Single-cell printer and cell printing method

Abstract

A single-cell printer and a cell printing method. The single-cell printer comprises a sample injection apparatus, a spraying apparatus, an optical detection apparatus, a sorting apparatus, and a control apparatus. A liquid inlet of the spraying apparatus is in communication with an outlet of the sample injection apparatus, and cell droplets are sprayed out by means of a liquid outlet of the spraying apparatus. The optical detection apparatus acquires droplet image information of the cell droplets in the spraying apparatus. The sorting apparatus is disposed at the liquid outlet. The control apparatus controls the spraying apparatus to form cell droplets and sorts the cell droplets into a preset well plate.

Description

Single cell printer and cell printing method

[0001] The present application claims priority to the Chinese patent application No. 2024108619954, filed on June 28, 2024, and entitled "Single cell printer and cell printing method", the whole content of which is incorporated herein by reference. TECHNICAL FIELD

[0002] The present application relates to the field of microfluidic technology, in particular to a single cell printer and a cell printing method. BACKGROUND

[0003] With the continuous development of cell therapy and gene therapy technology, cell-related research has gradually become a research hotspot in the field of current medicine and the whole life science, and cell culture has become a very important technology in scientific research. Cell sorting and purification are the key to obtaining relatively pure target cells.

[0004] Single cell sorting can realize the capture separation and analysis of any single type of cells or rare cells (such as circulating tumor cells, endothelial cells, stem cells, and related cells in blood and bone marrow) in a suspension system. Existing single cell sorting mostly adopts the separation dilution method, which disperses single cells in the culture medium by repeatedly diluting the cell suspension, continuously reduces the cell concentration, and finally realizes single cell sorting. This method requires a large amount of time, labor and resources, has high cost, low sorting efficiency, and may cause non-single sorting of cells.

[0005] In addition, flow cytometry is also a common cell sorting technology, which combines the specificity of fluorescently labeled cells to realize the sorting of single cells. However, cross contamination between cells may occur during the sorting process, which requires strict sample handling and instrument cleaning measures. At the same time, during the flow cytometric sorting process, cells may be exposed to mechanical stress and light damage from the fluorescence exciter, which may affect the cell state. The existing cell sorting method only screens droplets containing single cells into a well plate, and if the cells themselves do not meet the experimental requirements during the subsequent cell culture process, it will interfere with the experimental results and affect the efficiency of the experiment. SUMMARY

[0006] The application provides a single cell printer and a cell printing method, and aims to solve the problem that the existing single cell printer may cause cross contamination between cells in the cell sorting process, and needs strict sample processing and instrument cleaning measures. Meanwhile, in the flow cytometry sorting process, cells may be exposed to mechanical stress and light damage of a fluorescence exciter, which may affect the cell state, and the existing cell sorting method only screens droplets containing single cells into a well plate, and if the cells themselves do not meet the experimental requirements in the subsequent cell culture process, the experimental results will be disturbed, and the experimental efficiency is affected.

[0007] In a first aspect, the application provides a single cell printer, which comprises:

[0008] a sample inlet device, in which a cell suspension to be sorted is stored;

[0009] a jetting device, a liquid inlet of the jetting device being in communication with an outlet of the sample inlet device, the jetting device being used to control vibration of the cell suspension, form cell droplets to be sorted, and make the cell droplets be sprayed out through a liquid outlet of the jetting device;

[0010] an optical detection device, which is arranged towards the jetting device and is used to acquire droplet image information of the cell droplets to be sorted in the jetting device;

[0011] a sorting device, which is arranged at the liquid outlet;

[0012] a control device, which is electrically connected with the jetting device, the optical detection device and the sorting device, and is used to control the jetting device to form the cell droplets, acquire the droplet image information detected by the optical detection device, confirm whether the cell droplets are target droplets according to the droplet image information, and sort the target droplets to a preset well plate; wherein the target droplets are the cell droplets containing a preset number of cells meeting preset cell conditions.

[0013] In a second aspect, the application provides a cell printing method, which is applied to the single cell printer provided in any one of the embodiments of the application, and comprises the following steps:

[0014] controlling a jetting device of the single cell printer to form the cell droplets;

[0015] acquiring droplet image information of the cell droplets in the jetting device, which is collected by an optical detection device of the single cell printer;

[0016] According to the droplet image information, it is determined whether the cell droplet is a target droplet, the target droplet being the cell droplet containing a preset number of cells meeting a preset cell condition.

[0017] If the cell droplet is the target droplet, the sorting device is controlled to sort the target droplet to a preset well plate, and printing of the target droplet is completed.

[0018] The application provides a single-cell printer, which comprises a sample inlet device, a jetting device, an optical detection device, a sorting device and a control device. The sample inlet device stores a cell suspension to be sorted. The jetting device is connected to the outlet of the sample inlet device, and is used to control vibration of the cell suspension, form a cell droplet to be sorted, and make the cell droplet be sprayed out of the outlet of the jetting device. The optical detection device is arranged towards the jetting device, and is used to acquire droplet image information of the cell droplet to be sorted in the jetting device. The sorting device is arranged at the outlet. The control device is electrically connected to the jetting device, the optical detection device and the sorting device, and is used to control the jetting device to form the cell droplet, acquire the droplet image information detected by the optical detection device, determine whether the cell droplet is a target droplet according to the droplet image information, and sort the target droplet to a preset well plate; wherein the target droplet is the cell droplet containing a preset number of cells meeting a preset cell condition.

[0019] The single-cell printer provided is used to control the jetting device by the control device, so that the cell suspension in the jetting device is vibrated, the cell droplet to be sorted is formed and sprayed out, and the optical detection device is controlled to capture the droplet image information of the cell droplet to be sorted. The control device can control the sorting device to sort the target droplet to a preset well plate when it is determined that the cell droplet is the target droplet according to the droplet image information. The single-cell printer provided can reduce damage to cells and improve the accuracy of experiments in the process of cell sorting. Meanwhile, the single-cell printer provided can sort the target droplet of a single cell meeting the experimental requirements without affecting the state of the cell, improve the sorting precision and efficiency of single-cell sorting, reduce the equipment cost of the single-cell printer, and make the single-cell printer be widely applied and popularized in high-throughput sorting applications. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the technical solutions of the embodiments of the application, the following will briefly introduce the drawings needed in the embodiment description. Obviously, the drawings in the following description are some embodiments of the application, and other drawings can also be obtained by those skilled in the art without any creative effort.

[0021] FIG. 1 is a structural schematic diagram of a single-cell printer provided by an embodiment of the application;

[0022] Fig. 2 is a structural schematic diagram of a sorting device according to an embodiment of the present application;

[0023] Fig. 3 is a structural schematic diagram of another sorting device according to an embodiment of the present application;

[0024] Fig. 4 is a workflow schematic diagram of a cell detection model according to an embodiment of the present application;

[0025] Fig. 5 is a structural schematic diagram of a sample injection device according to an embodiment of the present application;

[0026] Fig. 6 is a structural schematic diagram of a jetting device according to an embodiment of the present application;

[0027] Fig. 7 is a step schematic flow chart of a cell printing method according to an embodiment of the present application;

[0028] Fig. 8 is a structural schematic diagram of a control device according to an embodiment of the present application.

[0029] Main elements and symbol explanations: 10, single cell printer; 11, sample injection device; 111, sample injection module; 1111, first pipeline; 1112, second pipeline; 112, mixing module; 1121, second air pump; 1122, second electromagnetic valve; 12, jetting device; 121, substrate; 1211, cell liquid cavity; 1212, first substrate; 1213, second substrate; 122, first electrode; 123, piezoelectric substrate; 124, second electrode; 13, optical acquisition device; 14, sorting device; 141, pipetting device; 1411, first air pump; 1412, first electromagnetic valve; 1413, positive electrode; 142, waste liquid collection area; 143, motion platform; 15, control device; 20, cell liquid droplet; 30, pre-set well plate.

[0030] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present application. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of protection of the present application.

[0032] The flow chart shown in the drawings is only an example, and does not necessarily include all the contents and operations / steps, nor does it necessarily be executed in the described order. For example, some operations / steps can be decomposed, combined or partially merged, so the actual execution order can be changed according to the actual situation.

[0033] It is to be understood that the terms used in the specification of the present application are only for the purpose of describing particular embodiments of the present application and are not intended to limit the present application. As used in the specification and the appended claims of the present application, the singular forms "a," "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0034] It should be understood that, in order to facilitate the clear description of the technical solutions of the embodiments of the present application, in the embodiments of the present application, the same items or similar items with basically the same functions and roles are distinguished by using "first", "second", etc. For example, the first support and the second support are only used to distinguish different supports, and do not limit the sequence. Those skilled in the art can understand that "first", "second", etc. do not limit the quantity and execution sequence, and "first", "second", etc. also do not necessarily mean different.

[0035] It should also be understood that the term "and / or" used in the specification and the appended claims means any combination of one or more of the associated listed items and all possible combinations, and includes these combinations.

[0036] Some embodiments of the present application will be described in detail below with reference to the accompanying drawings. The following embodiments and features in the embodiments can be combined with each other without conflict.

[0037] With the continuous development of cell therapy and gene therapy technology, cell-related research has gradually become a research hotspot in the field of current medicine and the entire field of life sciences. Cell culture has become an important technology in scientific research, and cell sorting and purification are the key to obtaining pure target cells.

[0038] Single cell sorting can realize the capture separation and analysis of any single type of cells or rare cells (such as circulating tumor cells, endothelial cells, stem cells, and related cells in blood and bone marrow) in a suspension system. Existing single cell sorting mostly uses the separation dilution method, which disperses single cells in the culture medium by repeatedly diluting the cell suspension, continuously reduces the cell concentration, and finally realizes single cell sorting. This method requires a large amount of time, labor and resources, has high cost, low sorting efficiency, and may cause non-single sorting of cells.

[0039] In addition, flow cytometry is also a common cell sorting technique that combines the specificity of fluorescently labeled cell sorting to sort individual cells. However, during the sorting process, cross-contamination between cells may occur, requiring strict sample handling and instrument cleaning measures. At the same time, during the flow cytometry sorting process, cells may be exposed to mechanical stress and light damage from the fluorescence exciter, which may affect the cell state, and the existing cell sorting method only screens droplets containing single cells into the well plate, and if the cells themselves do not meet the experimental requirements during subsequent cell culture, it will interfere with the experimental results and affect the efficiency of the experiment.

[0040] Single cell printers may have some drawbacks, including but not limited to:

[0041] 1. Technical limitations: Single cell sorting technology may be subject to technical limitations, such as sorting accuracy and sorting efficiency. Current technology may not fully meet the needs of all application scenarios.

[0042] 2. High cost: Single cell printers often require expensive equipment and consumables, making them costly and limiting their popularity and application in some laboratories or institutions.

[0043] 3. Complex operation: Operating a single cell printer may require specialized skills and training, as it involves fluid mechanics, optics, and computer control technologies that require certain professional knowledge.

[0044] 4. Sample damage: During the sorting process, cells may be subjected to mechanical damage or light damage, resulting in a decline in sample quality or cell death, affecting the accuracy of experimental results.

[0045] 5. Longer processing time: Sorting a large number of samples may take a long time, which limits the application of single cell sorting systems in high-throughput experiments.

[0046] 6. Data processing challenges: The amount of data generated by single cell printers can be large, requiring complex data processing and analysis to extract useful information from the vast amount of data, which can be a challenge.

[0047] 7. Difficulty in handling heterogeneity: The heterogeneity of cell populations may cause difficulties in the sorting process, as different types of cells may have similar characteristics in terms of phenotype, making it difficult to accurately sort target cells.

[0048] 8. Potential cross-contamination: During single cell sorting, there is a risk of cross-contamination, i.e. cells sorted from one sample may be mistakenly placed into another sample, affecting the reliability of experimental results.

[0049] To solve the above problems, please refer to Figure 1, which is a structural schematic diagram of a single-cell printer 10 provided by an embodiment of the present application. As shown in Figure 1, the provided single-cell printer 10 comprises a sample inlet device 11, a jet device 12, an optical detection device 13, a sorting device 14, and a control device 15. The sample inlet device 11 stores a cell suspension to be sorted. The liquid inlet of the jet device 12 is in communication with the outlet of the sample inlet device 11, and the jet device 12 is used to control the vibration of the cell suspension, form a cell droplet 20 to be sorted, and make the cell droplet 20 be ejected through the liquid outlet of the jet device. The optical detection device 13 is arranged towards the jet device 12, and is used to acquire droplet image information of the cell droplet 20 to be sorted in the jet device 12. The sorting device 14 is arranged at the liquid outlet. The control device 15 is electrically connected with the jet device 12, the optical detection device 13, and the sorting device 14, and is used to control the jet device 12 to form a cell droplet, acquire the droplet image information detected by the optical detection device 13, confirm whether the cell droplet 20 is a target droplet according to the droplet image information, and sort the target droplet to a preset well plate 30; wherein the target droplet is a cell droplet containing a preset number of cells meeting a preset cell condition.

[0050] Specifically, the control device 15 controls the jet device 12 to make the cell suspension in the jet device 12 vibrate, form a cell droplet 20 to be sorted to be ejected, and controls the optical detection device 16 to shoot the droplet image information of the cell droplet 20 to be sorted. The control device 15 can control the sorting device 14 to sort the target droplet to the preset well plate 30 when confirming that the cell droplet 20 is the target droplet according to the droplet image information. The provided single-cell printer 10 can reduce the damage to the cells in the process of cell sorting, and improve the accuracy of the experiment. At the same time, the provided single-cell printer 10 can sort out the target droplet containing a preset number of cells meeting the experimental requirements without affecting the state of the cells, improve the sorting precision and efficiency of single-cell sorting, reduce the equipment cost of the single-cell printer 10, and make the single-cell printer 10 popular in high-throughput sorting applications, so as to be widely applied and popularized.

[0051] In some embodiments, the preset number can be 1 or any number greater than 1. Through the provided single-cell printer 10, the target droplet containing a single or multiple cells can be sorted according to the experimental requirements, thereby improving the applicable range of the provided single-cell printer 10.

[0052] In some embodiments, the preset cell condition includes that the cell roundness of at least one cell in the cell droplet 20 is in a preset roundness range, and the size of the cell in the cell droplet is in a preset size range.

[0053] The preset circularity range of the cell circularity is generally 0.00 to 1.00, and the greater the value of the cell circularity is, the more round the cells in the cell droplet 20 are, and when the cell circularity is 1.00, it is a standard circle. The preset circularity is 0.8, and the preset size range can be 10-30 μm in diameter, and the target droplet of the single cell meeting the experimental requirements is sorted out by selecting the cell circularity and size of the cell droplet 20. At the same time, the preset circularity and the preset size can be adjusted according to the experimental requirements, and the preset circularity range and the preset size range are not limited in the embodiment of the present application.

[0054] It should be noted that in some embodiments, whether it is a target droplet can also be determined according to the selected range of parameters such as whether the cell printing is successful (whether the target droplet is ejected), cell area, cell perimeter, aspect ratio, roundness, etc. The type of parameters selected is set according to actual needs, and the embodiment of the present application does not limit this.

[0055] In some embodiments, please refer to FIG. 2, which is a structural schematic diagram of a sorting device 14 provided by the embodiment of the present application. As shown in FIG. 2, the sorting device 14 comprises a pipetting device 141 and a waste liquid collection area 142, and the pipetting device 141 is arranged at the liquid outlet. The waste liquid collection area 142 is arranged at the liquid outlet. The control device 15 is electrically connected with the pipetting device 141, and the control device 15 is used to acquire the cell number and the cell parameter information according to the droplet image information, so as to control the pipetting device 141 to move the cell droplet 20 with the cell number not being 1 and the cell parameter information not meeting the preset cell condition to the waste liquid collection area 142.

[0056] In the process of cell sorting of the single cell printer 10, when the control device 15 confirms that the cell droplet 20 ejected from the liquid outlet of the ejection device 12 is a cell droplet 20 not meeting the preset cell condition according to the droplet image information, the control device 15 can control the pipetting device 141 to timely move the cell droplet 20 to the waste liquid collection area 142, and at the same time, the cell droplet 20 meeting the preset cell condition is directly sorted into the preset well plate 30. Further, the provided single cell printer 10 can quickly realize the sorting of the cell droplet 20 of the single cell.

[0057] For example, the waste liquid collection area 142 can be a clean cotton or a waste liquid tank, and the type of the waste liquid collection area 142 is not limited in the embodiment of the present application.

[0058] As shown in FIG. 2, the pipetting device 141 includes a first air pump 1411, which is arranged at the liquid outlet relative to the waste liquid collection area 142; the control device 15 is electrically connected with the first air pump 1411, and when the first air pump 1411 is controlled to be turned on, the first air pump 1411 blows gas to the cell droplet 20 ejected by the ejection device 12, so that the cell droplet 20 moves to the waste liquid collection area 142; when the first air pump 1411 is turned off, the cell droplet 20 is ejected to the preset well plate 30.

[0059] When the first air pump 1411 is controlled to be turned on by the control device 15, gas is blown at the ejection port towards the waste liquid collection area 142, and the cell droplet 20 ejected through the ejection port is blown into the waste liquid collection area.

[0060] It should be noted that in some embodiments, the pipetting device 141 further includes a first electromagnetic valve 1412, the first air pump 1411 is connected with a first interface of the first electromagnetic valve 1412 through a first pipeline, a second interface of the first electromagnetic valve 1412 arranged opposite to the first interface is connected with a second pipeline, and the first electromagnetic valve 1412 is electrically connected with the control device 15. When the control device 15 controls the first interface and the second interface of the first electromagnetic valve to be conductive, the first air pump 1411 can blow gas to the cell droplet 20 ejected by the ejection device 12 through the first electromagnetic valve 1412.

[0061] As shown in FIG. 3, which is a structural schematic diagram of another sorting device 14 provided by the embodiment of the present application, the polarity of the cell suspension is negative; the pipetting device 141 includes a positive electrode 1413, which is arranged at the liquid outlet and located above the waste liquid collection area 142, and the positive electrode 1413 is electrically connected with the control device 15; when the control device 15 controls the positive electrode 1413 to be turned on, the cell droplet 20 ejected by the ejection device 12 moves towards the positive electrode 1413, so as to be ejected to the waste liquid collection area 142 (as trajectory 1 in FIG. 3); when the positive electrode is turned off, the cell droplet is ejected to the preset well plate (as trajectory 2 in FIG. 3).

[0062] In some embodiments, the sorting device 14 includes a liquid suction device arranged at the liquid outlet, and the liquid suction device is electrically connected with the control device 15; when the control device 15 controls the liquid suction device to be turned on, the liquid suction device sucks away the cell droplet 20 ejected by the ejection device 12; when the control device 15 controls the liquid suction device to be turned off, the cell droplet 20 is ejected to the preset well plate.

[0063] The liquid suction device can be a first air pump 1411 as shown in FIG. 2, which is used to suck air towards the liquid outlet of the jetting device 12 when turned on, thereby sucking away the cell droplet 20 that does not meet the preset cell condition. In some embodiments, as shown in FIG. 2, the sorting device 14 further comprises a moving platform 143, on which the preset well plate 30 is arranged, and the moving platform 143 is electrically connected with the control device 15, which is used to determine a target slot position for the target droplet jetting among a plurality of slot positions of the preset well plate 30 when the cell parameter information meets the preset cell condition, so as to control the moving platform 143 to move the preset well plate 20 according to the coordinate information of the target slot position, so that the liquid outlet of the jetting device 12 is aligned with the target slot position. Through the arrangement of the moving platform 143, the corresponding target slot position can be quickly aligned every time the cell droplet 20 is jetted out, thereby improving the efficiency of cell sorting.

[0064] For example, the moving platform 143 can be an X / Y two-axis moving platform or a single-axis moving platform, and the type of the moving platform 143 is not limited in the embodiments of the present application.

[0065] For example, the liquid suction device comprises a preset liquid suction area arranged on the moving platform 143, which is used to control the moving platform 143 to move the preset liquid suction area so that the liquid outlet of the jetting device 12 is aligned with the preset liquid suction area when the cell droplet 20 is not the target droplet. The preset liquid suction area can be a cleaning cotton or another well plate, and the type of the preset liquid suction area is not limited in the embodiments of the present application.

[0066] In some embodiments, the moving platform 142 is connected with the jetting device 12 through a fastener such as a clamp, thereby being able to control the liquid outlet of the jetting device 12 to be aligned with the waste liquid collection area 142 or the preset well plate 30.

[0067] In some embodiments, the control device comprises a microcontroller unit (MCU) or a terminal device carrying the MCU.

[0068] In some embodiments, as shown in FIG. 4, which is a workflow schematic diagram of a cell detection model provided by the embodiments of the present application, the control device 15 stores a preset cell detection model, which is used to input the droplet image information into the cell detection model to obtain cell number information corresponding to the droplet image information and the cell parameter information; wherein the cell number information is used to confirm the number of cells in the cell droplet.

[0069] The provided cell detection model mainly uses a YOLOv8 segmentation model based on visual imaging, as shown in FIG. 4. The model has strong feature extraction capability and can better capture the detailed features of the target. At the same time, the YOLOv8 algorithm uses a one-stage method for target detection, which has a faster detection speed. Therefore, the algorithm can meet the requirements of high recognition rate and fast printing of the single-cell printer. The droplet image information needs to be scaled proportionally and the background needs to be filled before being input into the model to adapt to the input size of the model. The calculation inside the model mainly includes feature extraction and feature fusion. Finally, the position, category and segmentation map of the target are output. Some targets with low probability are filtered out through confidence filtering, and redundant items are removed through maximum value suppression. Finally, the output segmentation map is restored to the original size, the image is binarized through a threshold, and the contour of the cell can be found.

[0070] It should be noted that in some embodiments, the latest model currently trained has accumulated 3000 labeled droplet image information. The training results show that the recognition rate of the cells can reach 97%.

[0071] In some embodiments, please refer to FIG. 5, which is a structural schematic diagram of a sample injection device 11 provided by an embodiment of the present application. The sample injection device 11 includes a sample injection module 111 and a mixing module 112. The sample injection module 111 includes a first pipeline 1111 and a second pipeline 1112. The inlet of the first pipeline 1111 is used for inputting a cell suspension. The outlet of the first pipeline 1111 is in communication with the liquid inlet of the jet device 12. One side of the first pipeline 1111 is in communication with the outlet of the second pipeline 1112. The mixing module 112 is connected with the inlet of the second pipeline 1112. The mixing module 112 is also electrically connected with the control device 15. The control device 15 is used for determining the number of cells in the cell suspension in the jet device 12 according to the droplet image information, so as to control the mixing module 112 to reciprocally suck gas at the outlet of the second pipeline according to the number of cells, so as to ensure that the cell suspension flowing into the jet device 12 is in a mixed state. In turn, the single-cell printer 10 provided by the present application can avoid the problem that the cells in the cell suspension are prone to sedimentation, which can cause blockage in the jet device 12, thereby affecting the normal operation of the cell sorting process and the efficiency and accuracy of the optical detection result of the sorted cell droplets.

[0072] Exemplarily, as shown in FIG. 5, the mixing module 112 includes a second air pump 1121 connected with the inlet of the second pipeline 1112, and the control device 15 is electrically connected with the second air pump 1121. The control device 15 is configured to determine a control parameter of the second air pump 1121 according to the cell number of the cell suspension in the injection device 12, so as to control the second air pump 1121 to reciprocally suck the outlet of the second pipeline 1112 according to the control parameter, so as to ensure that the cell suspension flowing into the injection device 12 is in a mixed state.

[0073] Exemplarily, the control parameter includes the suction power of the second air pump; when the cell number is less than a preset number, the control device adjusts the control parameter to reduce the suction power of the second air pump, so as to increase the cell number; when the cell number is greater than the preset number, the control device adjusts the control parameter to increase the suction power of the second air pump, so as to reduce the cell number; and when the cell number is equal to the preset number, the control device does not adjust the control parameter.

[0074] In some embodiments, as shown in FIG. 6, which is a structural schematic diagram of an injection device 12 provided by an embodiment of the present application, the injection device 12 includes a substrate 121, a first electrode 122, a piezoelectric substrate 123 and a second electrode 124. The substrate 121 includes a cell liquid cavity 1211 for placing a cell suspension to be sorted, the cell liquid cavity 1211 includes an inlet and an outlet, the inlet is communicated with the sample injection device 11; the optical detection device 13 faces the substrate 121 and is opposite to the cell liquid cavity 1211. The first electrode 122 is arranged on the substrate 121 and located outside the cell liquid cavity 1211. The piezoelectric substrate 123 is made of a piezoelectric material, and the piezoelectric substrate 123 is arranged on the first electrode 122. The second electrode 124 is arranged on the piezoelectric substrate 123, and the polarity of the second electrode 124 is opposite to that of the first electrode 122. The second electrode 124 and the first electrode 122 are electrically connected with the control device 15, and the control device 15 is configured to control the first electrode 122 and the second electrode 124 to drive the piezoelectric substrate 123 to vibrate to propagate an acoustic pulse wave in the cell suspension in the cell liquid cavity 1211, so as to drive the cell suspension to vibrate to form a cell droplet 20, and make the cell droplet 20 sprayed out through the outlet. Further, the single-cell printer 10 provided by the present application can reduce the damage to the cells in the process of cell sorting, and improve the accuracy of the experiment.

[0075] Exemplarily, as shown in FIG. 6, the substrate 121 comprises a first substrate 1212 and a second substrate 1213, the first electrode 122 is arranged on a first side of the first substrate 1212, and a second side of the first substrate 1212 and a first side of the second substrate 1213 are connected to form the cell solution cavity 1211. The light transmittance of the first substrate 1212 is greater than a preset light transmittance, and the optical detection device 13 faces the first substrate 1212 and is opposite to the cell solution cavity 1211.

[0076] It should be noted that, in some embodiments, the first substrate 1212 can be a light-transmitting material such as glass, and the second substrate can be silicon. In this case, the light of the optical detection device 13 enters the cell solution cavity 1211 through the first substrate 1212 and is reflected back by the second substrate 1213. If the second substrate 1213 is also a light-transmitting material, the optical detection device needs to be arranged opposite to the second substrate 1213 to set a receiving device.

[0077] It should be noted that, in some embodiments, the first side of the second substrate 1213 is provided with a solution groove, and when the second side of the first substrate 1212 and the first side of the second substrate 1213 are connected, the solution groove and the second side of the first substrate 1212 form the cell solution cavity 1211.

[0078] It should be noted that, in some embodiments, the liquid inlet is arranged on the second side of the second substrate 1213; the solution groove comprises a liquid outlet groove, and the liquid outlet groove and the second side of the first substrate 1212 form the liquid outlet.

[0079] It should be noted that, in some embodiments, the length of the first electrode 122 relative to the flow direction of the cell suspension is a first preset length, and the length of the piezoelectric substrate 123 relative to the flow direction of the cell suspension is a second preset length.

[0080] In the method provided in the present application, the first electrode 122 must cover the piezoelectric material 123 relative to the flow direction of the cell suspension, so as to ensure that the piezoelectric material 123 can vibrate sufficiently. If the first preset length is equal to or slightly less than the second preset length, the scheme provided in the embodiments of the present application can also be realized, but the vibration effect of the piezoelectric material 123 on the cell solution cavity 16 will be affected.

[0081] Exemplarily, the length of the second electrode 124 relative to the flow direction of the cell suspension can be less than the second preset length, or greater than or equal to the second preset length, and the embodiments of the present application do not limit the length of the second electrode 124 relative to the flow direction of the cell suspension.

[0082] It should be noted that, in some embodiments, the second preset length is greater than the length of the cell solution cavity 1211 relative to the flow direction of the cell suspension; or, the second preset length is equal to the length of the cell solution cavity 1211 relative to the flow direction of the cell suspension.

[0083] It should be noted that in some embodiments, the shape of the cross section of the first electrode 122, the second electrode 124 and the piezoelectric substrate 123 relative to the direction of the cell suspension flow is the same as the shape of the cross section of the cell liquid cavity 1211 relative to the direction of the cell suspension flow.

[0084] The single-cell printer provided by the embodiment of the application controls the ejection device to vibrate the cell suspension in the ejection device, so that the cell droplet to be sorted is ejected, and controls the optical detection device to capture droplet image information of the cell droplet to be sorted. The control device can control the sorting device to sort the target droplet to the preset well plate when it is confirmed that the cell droplet is the target droplet according to the droplet image information. The single-cell printer provided by the application does not apply any external force to the cell during the cell sorting process, reduces the damage to the cell, and improves the accuracy of the experiment. At the same time, the single-cell printer provided by the application can sort the target droplet of the single cell meeting the requirements of the experiment without affecting the state of the cell, improve the sorting accuracy and efficiency of the single-cell sorting, reduce the equipment cost of the single-cell printer, and make the single-cell printer widely used in high-throughput sorting applications and be widely applied.

[0085] Please refer to FIG. 7, which is a step schematic flow chart of a cell printing method provided by an embodiment of the application, applied to the single-cell printer provided by any one of the embodiments corresponding to FIGS. 1 to 6.

[0086] As shown in FIG. 7, the cell printing method provided by the application includes steps S401 to S404.

[0087] S401. Control the ejection device of the single-cell printer to form the cell droplet.

[0088] Specifically, the ejection device of the single-cell printer is controlled to form the cell droplet, for example, by transmitting an acoustic pulse wave to the cell liquid cavity, the cell droplet to be sorted can be formed.

[0089] S402. Obtain the droplet image information of the cell droplet in the ejection device collected by the optical detection device of the single-cell printer.

[0090] Specifically, by obtaining the droplet image information collected by the optical detection device when the cell droplet is ejected from the ejection device, the state of the cell droplet can be determined.

[0091] S403. Confirm whether the cell droplet is a target droplet according to the droplet image information, the target droplet being the cell droplet containing a single cell meeting the preset cell condition.

[0092] Specifically, when it is confirmed according to the droplet image information that the cell droplet is a target droplet containing a single cell meeting the preset cell condition, such as the cell roundness of the cell droplet being greater than a preset roundness and the size of the cell of the cell droplet being greater than a preset size, sorting of the cell droplet can be quickly completed.

[0093] S404. If the cell droplet is the target droplet, the sorting device is controlled to sort the target droplet to a preset well plate, and printing of the target droplet is completed.

[0094] Specifically, after confirming that the cell droplet is the target droplet, the cell droplet is quickly sorted into a target groove corresponding to the preset well plate by the sorting device, which can reduce damage to the cell in the process of cell sorting and improve the accuracy of the experiment. Meanwhile, the single-cell printer provided can sort the target droplet of a single cell meeting the requirements of the experiment without affecting the state of the cell, improve the sorting precision and efficiency of single-cell sorting, reduce the equipment cost of the single-cell printer, and make the single-cell printer popular in high-throughput sorting applications and be widely applied and popularized.

[0095] Please refer to FIG. 8, which is a structural schematic diagram of the control device provided by the embodiment of the present application.

[0096] Referring to FIG. 8, the control device includes a processor, a memory and a network interface connected through a system bus, wherein the memory can include a non-volatile storage medium and an internal memory.

[0097] The non-volatile storage medium can store an operating system and a computer program. The computer program includes program instructions which, when executed, can cause the processor to execute the cell printing method provided in FIG. 7 and any corresponding embodiment thereof.

[0098] The processor is used to provide computing and control capabilities to support the operation of the entire computer device.

[0099] The internal memory provides an environment for the execution of the computer program in the non-volatile storage medium, which, when executed by the processor, can cause the processor to execute the cell printing method provided in FIG. 7 and any corresponding embodiment thereof.

[0100] The network interface is used for network communication, such as sending assigned tasks. Those skilled in the art can understand that the structure shown in FIG. 8 is only a block diagram of part of the structure related to the scheme of the present application, and does not constitute a limitation on the control device to which the scheme of the present application is applied. Specifically, the control device can include more or fewer components than those shown in the figure, or combine certain components, or have a different component arrangement.

[0101] It should be appreciated that the processor can be a central processing unit (CPU), the processor can also be other general-purpose processors, a digital signal processor (DSP), an application specific integrated circuit (ASIC), a field-programmable gate array (FPGA) or other programmable logic device, discrete gate or transistor logic device, discrete hardware component, etc. Among them, the general-purpose processor can be a microprocessor or the processor can also be any conventional processor, etc.

[0102] It should be noted that the specific working process of the cloud can be clearly understood by those skilled in the art, and for the convenience and brevity of description, the specific working process of the cloud can be referred to the corresponding process in the cell printing method and any one of the corresponding embodiments provided in FIG. 6, which will not be repeated here.

[0103] The embodiment of the application also provides a computer readable storage medium, which stores a computer program, and the computer program includes program instructions. The processor executes the program instructions to realize the steps of the cell printing method provided in the above embodiment. For example, the computer program is loaded by the processor, and the following steps can be executed:

[0104] The ejection device of the single-cell printer is controlled to form the cell droplet;

[0105] The droplet image information of the cell droplet in the ejection device collected by the optical detection device of the single-cell printer is acquired;

[0106] According to the droplet image information, it is determined whether the cell droplet is a target droplet, and the target droplet is the cell droplet containing a single cell meeting a preset cell condition;

[0107] If the cell droplet is the target droplet, the sorting device is controlled to sort the target droplet to a preset well plate, and the printing of the target droplet is completed.

[0108] The specific implementation of each operation can be referred to the above embodiment, which will not be repeated here.

[0109] The computer readable storage medium can be an internal storage unit of the computer device, such as a hard disk or a memory of the computer device. The computer readable storage medium can also be an external storage device of the computer device, such as a plug-in hard disk, a smart media card (SMC), a secure digital (SD) card, a flash card, and the like.

[0110] The computer program stored in the computer readable storage medium can execute any of the cell printing methods provided by the embodiments of the present application, thus achieving the beneficial effects of any of the cell printing methods provided by the embodiments of the present application. Details are described in the foregoing embodiments, which will not be repeated here.

[0111] The above sequence numbers of the embodiments of the present application are only for description, and do not represent the advantages and disadvantages of the embodiments. The above is only a specific implementation of the present application, but the protection scope of the present application is not limited to this. Any person skilled in the art can easily think of various equivalent modifications or replacements within the technical range disclosed by the present application, and these modifications or replacements should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A single-cell printer, wherein, The single-cell printer includes: A sample introduction device, wherein the sample introduction device stores a cell suspension to be sorted; The spraying device has its inlet connected to the outlet of the sample injection device. The spraying device is used to control the vibration of the cell suspension to form cell droplets to be sorted, and to spray the cell droplets out through the outlet of the spraying device. An optical detection device is provided, which is positioned toward the jetting device, and is used to acquire droplet image information of the cell droplets to be sorted in the jetting device; A sorting device is provided at the liquid outlet; A control device, electrically connected to the jetting device, the optical detection device, and the sorting device, is used to control the jetting device to form the cell droplets, acquire the droplet image information detected by the optical detection device, and confirm whether the cell droplet is a target droplet based on the droplet image information, so as to sort the target droplet to a preset well plate; wherein, the target droplet is the cell droplet containing a preset number of cells that meet preset cell conditions.

2. The single-cell printer according to claim 1, wherein, The preset cell conditions include: the roundness of at least one cell in the cell droplet is within a preset roundness range and the size of the cells in the cell droplet is within a preset size range.

3. The single-cell printer according to claim 1, wherein, The sorting device includes: A pipetting device, wherein the pipetting device is located at the outlet; Waste liquid collection area, wherein the waste liquid collection area is located at the outlet; The control device is electrically connected to the pipetting device. The control device is used to obtain the number of cells and cell parameter information based on the droplet image information, so as to control the pipetting device to move the cell droplets whose number of cells is not the preset number and whose cell parameter information does not meet the preset cell conditions to the waste liquid collection area.

4. The single-cell printer according to claim 3, wherein, The pipetting device includes a first air pump, which is located at the outlet relative to the waste liquid collection area. The control device is electrically connected to the first air pump and is used to blow gas from the first air pump onto the cell droplets ejected by the spraying device when the first air pump is turned on, so that the cell droplets move to the waste liquid collection area; when the first air pump is turned off, the cell droplets are sprayed onto the preset orifice plate.

5. The single-cell printer according to claim 1, wherein, The sorting device further includes: A motion platform is provided, wherein the preset orifice plate is disposed on the motion platform and the motion platform is electrically connected to the control device. When the cell parameter information meets the preset cell conditions, the control device determines the target orifice position for the target droplet injection among multiple orifices of the preset orifice plate, and controls the motion platform to move the preset orifice plate according to the coordinate information of the target orifice position, so that the liquid outlet of the injection device is aligned with the target orifice position.

6. The single-cell printer according to claim 1, wherein, The sample introduction device includes: The sample introduction module includes a first pipeline and a second pipeline. The inlet of the first pipeline is used to input cell suspension, the outlet of the first pipeline is connected to the liquid inlet of the spraying device, and the outlet of the second pipeline is connected to one side of the first pipeline. A mixing module is connected to the inlet of the second pipeline and is also electrically connected to the control device. The control device is used to determine the number of cells in the cell suspension in the jetting device based on the droplet image information, and to control the mixing module to perform reciprocating gas suction at the outlet of the second pipeline based on the number of cells, so as to ensure that the cell suspension flowing into the jetting device is in a mixed state.

7. The single-cell printer according to claim 6, wherein, The mixing module includes: The second air pump is connected to the inlet of the second pipeline, and the control device is electrically connected to the second air pump. The control device is used to determine the control parameters of the second air pump based on the number of cells in the cell suspension in the spraying device, so as to control the second air pump to perform reciprocating gas suction at the outlet of the second pipeline according to the control parameters, so as to ensure that the cell suspension flowing into the spraying device is in the mixed state.

8. The single-cell printer according to claim 1, wherein, The spraying device includes: A substrate, the substrate including a cell fluid cavity for holding a cell suspension to be sorted, the cell fluid cavity including an inlet and an outlet, the inlet being connected to the sample introduction device; the optical detection device facing the substrate and opposite to the cell fluid cavity; The first electrode is disposed on the substrate and located outside the cell fluid cavity; A piezoelectric substrate, wherein the piezoelectric substrate is made of a piezoelectric material and is disposed on the first electrode; The second electrode is disposed on the piezoelectric substrate, and the polarity of the second electrode is opposite to that of the first electrode; The second electrode and the first electrode are electrically connected to the control device, which controls the first electrode and the second electrode to drive the piezoelectric substrate to vibrate so as to propagate acoustic pulse waves in the cell suspension in the cell fluid cavity, thereby driving the cell suspension to vibrate and form the cell droplets, and causing the cell droplets to be ejected through the outlet.

9. The single-cell printer according to claim 8, wherein, The length of the first electrode relative to the direction of cell fluid flow is a first preset length, and the length of the piezoelectric material relative to the direction of cell fluid flow is a second preset length.

10. The single-cell printer according to claim 9, wherein, The first preset length is greater than the second preset length.

11. The single-cell printer according to claim 8, wherein, The cross-sectional shapes of the first electrode, the second electrode, and the piezoelectric substrate relative to the flow direction of the cell suspension are the same as the cross-sectional shapes of the cell fluid cavity relative to the flow direction of the cell suspension.

12. The single-cell printer according to claim 10, wherein, The second preset length is greater than the length of the cell fluid cavity relative to the flow direction of the cell suspension; or, the second preset length is equal to the length of the cell fluid cavity relative to the flow direction of the cell suspension.

13. The single-cell printer according to claim 8, wherein, The substrate includes a first substrate and a second substrate, the first electrode is disposed on a first side of the first substrate, and the second side of the first substrate is connected to the first side of the second substrate to form the cell fluid cavity.

14. The single-cell printer according to claim 13, wherein, The light transmittance of the first substrate is greater than the preset light transmittance, and the optical detection device faces the first substrate and is opposite to the cell fluid cavity.

15. The single-cell printer according to claim 13, wherein, The first side of the second substrate is provided with a solution tank. When the second side of the first substrate is connected to the first side of the second substrate, the solution tank and the second side of the first substrate constitute the cell fluid cavity.

16. The single-cell printer according to claim 15, wherein, The liquid inlet is located on the second side of the second substrate; The solution tank includes an outlet tank, and the outlet tank and the second side of the first substrate form an outlet.

17. The single-cell printer according to claim 13, wherein, The first substrate is a light-transmitting material, and the second substrate is silicon; the light from the optical detection device enters the cell fluid cavity through the first substrate and is reflected back by the second substrate.

18. The single-cell printer according to claim 13, wherein, Both the first substrate and the second substrate are light-transmitting materials, and the optical detection device needs to be equipped with a receiving device relative to the second substrate.

19. A cell printing method, wherein, The method is applied to the single-cell printer according to any one of claims 1-18; the method includes: The jetting device of the single-cell printer is controlled to form the cell droplets; The droplet image information of the cell droplets in the jetting device is acquired by the optical detection device of the single-cell printer; The cell droplet is confirmed to be a target droplet based on the droplet image information. The target droplet is the cell droplet containing a preset number of cells that meet the preset cell conditions. If the cell droplet is the target droplet, the sorting device is controlled to sort the target droplet into a preset well plate, thus completing the printing of the target droplet.

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