the target value deviation are within the allowable error range. At the same time, the multi-stage flow stabilizing structure makes the liquid flow rate decrease in a gradient, and the cell suspension can enter the culture hole almost without impact, effectively avoiding the accumulation of cells at the center of the hole bottom due to inertia, and realizing the uniform distribution of cells in the hole.

[0028] At the same time, the positioning module 4 is set to position the intermediate layer and the culture dish 6, so as to ensure the alignment of the upper and lower hole positions, which can improve the stability of the experiment of the operation device. A locking mechanism is also provided to lock after positioning, so that the liquid outlet channel 107 corresponds to the culture hole, further ensuring the positioning effect.

[0029] Overall, as shown in Figure 1 The base and the workbench are provided as the support structure of the device, the workbench is provided with a protective cover for isolating the external environment during the work process, which plays a role in sterile operation. The embodiment also provides a servo motor for driving the rotation of the liquid distribution module 1, a drive shaft, a driving gear provided on the drive shaft, a positioning shaft connected to the workbench, a driven gear pivotally connected to the positioning shaft, an installation sleeve sleeved on the positioning shaft and connected to the driven gear, and the installation sleeve is connected to the positioning seat 403 described below. The servo motor drives the positioning seat 403 to drive the flow distribution module to rotate, which is convenient for replacing or flushing the flow distribution module.

[0030] As a preferred embodiment, as shown in Figures 1 to 3As shown, the liquid separation module 1 is made of PEEK material, the inner cavity of the liquid separation module 1 is coated with a polytetrafluoroethylene hydrophobic coating, the bottom of the liquid storage cavity 101 is connected with a plurality of shunt pipes 102, the shunt pipes 102, the buffer groove 103, the fine liquid cavity 104, the liquid separation cavity 105 and the liquid outlet channel 107 are arranged one-to-one corresponding to the culture wells. The liquid separation module 1 is made of PEEK material, which has excellent biocompatibility, chemical corrosion resistance and mechanical stability, avoids the reaction between the material and the cell suspension, and protects the cell activity. At the same time, the inner cavity is coated with a polytetrafluoroethylene hydrophobic coating, which significantly reduces the adsorption probability of the cell suspension on the inner wall of the flow channel, and reduces the cell loss. The shunt pipe 102, the buffer groove 103, the fine liquid cavity 104, the liquid separation cavity 105 and the liquid outlet channel 107 are arranged one-to-one corresponding to the culture wells, forming an independent shunt path, ensuring the consistency of each liquid distribution, avoiding the mutual interference between the multi-channel, and adapting to the batch inoculation demand.

[0031] Further, as shown in the buffer groove 103, the buffer groove 103 is in a semispherical structure, a protrusion 1031 protruding upward is formed at the center of the bottom of the buffer groove 103, a plurality of annular through holes 1032 are arranged on the protrusion 1031, and the through holes 1032 are used for the communication of the fine liquid cavity 104. Figure 2 and Figure 3 As shown, the buffer groove 103 is in a semispherical structure, which optimizes the liquid flow trajectory, reduces the vortex and retention of the liquid in the groove, makes the cell suspension flow smoothly, and avoids the damage of the cells caused by turbulence. Moreover, the protrusion 1031 at the center of the bottom of the buffer groove 103 and the annular through holes 1032 are designed, on the one hand, to guide the liquid to converge to the center, realize the uniform shunting to the fine liquid cavity 104, and on the other hand, the annular through holes 1032 can further refine the liquid flow, improve the uniformity of the secondary shunting, and ensure the consistency of the cell content of each path.

[0032] Preferably, the inoculation needle assembly 2 includes an inoculation needle body and a micro injection pump, the micro injection pump is in communication with the inoculation needle body, and is used for injecting the cell suspension into the liquid storage cavity 101. The inoculation needle assembly 2 further includes an electric cylinder for driving the inoculation needle body to move up and down and a bracket for fixing the inoculation needle body. The micro injection pump is provided with a precise driving mechanism such as a stepping motor driving screw rod, according to the injection speed and volume parameters preset by the control system, the cell suspension in the liquid storage pool is quantitatively extracted, and the micro injection pump realizes the leakage-free communication with the inoculation needle body through a sealed pipeline; when working, after receiving the start signal of the control system, the driving mechanism pushes the liquid at a set rate, so that the cell suspension is smoothly output along the internal flow channel of the inoculation needle body, accurately injected into the liquid storage cavity 101 of the liquid separation module 1, and through the closed-loop control of the micro injection pump during the injection process, the pushing force and speed are adjusted in real time, so as to avoid the splashing or flow rate mutation of the liquid, and to protect the stable rising of the liquid level of the liquid storage cavity 101, thereby providing a prerequisite for the uniform shunting of the subsequent liquid separation module 1.

[0033] Preferably, as shown in Figures 2 to 4As shown, the buffer tank 103 is spaced below to form an annular mounting groove 108, the shock pad is arranged in the mounting groove 108, and a lithium battery 109 for driving the shock pad to vibrate is also arranged in the mounting groove 108. The mounting groove 108 is provided with a blocking plate 110 at the end, the blocking plate 110 blocks the outlet of the mounting groove 108, and the blocking plate 110 is formed with a liquid flow hole coaxially arranged with the liquid outlet channel 107. Specifically, the shock pad is made of silicone rubber, with a thickness of 2-3 mm, a vibration frequency of 12-15 Hz, and a vibration amplitude of 0.2 mm, which is matched with the liquid outlet speed of 8-15 mL / min of the liquid separation cavity 105. The inner wall of the annular mounting groove 108 is coated with insulating and heat-conducting glue. The matching design of the vibration frequency and the liquid outlet speed not only ensures the cell dispersion effect, but also avoids liquid droplets from splashing; the insulating and heat-conducting glue improves the vibration transmission efficiency and prolongs the service life.

[0034] In addition, the control system includes a microcontroller, a relay, a time relay, and a liquid level sensor. The lower part of the liquid separation cavity 105 is provided with a liquid outlet valve 5 for limiting the flow rate of the outlet of the liquid separation cavity 105. The liquid level sensor is arranged in the liquid storage cavity 101 and each buffer tank 103, and the microcontroller is signal connected with the relay, the time relay, the liquid level sensor, the micro injection pump, the liquid outlet valve 5 of the liquid separation cavity 105, and the positioning module 4.

[0035] The liquid level sensor monitors the liquid level in real time, the microcontroller realizes the synchronous linkage of multiple components, the time relay accurately controls the vibration and liquid outlet time, ensures the cooperation of liquid separation, vibration, displacement and other actions, and further improves the experimental repeatability. The model of the microcontroller is STM32F103, the vibration duration of the time relay is set to 1.5-2.0s, which is synchronized with the opening time of the liquid outlet valve 5, the injection speed range of the micro injection pump is 0.1-10 mL / min, the injection volume accuracy is ±0.5%, and the accurate control of the injection speed and time makes the device adapt to the inoculation requirements of cell suspension of different concentrations and different volumes, and improves the universality.

[0036] Preferably, the bottom end of the liquid outlet channel 107 is provided with a 45° chamfer which gradually expands outward, the spacing between the liquid outlet channel 107 and the culture hole can be adjusted, and the adjustment range is 2-3 mm, and the spacing between the bottom of the liquid outlet channel 107 and the target container inoculation hole can be adjusted, and the range is 2-3 mm. The chamfer design of the liquid outlet channel 107 combined with the hydrophobic coating avoids liquid adhesion to the wall of the culture dish 6, the whole channel hydrophobic coating makes the liquid residue ≤0.02 mL / time, solves the problem of wall hanging, high-precision alignment and adjustable spacing, ensures accurate liquid drop into the target hole, and improves the inoculation effect.

[0037] As Figures 5 to 6As shown, the positioning module 4 includes a first driving part 401 provided on the workbench, a positioning cylinder 402 connected to the power output end of the first driving part 401, a positioning seat 403 provided on one side of the dispensing module 1, a positioning column 404 provided on the positioning cylinder 402 and protruding upward, and the first driving part 401 drives the positioning column 404 to insert into or separate from the positioning hole of the positioning seat 403. The first driving part 401 is an electric cylinder, the positioning column 404 is in positioning connection with the positioning hole, the rapid and accurate alignment of the dispensing module 1 and the culture dish 6 is realized, the deviation caused by manual alignment is avoided, the one-to-one correspondence between the liquid outlet channel 107 and the culture hole is ensured, and the inoculation accuracy is improved. The positioning column 404 and the positioning hole are matched in a convenient disassembly and assembly manner, the device debugging time is shortened, the experimental efficiency is improved, the displacement of the dispensing module 1 during inoculation is avoided, and the consistency of batch inoculation is ensured.

[0038] Further, as shown in Figures 5 to 6 The positioning module 4 further includes a locking assembly, the locking assembly includes a locking seat 405 connected to the workbench, extension shafts 406 extending outwardly provided at both ends of the dispensing module 1, a second driving part 409 connected to the locking seat 405, and a locking shaft 407 connected to the power output end of the second driving part 409. The locking seat 405 is provided with a support plate 408, when the extension shaft 406 is arranged above the support plate 408, the second driving part 409 drives the locking shaft 407 to extend outwardly, and the extension shaft 406 is locked on the support plate 408.

[0039] As shown in Figure 5 The power output shaft of the second driving part 409 is connected with a rod end bearing, the rod end bearing is pivotally connected to the locking shaft 407, the lower end of the locking shaft 407 is an inclined surface, and the extension shaft 406 that is not rotated in place can be gradually locked, so that jamming is prevented.

[0040] In addition, the present embodiment also relates to a cell inoculation dispensing method, which includes the following steps: Step S1, device pretreatment: check the sealing performance of the device, adapt the multi-specification channels according to the target container type, set the experimental parameters and calibrate each component; Step S2, sample preparation: load the cell suspension into the liquid storage pool of the micro-injection pump, and set the injection speed and volume; Step S3, accurate injection: the control system controls the micro-injection pump to start, and the inoculation needle injects the preset volume of cell suspension into the storage cavity 101; S31, primary flow distribution and flow stabilization: the cell suspension in the storage cavity 101 flows into each buffer groove 103 through the flow distribution pipe 102, The excess liquid in each buffer groove 103 flows back to the bottom of the storage cavity 101 through the corresponding independent overflow channel 106, so that the liquid level is stabilized; S32, secondary split homogenization: the cell suspension in the buffer tank 103 flows into the split cavity 105 through the fine liquid tube, the fine liquid cavity 104 precisely controls the speed, the split cavity 105 temporarily stores the homogenate, and the cell content is uniform; Step S4, synchronous operation: the control system controls the opening of the outlet valve 5, and simultaneously triggers the start of the vibration module 3, and the cell suspension is dropped into the target container through the outlet channel 107; Step S5, operation cycle: the outlet valve 5 is closed, and the time relay triggers the stop of the vibration module 3, and the batch inoculation is completed; Step S6, effect verification: detection from four dimensions of split precision, cell distribution uniformity, cell survival rate and anti-overflow condition.

[0041] Among them, the injection control logic of the embodiment includes the following stages: Injection stage: after the microcontroller receives the start instruction, it controls the micro-injection pump to inject cell suspension into the buffer tank 103, and the liquid level sensor feeds back the liquid level in real time. The microcontroller of the control system sends a signal to the micro-injection pump according to the preset parameters, with an injection volume of 12-20mL and an injection speed of 5mL / min; after the injection pump starts, the cell suspension is slowly injected into the buffer tank 103 through the inoculation needle, and the liquid level sensor collects liquid level data in real time. When the liquid level reaches the preset value, the injection pump automatically stops, and if the liquid level is below the threshold, it triggers a refill to ensure the stability of the liquid level in the buffer tank 103.

[0042] Transport stage: the microcontroller controls the opening of the buffer tank 103 liquid delivery valve, and the cell suspension flows into the split cavity 105 through the fine liquid tube; after the split cavity 105 liquid level meets the standard, the liquid delivery valve is closed; Split vibration stage: the microcontroller sends an opening signal to the outlet valve 5, and simultaneously triggers the relay to close, and the lithium battery 109 supplies power to the vibration pad, and the vibration starts; after the time relay counts for 1.5-2.0 seconds, it feeds back the signal to the microcontroller, controls the outlet valve 5 to close and the vibration to stop; It should be noted that the vibration module 3 is only started when the split cavity 105 discharges, with low-intensity vibration, specifically 12-15Hz and 0.2mm. Such setting can disperse cell aggregation without affecting the stability of the liquid in the buffer tank 103 and the fine liquid tube.

[0043] Taking the low-throughput scenario as an example, the cell inoculation split method of the embodiment is introduced in the cell inoculation experiment adapted to a 6-well plate: Step S1, device pretreatment: check the sealing of the device, adapt to multiple specifications of channels according to the type of target container, set experimental parameters and calibrate each component; open the clean bench, place the device, 6-well plate and cell suspension on the workbench, and close after ultraviolet disinfection for 30 minutes; Experimental parameter setting: Inoculation needle: injection speed 5 mL / min, injection volume 12 mL, needle tip to buffer tank 103 liquid level distance 8 mm; Outlet speed: 15 mL / min; Vibration parameters: frequency 12 Hz, amplitude 0.2 mm, duration 2.0 s; Buffer tank 103 liquid level: 12 mL; The liquid module 1 is a 6-channel adapter, the inoculation needle assembly 2 is 0.9 mm in diameter and 60 mm in length, and is sterilized in advance with 75% alcohol and sterilized with ultraviolet light for 30 minutes; 12 mL of sterile deionized water was injected into the buffer tank 103 and left for 30 minutes, and no leakage or bubbles were observed; the inoculation needle was calibrated: the height of the support was adjusted to ensure that the needle tip was 8 mm from the liquid level of the buffer tank 103; Calibrate the vibration module 3: the actual vibration frequency is 12.2 Hz and the amplitude is 0.19 mm detected by the vibration tester; Debug the control system: simulate the injection-liquid separation-vibration-shifting process to verify that the synchronization error is ≤0.1 s.

[0044] Step S2, sample preparation: load the cell suspension into the micro-syringe pump reservoir, and set the injection speed and volume; Take out the HUVEC cell suspension, gently blow 5 times, and use a pipette to take 12 mL of cell suspension and load it into the syringe pump reservoir, set the injection parameters, and stand by.

[0045] S31, primary flow stabilization: the cell suspension in the storage cavity 101 flows into each buffer tank 103 through the flow divider 102 Start the syringe pump, and the inoculation needle injects the cell suspension into the buffer tank 103 at a speed of 5 mL / min, and when the liquid level sensor displays 12 mL, the syringe pump automatically stops, and stands for 5 minutes to stabilize the liquid level.

[0046] Fractional delivery: the microcontroller controls the infusion valve to open, and the cell suspension flows into the six liquid separation cavities 105 through the fine liquid tube, and the infusion valve is closed after the liquid level meets the standard.

[0047] Synchronous operation: the microcontroller controls the outlet valve 5 to open, and the vibration pad starts at the same time; the cell suspension is dropped into the six-well plate through the outlet, and the vibration disperses the cell aggregation.

[0048] Operation cycle: after 2.0 seconds, the outlet valve 5 is closed, the vibration stops, and the XY axis platform drives the six-well plate to shift to the end point, and a single inoculation is completed.

[0049] Effect verification: the average error of liquid separation accuracy is ±1.5%; the cell distribution CV value is 2.8%; the cell survival rate is 96.5%; there is no overflow or splashing.

[0050] The cell inoculation distribution method of the present application lays a foundation for subsequent accurate operation by pre-treating the equipment to exclude faults, adapting multi-channel specifications and calibrating parameters in the early experimental stage; the combination of sample preparation and injection with quantitative control of the micro-injection pump, together with the double design of one-stage distribution steady flow and two-stage distribution homogenization, ensures the uniformity of the volume and concentration of the cell suspension in each channel, greatly improving the inoculation precision; the liquid outlet valve 5 and the vibration module 3 are started simultaneously in synchronous operation, effectively avoiding channel blockage and liquid residue, ensuring uniform cell dripping while reducing cell damage and improving cell survival rate; and one injection is used to realize the automation of multi-channel inoculation, which not only reduces manual intervention and improves batch inoculation efficiency, but also ensures experimental repeatability through standardized processes.

[0051] The above only describes the preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. An automated cell seeding device, characterized in that: It includes a liquid dispensing module (1), an inoculation needle assembly (2), a vibration module (3), a positioning module (4), and a control system; The liquid separation module (1) has, from top to bottom, a liquid storage chamber (101), a diversion pipe (102), a buffer tank (103), a fine liquid chamber (104), and a liquid separation chamber (105). The inoculation needle assembly (2) is located above the reservoir (101) and is used to inject cell suspension into the reservoir (101); The buffer tank (103) is provided with an overflow channel (106) at the top. The inlet of the overflow channel (106) is higher than the highest liquid level of the buffer tank (103). The end of the overflow channel (106) is connected to the bottom of the liquid storage chamber (101). The highest point of the overflow channel (106) is located above the liquid surface of the liquid storage chamber (101). The liquid distribution chamber (105) is provided with a liquid outlet channel (107) below it. The liquid outlet channel (107) is provided in correspondence with the culture holes of the culture dish (6). The vibration module (3) includes a vibration pad (301) that is annularly arranged around the end of the liquid outlet channel (107). The positioning module (4) is used to position the dispensing module (1) above the culture dish (6), and the control system is electrically connected to the inoculation needle assembly (2), the dispensing module (1), the vibration module and the positioning module (4) respectively.

2. The automated cell seeding device according to claim 1, characterized in that: The liquid separation module (1) is made of PEEK material, and the inner cavity of the liquid separation module (1) is coated with a hydrophobic polytetrafluoroethylene coating. The bottom of the liquid storage chamber (101) is connected to several of the diversion tubes (102), and the diversion tubes (102), buffer tank (103), fine liquid chamber (104), liquid separation chamber (105) and liquid outlet channel (107) are arranged in a one-to-one correspondence with the culture well.

3. The automated cell seeding device according to claim 2, characterized in that: The buffer groove (103) has a hemispherical structure. The bottom center of the buffer groove (103) has an upward protrusion (1031). The protrusion (1031) is provided with several annular through holes (1032), which are used to connect the fine liquid cavity (104).

4. The automated cell seeding device according to claim 3, characterized in that: The inoculation needle assembly (2) includes an inoculation needle body, a micro-injection pump, and a support; The micro-injection pump is connected to the inoculation needle body and is used to inject the cell suspension into the reservoir (101).

5. The automated cell seeding device according to claim 3, characterized in that: The buffer groove (103) is formed with annular mounting grooves (108) at intervals below it. The vibration pad is disposed in the mounting groove (108). The mounting groove (108) is also provided with a lithium battery (109) that drives the vibration pad to vibrate. The mounting groove (108) is provided with a blocking plate (110) at its end. The blocking plate (110) blocks the outlet of the mounting groove (108). The blocking plate (110) has a flow hole formed on it that is coaxially arranged with the liquid outlet channel (107).

6. The automated cell seeding device according to claim 1, characterized in that: The control system includes a microcontroller, relays, time relays, and a liquid level sensor; The lower part of the liquid separation chamber (105) is provided with a liquid outlet valve (5), which is used to limit the flow rate of the liquid separation chamber (105) outlet; The liquid level sensor is located in the liquid storage chamber (101) and each buffer tank (103). The microcontroller is connected to the relay, time relay, liquid level sensor, micro injection pump, liquid dispensing chamber (105) outlet valve (5) and positioning module (4) respectively.

7. The automated cell seeding device according to claim 5, characterized in that: The bottom end of the liquid outlet channel (107) is provided with a 45° chamfer that gradually expands outward. The distance between the liquid outlet channel (107) and the culture well is adjustable, with an adjustment range of 2~3mm.

8. The automated cell seeding device according to claim 1, characterized in that: The positioning module (4) includes a first driving part (401) and a positioning cylinder (402) connected to the power output end of the first driving part (401). A positioning seat (403) is provided on one side of the liquid separation module (1). A positioning post (404) protruding upward is provided on the positioning cylinder (402). The first driving part (401) drives the positioning post (404) to insert into or disengage from the positioning hole of the positioning seat (403).

9. The automated cell seeding device according to claim 8, characterized in that: The positioning module (4) also includes a locking assembly, which includes a locking seat (405) connected to the workbench, and extension shafts (406) extending outward at both ends of the liquid dispensing module (1). The locking seat (405) is connected to the second drive unit (409), and a locking shaft (407) is connected to the power output end of the second drive unit (409). The locking seat (405) is provided with a support plate (408). When the extension shaft (406) is located above the support plate (408), the second driving part (409) drives the locking shaft (407) to extend outward and lock the extension shaft (406) on the support plate (408).

10. A cell seeding and dispensing method using an automated cell seeding operating device according to any one of claims 1 to 9, characterized in that, Includes the following steps: Step S1, Equipment Pre-processing: Check the sealing of the device, adapt multiple channel specifications according to the target container type, set experimental parameters and calibrate each component; Step S2, Sample preparation: Load the cell suspension into the reservoir of the micro-injection pump and set the injection rate and volume; Step S3, Precise Injection: The control system controls the micro-injection pump to start, and the inoculation needle injects a preset volume of cell suspension into the reservoir (101); S31, First-stage diversion and steady flow: The cell suspension in the storage chamber (101) flows into each buffer tank (103) through the diversion pipe (102). Excess liquid in each buffer tank (103) flows back to the bottom of the storage chamber (101) through the corresponding independent overflow channel (106) to achieve liquid level stability; S32, Secondary Homogenization: The cell suspension in the buffer tank (103) flows into the dispensing chamber (105) through the thin liquid tube. The speed is precisely controlled in the thin liquid chamber (104), and the dispensing chamber (105) temporarily stores the homogenized cells to ensure uniform cell content. Step S4, synchronous operation: The control system controls the liquid outlet valve (5) to open, and at the same time triggers the vibration module (3) to start, and the cell suspension drips into the target container through the liquid outlet channel (107); Step S5, Operation Cycle: The liquid outlet valve (5) is closed, the time relay triggers the vibration module (3) to stop, and the batch inoculation is completed; Step S6, Effect Verification: The results are tested from four dimensions: dispensing accuracy, cell distribution uniformity, cell viability, and overflow prevention.

Images