WO2023029730A1 - 一种单细胞分选与鉴定方法及装置 - Google Patents

一种单细胞分选与鉴定方法及装置 Download PDF

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WO2023029730A1
WO2023029730A1 PCT/CN2022/103658 CN2022103658W WO2023029730A1 WO 2023029730 A1 WO2023029730 A1 WO 2023029730A1 CN 2022103658 W CN2022103658 W CN 2022103658W WO 2023029730 A1 WO2023029730 A1 WO 2023029730A1
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cell
nozzle
single cell
cells
sorting
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PCT/CN2022/103658
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English (en)
French (fr)
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王梦琪
杨苗苗
江巍
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上海傲睿科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
    • G01N15/10—Investigating individual particles

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  • the invention belongs to the fields of microfluidics, cell line development, monoclonal antibody screening, life science instruments and the like, and relates to a single cell sorting and identification method and device.
  • Single cell isolation mainly includes microneedle aspiration, limiting dilution method, microwell array and microfluidic-based sorting methods.
  • the dilution ratio of the limited dilution method needs to be optimized manually, so it has the disadvantages of heavy workload and poor stability; the cost of flow cytometry equipment is high, and it uses electrical signals to sort cells, which will cause great damage to cells ; Microwell array single cell capture relies on Poisson distribution, so its efficiency is very low; other microfluidic-based single cell sorting methods also have low throughput, high cost of consumables, low single cell rate, and low single cell activity And other issues.
  • the object of the present invention is to provide a single cell sorting and identification method and device for solving the problems of low throughput, high cost of consumables, low single cell rate, low single-cell viability issues.
  • the present invention provides a single cell sorting and identification method, comprising the following steps:
  • Liquid supply step applying a solution containing cells to multiple nozzles of the thermal bubble printing chip
  • the first nozzle photographing and identification step using an optical module to photograph the side of the thermal bubble printing chip with the nozzle to obtain the nozzle image before single-cell printing, and obtain the nozzle image with the nozzle image before single-cell printing the number of the single-cell nozzle;
  • Single-cell printing and photographing steps based on the obtained nozzle numbers with single cells, the single cells in the corresponding nozzles are exported to the cell receiving device in the form of printing according to the preset order, wherein, each time a single cell in a nozzle is printed, that is Using the optical module to take pictures of the single cells printed into the cell receiving device to obtain the zero-day image of the printed single cells;
  • the second nozzle photographing step using the optical module to photograph the side of the thermal bubble printing chip with the nozzle, so as to obtain the nozzle image after single-cell printing.
  • the liquid supply step includes making the nozzle perform empty spraying to refresh the solution at the front end of the nozzle.
  • the step of photographing and identifying nozzles for the first time includes:
  • Nozzle identification step searching and selecting nozzles according to the image characteristics of the nozzles
  • Single cell identification step perform at least one single cell identification on the selected nozzle.
  • the single cell identification step includes:
  • the first screening step Use the preset image algorithm to identify the cells in the nozzle for the first time. If it is qualified, proceed to the next second screening step. If it is unqualified, the corresponding nozzle will be eliminated without further identification;
  • the second screening step Use the pre-trained neural network to identify the cells in the nozzle for the second time to exclude impurities, cell debris, and non-cells. If it is judged that the nozzle is a single cell, calculate the number of the corresponding nozzle , if it is judged that there are non-single cells in the nozzle, the corresponding nozzle is eliminated.
  • the identification result shows that there is no single cell in all nozzles, return to the liquid supply step.
  • the optical module is focused on the expected landing point of the single cell to be printed.
  • the culture medium is applied in the cavity of the cell receiving device for receiving the printed single cell.
  • the second nozzle photographing step it is judged whether the number of single cells in the cell receiving device satisfies the requirements, if so, then end the single cell sorting, and if not, return to the liquid supply step.
  • a nozzle soaking treatment step is also included.
  • the cell receiving device comprises a well plate.
  • a cell state monitoring step includes: after printing a preset number of single cells in the cell receiving device, using the optical module to scan and take pictures of the cell receiving device to monitor cell status.
  • a secretion detection step includes: after printing a preset number of single cells in the cell receiving device, using the optical module to photograph the secretion of cells in the cell receiving device Fluorescence images of objects to screen target cells.
  • the present invention also provides a single cell sorting and identification device, including thermal bubble printing chips, optical modules and control components, characterized in that: the single cell sorting and identification device is used to perform any one of the above Single cell sorting and identification methods.
  • the single cell sorting and identification method and device of the present invention are based on the microfluidic characteristics of the high-throughput thermal bubble printing chip, and use the method of image recognition to export single cells efficiently and gently, so that it can quickly and Single cells with high single cell rate and high activity can be obtained in batches.
  • the method identifies single cells based on their morphological characteristics, and the resulting single cells are characterized by high viability. And this method can provide the zero-day appearance of cells, which is an important criterion for single cells.
  • the single cell sorting and identifying device of the present invention can monitor the growth status of cells after single cell sorting, and screen target cells according to fluorescent markers, providing a comprehensive solution for cell experiments.
  • Fig. 1 shows a flowchart of the method for sorting and identifying single cells of the present invention.
  • Fig. 2 shows an image recognition logic diagram used in the single cell sorting and identification method of the present invention.
  • Fig. 3 shows a flow chart of a single cell printing cycle adopted by the single cell sorting and identification method of the present invention.
  • FIG. 4 is a schematic diagram of the assembly of thermal foam printing chips in the present invention on an automatic instrument including control components.
  • FIG. 5 is a schematic diagram of the present invention using an optical module to take pictures of the side of the thermal foam printing chip with nozzles.
  • Fig. 6 shows a schematic diagram of the nozzle image obtained by photographing a single cell before printing.
  • FIG. 7 shows a schematic diagram of printing a solution containing single cells into an orifice plate for the thermal bubble printing chip and focusing the optical module under the orifice plate to the expected landing point of the printed single cell.
  • FIG. 8 is a schematic diagram of an image of a droplet containing a single cell obtained by taking a photo of the printed single cell in real time.
  • FIG. 9 is a schematic diagram of a monitoring interface used in the detection of cell growth status.
  • Figure 10 shows a schematic diagram of a day 0 image of cells in a well.
  • Figure 11 shows a schematic representation of day one images of cells in a well.
  • Figure 12 shows a schematic diagram of day 3 images of cells in one well.
  • This embodiment provides a single cell sorting and identification method, please refer to Figure 1, which shows a flow chart of the method, including the liquid supply step, the first nozzle photographing and identification step, the single cell printing and photographing step, and the second Secondary nozzle photographing steps.
  • the liquid supply step is to apply the cell-containing solution to multiple nozzles of the thermal bubble printing chip.
  • the thermal bubble printing nozzle uses the instantaneous high temperature of the heating film to vaporize the liquid above, and generates bubbles to push the liquid to flow and eject from the nozzle, and then the follow-up liquid is replenished under the action of capillary force, thus providing a continuous flow of liquid. power.
  • Thermal printing nozzles are controlled by the underlying circuitry.
  • the liquid supplying step includes emptying the nozzle to refresh the solution at the front end of the nozzle.
  • a flow channel communicating with the nozzle is provided in the thermal bubble printing chip, and a solution containing cells can be supplied to the nozzle through the flow channel.
  • the layout of the flow channel and the size of the nozzle can be adjusted according to the size of the cells to be sorted.
  • an appropriate volume of cells with an appropriate concentration is added to the thermal bubble printing chip, and a large number of single cells can be naturally formed in the nozzle at one time by utilizing the high-throughput and single-cell-level microfluidic design of the thermal bubble printing chip.
  • a nozzle infiltration treatment step is also included to reduce the impact on cell activity.
  • the soaking treatment may include applying a surfactant in the nozzle to wet the nozzle, and applying a buffer in the nozzle to clean the nozzle by printing, and the buffer may be water or culture medium.
  • the first nozzle photographing and identification step is to use an optical module to photograph the side of the thermal bubble printing chip with the nozzle to obtain the nozzle image before single-cell printing, and based on the The nozzle image is obtained with the numbering of the nozzles of the single cells.
  • the optical module includes an objective lens with high resolution, low magnification, and long working distance, a barrel mirror, a coaxial collimated light source, a multi-channel laser light source, a multi-channel filter, a large target surface camera, etc., which are connected to The relationship and parameters can be adjusted as needed to achieve large field of view and high resolution imaging performance.
  • the obtaining the number of the nozzle with the single cell based on the nozzle image of the single cell before printing includes using an image algorithm to preferentially select the single cell in the image, and calculating the nozzle number of the print head to ensure that the single cell is correctly exported for printing head.
  • the image algorithm here can use a combination of traditional image recognition and deep learning to adopt more effective solutions for different recognition targets.
  • Figure 2 shows an image recognition logic diagram used in this embodiment, which sequentially performs nozzle recognition and single cell recognition based on the nozzle image before printing, wherein the nozzle recognition step performs nozzle search according to the image characteristics of the nozzle and selection; the single cell identification step performs at least one single cell identification on the selected nozzle.
  • the printing nozzles will be arranged in a special way, so the nozzle numbers of the print head will form an incompletely regular distribution.
  • the vibration caused by the actual automatic movement, and the error of optical imaging and other comprehensive reasons will lead to micron-level errors, which may make the calculation of the nozzle number where the single cell is located wrong.
  • the nozzle identification step the nozzle is first searched according to the image characteristics of the nozzle, and the real nozzle is repaired and selected according to the morphological characteristics. The obtained true calculation value is used to calculate the nozzle number, which can drive the printing single cell correctly.
  • conventional algorithms or other suitable algorithms may be employed to identify nozzles.
  • the traditional algorithms include screening based on morphological features, filtering algorithms, and physical position screening combined with print head layout.
  • the physical position combined with the print head layout can remove non-nozzles, and the morphological screening and filtering algorithm can remove abnormal nozzles and nozzles that do not contain cells.
  • a single cell belongs to a living body, there are individual differences in the appearance of the living body, and the cell is a transparent body, and the cell solution is also a transparent solution. Therefore, it is very difficult to identify single cells.
  • a combination of traditional algorithms and deep learning can be used for single cell recognition.
  • the single cell identification step includes a first screening step and a second screening step.
  • the first screening step uses a preset image algorithm to identify the cells in the nozzle for the first time. If it is qualified, it will go to the next second screening step. If it is unqualified, the corresponding nozzle will be eliminated without further identification.
  • the traditional algorithm is preferably used in the first screening step to quickly and roughly judge whether there are single cells in the nozzle.
  • the second screening step uses a pre-trained neural network to identify the cells in the nozzle for the second time to exclude impurities, cell debris, and non-cellular conditions. If it is judged that the nozzle is a single cell, the corresponding nozzle is calculated.
  • the algorithm used in the second screening can select single cells with a moderate size and a good active state according to the state of the cells. Under this screening mechanism, the single cell rate can reach 95%, and the single cell viability can reach 90%.
  • the recognition result is that there is no single cell in all the nozzles, return to the liquid supply step.
  • the single-cell printing and photographing steps are based on the obtained numbers of the nozzles with single cells, and sequentially export the single cells in the corresponding nozzles to the cell receiving device in a preset order, wherein each print in a nozzle
  • the single cells printed into the cell receiving device are photographed by using the optical module to obtain the zero-day image of the printed single cells.
  • the optical module is focused on the expected landing point of the single cell to be printed, at this time, the Accurately capture the single cell just printed, which is the zero-day image of the cell.
  • the zero-day image of cells is an important criterion for single cells. The results of cell printing can be completely verified by the zero-day images of cells when needed, and this image is crucial for single-cell-related research and development.
  • the cell receiving device includes a well plate, which includes a plurality of wells arranged according to preset rules, and each well is used to receive a single cell.
  • the bottom of the well is transparent, and the lens of the optical module is located under the well plate, and focuses on the expected landing point of the single cell to be printed through the bottom of the well receiving the single cell.
  • a culture medium is applied in the cavity of the cell receiving device to receive the printed single cell, so as to improve the activity of the printed single cell .
  • the second nozzle photographing step is to use the optical module to photograph the side of the thermal bubble printing chip with the nozzle, so as to obtain a single-cell printed nozzle image. It should be pointed out that the second nozzle photography is performed after the single cells identified after the first liquid supply are printed, and the printing status of the single cells can be confirmed by comparing with the image obtained by the first nozzle photography.
  • the single cell sorting is ended, and if not, it is returned to the liquid supply step .
  • the single cell sorting and identification method may further include a cell state monitoring step, the cell state monitoring step includes: after printing a preset number of single cells in the cell receiving device, using the optical module The cell receiving device is scanned and photographed to monitor the cell state. For example, the entire well plate can be scanned and photographed at any time to monitor cell growth.
  • the diameter of each hole in the orifice plate is relatively large and the photographic field of view is relatively small, for one hole, multiple partial images of the hole can be captured by scanning and combined into one overall image of the hole.
  • the single cell sorting and identification method may further include a secretion detection step, and the secretion detection step includes: after printing a preset number of single cells in the cell receiving device, using the optical module Taking fluorescent images of cell secretions in the cell receiving device to screen target cells.
  • the optimal automatic control strategy is adopted to efficiently export the single cells in the thermal foam print head. Using this optimal control strategy, the printing time of a single cell in a single 96-well plate can be achieved in less than 5 minutes.
  • FIG. 3 shows a single-cell printing beat flow chart adopted in this embodiment.
  • the air spraying is carried out in sequence, and the thermal bubble printing chip is photographed to check the single cell for the first time, and it is judged whether there is a single cell. If there is no single cell, return to the empty spray beat. Take photos of the cells to check the single cells for the second time when needed in the future, and then take photos of the thermal bubble printing chip again, and finally judge whether the required number of single cells is satisfied, if not, return to the empty spraying step, if satisfied, then end .
  • the nozzle of the thermal bubble printing chip sprays an appropriate amount in the waste liquid collection area, so that the cell solution at the front end of the nozzle is renewed.
  • a high-resolution large-field optical module is selected to take pictures, and the results are calculated and fed back through image algorithms, and the images are stored.
  • focus the above optical module on the bottom of the well plate to capture the image of the cells falling into the bottom of the well plate, and use the image algorithm to identify and confirm the single cell of the result. Then use a liquid-adding probe to add an appropriate amount of medium into the wells of the single-cell plate to ensure high cell activity.
  • the single cell sorting and identification method of this example utilizes the high-throughput microfluidic design of the thermal bubble printing chip, which can form several single cells at a time, and can export cells quickly and gently.
  • multiple optical photography low magnification, high resolution, and long working distance optical modules are used to achieve large field of view and high resolution imaging, achieving high-efficiency single-cell imaging and imaging of single-cell printing results.
  • image algorithm superimposed by multiple methods to identify the single cells in the thermal bubble print head, and confirm the printing results of the single cells the double identification of the exported single cells is completed, and the result of ultra-high single cell rate is obtained.
  • the optimal automatic control strategy the single cells in the thermal foam print head are exported with high efficiency, ensuring high cell activity.
  • This embodiment provides a single cell sorting and identification device, including thermal bubble printing chips, optical modules and control components, the single cell sorting and identification device can be used to perform the single cell sorting described in the first embodiment And the authentication method, wherein, the control component is used to execute the calculation process of the above-mentioned image algorithm calculation and feedback results, image storage and so on.
  • FIG. 4 is a schematic diagram showing that the thermal foam printing chip 1 is assembled on the automation instrument 2 including the control component, wherein the automation instrument 2 includes a base 201, the control component 202 and A liquid reservoir 203 for storing cell solution, the thermal bubble printing chip 1 is assembled on the base 201 and electrically connected to the control assembly 202 .
  • the thermal bubble printing chip 1 is assembled on the base 201 and electrically connected to the control assembly 202 .
  • FIG. 5 shows a schematic diagram of using the optical module 3 to take pictures of the side of the thermal foam printing chip 1 having the nozzles 101 .
  • FIG. 6 shows a schematic diagram of a nozzle image of a single cell before printing obtained by photographing, which can present the distribution of cells 4 in multiple nozzles 101 .
  • FIG. 7 shows that the thermal bubble printing chip 1 exports the solution 5 containing single cells to the orifice plate 6 by printing, and the optical module 3 focuses on the printed single cell under the orifice plate 6.
  • FIG. 7 shows that the thermal bubble printing chip 1 exports the solution 5 containing single cells to the orifice plate 6 by printing, and the optical module 3 focuses on the printed single cell under the orifice plate 6.
  • FIG. 8 shows a schematic diagram of an image of a droplet 7 containing a single cell 8 obtained by taking a photo of the printed single cell in real time.
  • Fig. 9 shows a schematic diagram of a monitoring interface used in the detection of cell growth status, wherein each circle represents a well in the well plate, and its position can be numbered by row (for example, as shown in the figure).
  • A, B, C, D, E, F, G, H) and column numbers eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 as shown in the figure.
  • different color fillings in the circle represent different conditions of cells in the wells, for example, gray (horizontal stripes) represents no cells in the wells, orange (vertical stripes) represents abnormal cells in the wells, green (grid pattern) represents normal cells in the well.
  • FIG. 10 , FIG. 11 and FIG. 12 respectively show schematic diagrams of the zero-day image, the first-day image and the third-day image of cells presented after selecting one of the wells through the interface shown in FIG. 9 .
  • the single cell sorting and identification device of this embodiment matches the optical module with large field of view and high resolution imaging performance with the high-throughput nozzle to achieve high-efficiency and high-activity single-cell printing, and can perform multiple The second identification obtained the result of ultra-high single cell rate.
  • the optical module takes into account the multiple tasks of single cell identification, single cell result identification, and cell growth status detection, and can monitor the entire process of single cell sorting, growth, and characteristic screening.
  • the single cell sorting and identification device in Example 2 is used to sort Chinese hamster ovary (CHO) cells.
  • the thermal bubble printing head on the automatic instrument including the control component, perform nozzle pretreatment operation, add the prepared cell suspension into the thermal bubble printing chip, and use the automatic control scheme to perform optical module photography, Single cell identification, single cell printing, until the single cell printing of the entire 96-well plate is completed. It takes less than 5 minutes to print a single well plate, the rate of single cells in the 96-well plate is greater than 90%, and the activity of single cells can reach 80% after culture.
  • relevant reagents can be added, and fluorescence imaging can be used for target detection.
  • the single cell sorting and identification method and device of the present invention are based on the microfluidic characteristics of the high-throughput thermal bubble printing chip, and use image recognition to export single cells efficiently and gently, thereby enabling fast, Large quantities of single cells with high single cell rate and high activity can be obtained.
  • the method identifies single cells based on their morphological characteristics, and the resulting single cells are characterized by high viability. And this method can provide the zero-day appearance of cells, which is an important criterion for single cells.
  • the single cell sorting and identifying device of the present invention can monitor the growth status of cells after single cell sorting, and screen target cells according to fluorescent markers, providing a comprehensive solution for cell experiments. Therefore, the present invention effectively overcomes various shortcomings in the prior art and has high industrial application value.

Abstract

提供一种单细胞（8）分选与鉴定方法及装置，单细胞（8）分选与鉴定方法包括供液步骤、第一次喷嘴（101）拍照与识别步骤、单细胞（8）打印与拍照步骤及第二次喷嘴（101）拍照步骤。单细胞（8）分选与鉴定方法及装置基于高通量热泡打印芯片（1）的微流控特性，采用图像识别的方法将单细胞（8）高效率、轻柔导出，可以快速、大批量得到高单细胞率、高活性的单细胞（8）。单细胞（8）分选与鉴定方法基于细胞形态学特征识别单细胞（8），得到的单细胞（8）具备高活性特点；且可以提供细胞第零天样貌，是单细胞（8）的重要判据。单细胞（8）分选与鉴定装置在完成单细胞（8）分选后，可监测细胞成长状态，并根据荧光标记筛选目标细胞，为细胞实验提供了全面的解决方案。

Description

一种单细胞分选与鉴定方法及装置 技术领域
本发明属于微流控、细胞系开发、单克隆抗体筛选、生命科学仪器等领域，涉及一种单细胞分选与鉴定方法及装置。
背景技术
细胞是生命活动的基本单位，基于单细胞水平的研究能够在更深层次上揭示生命活动的发展规律，在单克隆抗体筛选、细胞系培养等领域具有广泛的应用。目前，单细胞分离主要有微针吸取法、有限稀释法、微孔阵列和基于微流控的分选方法。其中，有限稀释法的稀释倍率需要人工单独优化，因此存在工作量大、稳定性差的缺点；流式细胞仪仪器成本造价高，其使用电信号对细胞进行分选，会对细胞造成很大损伤；微孔阵列法单细胞捕获依赖泊松分布，因此其效率很低；其他基于微流控的单细胞分选方法也都存在低通量、高耗材成本、低单细胞率、低单细胞活性等问题。
发明内容
鉴于以上所述现有技术的缺点，本发明的目的在于提供一种单细胞分选与鉴定方法及装置，用于解决现有单细胞分选方法存在低通量、高耗材成本、低单细胞率、低单细胞活性的问题。
为实现上述目的及其他相关目的，本发明提供一种单细胞分选与鉴定方法，包括以下步骤：
供液步骤：将包含细胞的溶液施加于热泡打印芯片的多个喷嘴内；
第一次喷嘴拍照与识别步骤：采用光学模组对所述热泡打印芯片具有所述喷嘴的一面进行拍照，以得到单细胞打印前的喷嘴图像，并基于单细胞打印前的喷嘴图像获得具有单细胞的喷嘴的编号；
单细胞打印与拍照步骤：基于获得的具有单细胞的喷嘴的编号按照预设顺序依次将对应喷嘴内的单细胞以打印方式导出至细胞接收装置，其中，每打印一个喷嘴内的单细胞，即采用所述光学模组对打印至所述细胞接收装置中的单细胞进行拍照以获得被打印单细胞的第零天图像；
第二次喷嘴拍照步骤：采用所述光学模组对所述热泡打印芯片具有所述喷嘴的一面进行拍照，以得到单细胞打印后的喷嘴图像。
可选地，所述供液步骤包括使所述喷嘴进行空喷以使所述喷嘴前端的溶液进行更新。
可选地，所述第一次喷嘴拍照与识别步骤包括：
喷嘴识别步骤：根据喷嘴的图像特性进行喷嘴寻找与选择；
单细胞识别步骤：对被选择的喷嘴进行至少一次单细胞识别。
可选地，所述单细胞识别步骤包括：
第一次筛选步骤：采用预设图像算法对喷嘴内的细胞情况进行第一次识别，若合格，则进行下一步第二次筛选步骤，若不合格，则剔除对应喷嘴，不进一步识别；
第二次筛选步骤：采用预先训练好的神经网络对喷嘴内的细胞情况进行第二次识别以排除杂质、细胞碎片、非细胞的情况，若判断喷嘴内为单细胞，则计算对应喷嘴的编号，若判断喷嘴内为非单细胞，则剔除对应喷嘴。
可选地，在所述第一次喷嘴拍照与识别步骤中，若识别结果为所有喷嘴内均无单细胞，则返回所述供液步骤。
可选地，在所述单细胞打印与拍照步骤中，在将单细胞导出至所述细胞接收装置中之前，将所述光学模组对焦至即将被打印的单细胞的预计落点。
可选地，在所述单细胞打印与拍照步骤中，每打印一个喷嘴内的单细胞，即在所述细胞接收装置接收被打印单细胞的腔体内施加培养基。
可选地，在所述第二次喷嘴拍照步骤之后，判断所述细胞接收装置中的单细胞数量是否满足要求，若满足，则结束单细胞分选，若不满足，则返回所述供液步骤。
可选地，在所述供液步骤之前，还包括喷嘴浸润处理步骤。
可选地，所述细胞接收装置包括孔板。
可选地，还包括细胞状态监测步骤，所述细胞状态监测步骤包括：在所述细胞接收装置内打印预设数量的单细胞之后，采用所述光学模组对所述细胞接收装置进行扫描拍照以监测细胞状态。
可选地，还包括分泌物检测步骤，所述分泌物检测步骤包括：在所述细胞接收装置内打印预设数量的单细胞之后，采用所述光学模组拍摄所述细胞接收装置内细胞分泌物的荧光图像以筛选目标细胞。
本发明还提供一种单细胞分选与鉴定装置，包括热泡打印芯片、光学模组及控制组件，其特征在于：所述单细胞分选与鉴定装置用于执行如上任意一项所述的单细胞分选与鉴定方法。
如上所述，本发明的单细胞分选与鉴定方法及装置基于高通量热泡打印芯片的微流控特性，采用图像识别的方法将单细胞高效率、轻柔导出，由此可以快速、大批量得到高单细胞 率、高活性的单细胞。该方法基于细胞形态学特征识别单细胞，由此得到的单细胞具备高活性特点。并且该方法可以提供细胞第零天样貌，是单细胞的重要判据。此外本发明的单细胞分选与鉴定装置在完成单细胞分选后，可监测细胞成长状态，并根据荧光标记筛选目标细胞，为细胞实验提供了全面的解决方案。
附图说明
图1显示为本发明的单细胞分选与鉴定方法的流程图。
图2显示为本发明的单细胞分选与鉴定方法采用的一种图像识别逻辑图。
图3显示为本发明的单细胞分选与鉴定方法采用的一种单细胞打印节拍流程图。
图4显示为本发明中热泡打印芯片组装于包含控制组件的自动化仪器上的示意图。
图5显示为本发明采用光学模组对热泡打印芯片具有喷嘴的一面进行拍照的示意图。
图6显示为拍照得到的单细胞打印前的喷嘴图像的示意图。
图7显示为热泡打印芯片将包含单细胞的溶液采用打印方式导出至孔板中及光学模组在孔板下方对焦至被打印单细胞预计落点的示意图。
图8显示为对被打印单细胞即时拍照所获得的含有单细胞的液滴的图像示意图。
图9显示为在细胞成长状态检测中所使用的一种监测界面示意图。
图10显示为一个孔内细胞的第零天图像示意图。
图11显示为一个孔内细胞的第一天图像示意图。
图12显示为一个孔内细胞的第三天图像示意图。
元件标号说明
1                      热泡打印芯片
101                    喷嘴
2                      自动化仪器
201                    基座
202                    控制组件
203                    储液槽
3                      光学模组
4                      细胞
5                      溶液
6                      孔板
7                      液滴
8                      单细胞
具体实施方式
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。
请参阅图1至图12。需要说明的是，本实施例中所提供的图示仅以示意方式说明本发明的基本构想，遂图式中仅显示与本发明中有关的组件而非按照实际实施时的组件数目、形状及尺寸绘制，其实际实施时各组件的型态、数量及比例可为一种随意的改变，且其组件布局型态也可能更为复杂。
实施例一
本实施例中提供一种单细胞分选与鉴定方法，请参阅图1，显示为该方法的流程图，包括供液步骤、第一次喷嘴拍照与识别步骤、单细胞打印与拍照步骤及第二次喷嘴拍照步骤。
具体的，所述供液步骤是将包含细胞的溶液施加于热泡打印芯片的多个喷嘴内。其中，热泡打印喷嘴是利用加热膜的瞬间高温使上方的液体气化，产生气泡推动液体流动并从喷嘴喷出，接着后续液体在毛细力的作用下进行补充，从而为液体的持续流动提供动力。热泡打印喷嘴由底层电路控制。
作为示例，所述供液步骤包括使所述喷嘴进行空喷以使所述喷嘴前端的溶液进行更新。
作为示例，所述热泡打印芯片中设有与所述喷嘴连通的流道，包含细胞的溶液可经由所述流道供应至所述喷嘴中。所述流道的布局及所述喷嘴的尺寸可以根据所要分选的细胞尺寸进行调整。本发明在所述热泡打印芯片内加入适量体积的适量浓度的细胞，利用热泡打印芯片的高通量、单细胞级别的微流控设计，可以在喷嘴内一次性自然形成大量单细胞。
作为示例，在所述供液步骤之前，还包括喷嘴浸润处理步骤以减少对细胞活性的影响。所述浸润处理可以包括施加表面活性剂于所述喷嘴内以浸润所述喷嘴，并施加缓冲液于所述喷嘴内，采用打印方式清洗所述喷嘴，所述缓冲液可以是水或培养基。
具体的，所述第一次喷嘴拍照与识别步骤是采用光学模组对所述热泡打印芯片具有所述喷嘴的一面进行拍照，以得到单细胞打印前的喷嘴图像，并基于单细胞打印前的喷嘴图像获得具有单细胞的喷嘴的编号。
作为示例，所述光学模组包含高解析度、低放大倍率、长工作距离的物镜、桶镜、同轴 准直光源、多通道激光光源、多通道滤波片、大靶面相机等，其连接关系及参数可以根据需要进行调整，以实现大视野、高分辨率的成像性能。
作为示例，所述基于单细胞打印前的喷嘴图像获得具有单细胞的喷嘴的编号包括使用图像算法对图像内的单细胞进行择优选择，并且计算打印头喷嘴编号，以确保将单细胞正确导出打印头。此处的图像算法可使用传统图像识别与深度学习相结合的方式，在不同的识别目标时，采用更有效的解决方法。
作为示例，请参阅图2，显示为本实施例采用的一种图像识别逻辑图，其基于打印前喷嘴图像依次进行喷嘴识别与单细胞识别，其中，喷嘴识别步骤根据喷嘴的图像特性进行喷嘴寻找与选择；单细胞识别步骤对被选择的喷嘴进行至少一次单细胞识别。
需要指出的是，由于热泡打印芯片的打印头对于打印单细胞应用的特殊设计需要，会以特殊方式排列打印喷嘴，因此打印头喷嘴编号会形成不完全规律的分布。加之实际自动化运动带来的振动，以及光学成像的误差等多方面综合原因，会导致微米级的误差，这种误差可能会使单细胞所在的喷嘴编号的计算错误。本实施例中，在所述喷嘴识别步骤中，首先根据喷嘴的图像特性进行喷嘴寻找，根据形态学特征修复并选择真实喷嘴。得到的真实计算值用于计算喷嘴编号，可以正确驱动打印单细胞。
作为示例，可采用传统算法或其它合适的算法来识别喷嘴。所述传统算法包括基于形态学特征的筛选、滤波算法、结合打印头版图的物理位置筛选。物理位置结合打印头版图可以去掉非喷嘴，形态学筛选和滤波算法可以去掉异常喷嘴，不含有细胞的喷嘴。
另外，由于单细胞属于生命体，生命体的外貌形态上存在个体差异，并且细胞是透明体，细胞溶液亦为透明溶液。因此对于单个细胞的识别存在很大的难度。本实施例中，为了提高识别率，在所述单细胞识别步骤中，可采用传统算法与深度学习相结合的方式进行单细胞识别。
作为示例，所述单细胞识别步骤包括第一次筛选步骤与第二次筛选步骤。所述第一次筛选步骤采用预设图像算法对喷嘴内的细胞情况进行第一次识别，若合格，则进行下一步第二次筛选步骤，若不合格，则剔除对应喷嘴，不进一步识别。本实施例中，第一次筛选步骤优选采用了传统算法，用于快速粗判断喷嘴内是否为单细胞。所述第二次筛选步骤采用了预先训练好的神经网络对喷嘴内的细胞情况进行第二次识别以排除杂质、细胞碎片、非细胞的情况，若判断喷嘴内为单细胞，则计算对应喷嘴的编号，若判断喷嘴内为非单细胞，则剔除对应喷嘴。本实施例中，第二次筛选使用的算法可根据细胞的状态选择尺寸适中、择优活性状态好的单细胞。在这种筛选机制下，得到单细胞率可以达到95％，单细胞的活率可以达到90％。
作为示例，在所述第一次喷嘴拍照与识别步骤中，若识别结果为所有喷嘴内均无单细胞， 则返回所述供液步骤。
具体的，所述单细胞打印与拍照步骤是基于获得的具有单细胞的喷嘴的编号按照预设顺序依次将对应喷嘴内的单细胞以打印方式导出至细胞接收装置，其中，每打印一个喷嘴内的单细胞，即采用所述光学模组对打印至所述细胞接收装置中的单细胞进行拍照以获得被打印单细胞的第零天图像。
作为示例，在所述单细胞打印与拍照步骤中，在将单细胞导出至所述细胞接收装置中之前，将所述光学模组对焦至即将被打印的单细胞的预计落点，此时可以准确的拍摄到刚刚打印的单细胞，即为细胞第零天图像。细胞第零天图像是单细胞的重要判据，可以在需要的时候由细胞第零天图像完整验证细胞打印的结果，且此图像对于单细胞相关的研发至关重要。
作为示例，所述细胞接收装置包括孔板，其包括多个按预设规则排列的孔，每个孔用于接收一个单细胞。孔的底部透明，所述光学模组镜头位于孔板下方，经由接收单细胞的孔的底部对焦至即将被打印的单细胞的预计落点。
作为示例，在所述单细胞打印与拍照步骤中，每打印一个喷嘴内的单细胞，即在所述细胞接收装置接收被打印单细胞的腔体内施加培养基，以提高被打印单细胞的活性。
具体的，所述第二次喷嘴拍照步骤是采用所述光学模组对所述热泡打印芯片具有所述喷嘴的一面进行拍照，以得到单细胞打印后的喷嘴图像。需要指出的是，第二次喷嘴拍照是在将一次供液后识别到的单细胞都打印完成后再进行，通过和第一次喷嘴拍照得到的图像对比，即可确定单细胞打印情况。
作为示例，在所述第二次喷嘴拍照步骤之后，判断所述细胞接收装置中的单细胞数量是否满足要求，若满足，则结束单细胞分选，若不满足，则返回所述供液步骤。
作为示例，所述单细胞分选与鉴定方法可进一步包括细胞状态监测步骤，所述细胞状态监测步骤包括：在所述细胞接收装置内打印预设数量的单细胞之后，采用所述光学模组对所述细胞接收装置进行扫描拍照以监测细胞状态。例如可在任意时间对整块孔板进行扫描拍照，监控细胞成长情况。当孔板中每一个孔的直径相对较大而拍照视野相对较小时，对于一个孔，可通过扫描拍摄多张孔的局部图像并组合成一张孔的整体图像。
作为示例，所述单细胞分选与鉴定方法可进一步包括分泌物检测步骤，所述分泌物检测步骤包括：在所述细胞接收装置内打印预设数量的单细胞之后，采用所述光学模组拍摄所述细胞接收装置内细胞分泌物的荧光图像以筛选目标细胞。
具体的，由于细胞在非培养环境下的活性直接受暴露时间长度影响，被识别到的单细胞，需要在短时间内打印到指定容器内以保持细胞活性，因此需要在所有环节选择最优节拍。本实施例中采用最优自动化控制策略，高效率将热泡打印头内单细胞导出。在使用此最优控制 策略下，可以实现单个96孔板的单细胞打印耗时小于5min。
作为示例，请参阅图3，显示为本实施例中采用的一种单细胞打印节拍流程图。其中，依次进行空喷，对热泡打印芯片拍照以对单细胞第一次核验，判断是否有单细胞，若无，返回到空喷节拍，若有，则打印单细胞，并对打印的单细胞拍照以在后续需要时对单细胞进行第二次核验，然后对热泡打印芯片再次拍照，最后判断所需单细胞数量是否满足，若不满足，则返回空喷步骤，若满足，则结束。
具体的，在所述空喷节拍，单细胞识别前，所述热泡打印芯片的喷嘴在废液收集区进行适量喷射，使得喷嘴前端的细胞溶液进行更新。在对热泡打印芯片拍照时，为了增加单细胞出现的概率，选择高解析度的大视野光学模组拍照，并通过图像算法计算与反馈结果、图像储存。在对打印的单细胞即时拍照时，将上述光学模组对焦至孔板底部，可以捕捉细胞落入孔板底部的图像，并采用图像算法识别确认结果的单细胞性。而后采用加液探头在结果为单细胞的孔板孔内加入适量培基，以保证高细胞活性。
本实施例的单细胞分选与鉴定方法利用热泡打印芯片的高通量微流控设计，单次即可形成若干个单细胞，并且可以快速、轻柔的导出细胞。利用多重光学拍照，采用低放大倍率、高解析度、长工作距离的光学模组实现大视野高分辨率成像，实现了高效率的单细胞成像，以及单细胞打印结果的成像。利用多重方法叠加的图像算法对热泡打印头内的单细胞进行择优识别，以及对单细胞的打印结果进行确认，完成了对导出的单细胞进行双重鉴定，得到超高单细胞率结果。利用最优自动化控制策略，高效率将热泡打印头内单细胞导出，保证了高细胞活性。
实施例二
本实施例提供一种单细胞分选与鉴定装置，包括热泡打印芯片、光学模组及控制组件，所述单细胞分选与鉴定装置可用于执行实施例一中所述的单细胞分选与鉴定方法，其中，所述控制组件用于执行上述图像算法计算与反馈结果、图像储存等计算过程。
作为示例，请参阅图4，显示为所述热泡打印芯片1组装于包含所述控制组件的自动化仪器2上的示意图，其中，所述自动化仪器2包括基座201、所述控制组件202及用于储存细胞溶液的储液槽203，所述热泡打印芯片1组装于所述基座201上并与所述控制组件202电连接。经由所述储液槽203往所述热泡打印芯片内加入适量体积的适量浓度的细胞，利用热泡打印芯片的高通量、单细胞级别的微流控设计，可以在喷嘴内一次性自然形成大量单细胞。
作为示例，请参阅图5，显示为所述采用所述光学模组3对所述热泡打印芯片1具有所 述喷嘴101的一面进行拍照的示意图。
作为示例，请参阅图6，显示为拍照得到的单细胞打印前的喷嘴图像示意图，其可呈现细胞4在多个喷嘴101内的分布情况。
作为示例，请参阅图7，显示为所述热泡打印芯片1将包含单细胞的溶液5采用打印方式导出至孔板6中及所述光学模组3在孔板6下方对焦至被打印单细胞预计落点的示意图。
作为示例，请参阅图8，显示为对被打印单细胞即时拍照所获得的含有单细胞8的液滴7的图像示意图。
作为示例，请参阅图9，显示为在细胞成长状态检测中所使用的一种监测界面的示意图，其中，每个圆代表孔板中的一个孔，其位置可通过行编号(例如图中所示A、B、C、D、E、F、G、H)和列编号(例如图中所示1、2、3、4、5、6、7、8、9、10、11、12)来确定，圆中不同颜色填充(图中采用不同图案代表不同颜色)代表孔中的细胞情况不同，例如灰色(横条纹)代表孔中无细胞，橙色(竖条纹)代表孔中细胞异形，绿色(网格纹)代表孔中细胞正常。通过选择对应编号的孔，可获取该孔对应的细胞图像。
作为示例，请参阅图10、图11及图12，分别显示为通过图9所示界面选择其中一个孔后所呈现的细胞第零天图像、第一天图像和第三天图像示意图。
本实施例的单细胞分选与鉴定装置通过大视野、高分辨率的成像性能的光学模组与高通量喷嘴相匹配，实现高效率、高活性的单细胞打印，可以对导出细胞进行多次鉴定，得到超高单细胞率结果。所述光学模组兼顾单细胞识别、单细胞结果鉴定、细胞成长状态检测多重工作，可监测单细胞分选、生长、特性筛选全过程。
实施例三
本实施例利用实施例二中的单细胞分选与鉴定装置进行中国仓鼠卵巢(CHO)细胞的单细胞分选。首先，将热泡打印头安装在包含所述控制组件的自动化仪器上，执行喷嘴预处理操作，制备好的细胞悬液加入所述热泡打印芯片，使用自动化控制方案分别执行光学模组拍照、单细胞识别、单细胞打印，直到完成整个96孔板的单细胞打印。打印单个孔板耗时小于5min，96孔板内的单细胞率大于90％，单细胞培养后活性可达到80％。当需要检测细胞特性时，例如细胞活死、蛋白表达等，可加入相关试剂，使用荧光成像方式进行目标检测。
综上所述，本发明的单细胞分选与鉴定方法及装置基于高通量热泡打印芯片的微流控特性，采用图像识别的方法将单细胞高效率、轻柔导出，由此可以快速、大批量得到高单细胞率、高活性的单细胞。该方法基于细胞形态学特征识别单细胞，由此得到的单细胞具备高活 性特点。并且该方法可以提供细胞第零天样貌，是单细胞的重要判据。此外本发明的单细胞分选与鉴定装置在完成单细胞分选后，可监测细胞成长状态，并根据荧光标记筛选目标细胞，为细胞实验提供了全面的解决方案。所以，本发明有效克服了现有技术中的种种缺点而具高度产业利用价值。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。

Claims (13)

  1. 一种单细胞分选与鉴定方法，其特征在于，包括以下步骤：
    供液步骤：将包含细胞的溶液施加于热泡打印芯片的多个喷嘴内；
    第一次喷嘴拍照与识别步骤：采用光学模组对所述热泡打印芯片具有所述喷嘴的一面进行拍照，以得到单细胞打印前的喷嘴图像，并基于单细胞打印前的喷嘴图像获得具有单细胞的喷嘴的编号；
    单细胞打印与拍照步骤：基于获得的具有单细胞的喷嘴的编号按照预设顺序依次将对应喷嘴内的单细胞以打印方式导出至细胞接收装置，其中，每打印一个喷嘴内的单细胞，即采用所述光学模组对打印至所述细胞接收装置中的单细胞进行拍照以获得被打印单细胞的第零天图像；
    第二次喷嘴拍照步骤：采用所述光学模组对所述热泡打印芯片具有所述喷嘴的一面进行拍照，以得到单细胞打印后的喷嘴图像。
  2. 根据权利要求1所述的单细胞分选与鉴定方法，其特征在于：所述供液步骤包括使所述喷嘴进行空喷以使所述喷嘴前端的溶液进行更新。
  3. 根据权利要求1所述的单细胞分选与鉴定方法，其特征在于，所述第一次喷嘴拍照与识别步骤包括：
    喷嘴识别步骤：根据喷嘴的图像特性进行喷嘴寻找与选择；
    单细胞识别步骤：对被选择的喷嘴进行至少一次单细胞识别。
  4. 根据权利要求3所述的单细胞分选与鉴定方法，其特征在于，所述单细胞识别步骤包括：
    第一次筛选步骤：采用预设图像算法对喷嘴内的细胞情况进行第一次识别，若合格，则进行下一步第二次筛选步骤，若不合格，则剔除对应喷嘴，不进一步识别；
    第二次筛选步骤：采用预先训练好的神经网络对喷嘴内的细胞情况进行第二次识别以排除杂质、细胞碎片、非细胞的情况，若判断喷嘴内为单细胞，则计算对应喷嘴的编号，若判断喷嘴内为非单细胞，则剔除对应喷嘴。
  5. 根据权利要求1所述的单细胞分选与鉴定方法，其特征在于：在所述第一次喷嘴拍照与识别步骤中，若识别结果为所有喷嘴内均无单细胞，则返回所述供液步骤。
  6. 根据权利要求1所述的单细胞分选与鉴定方法，其特征在于：在所述单细胞打印与拍照步骤中，在将单细胞导出至所述细胞接收装置中之前，将所述光学模组对焦至即将被打印的 单细胞的预计落点。
  7. 根据权利要求1所述的单细胞分选与鉴定方法，其特征在于：在所述单细胞打印与拍照步骤中，每打印一个喷嘴内的单细胞，即在所述细胞接收装置接收被打印单细胞的腔体内施加培养基。
  8. 根据权利要求1所述的单细胞分选与鉴定方法，其特征在于：在所述第二次喷嘴拍照步骤之后，判断所述细胞接收装置中的单细胞数量是否满足要求，若满足，则结束单细胞分选，若不满足，则返回所述供液步骤。
  9. 根据权利要求1所述的单细胞分选与鉴定方法，其特征在于：在所述供液步骤之前，还包括喷嘴浸润处理步骤。
  10. 根据权利要求1所述的单细胞分选与鉴定方法，其特征在于：所述细胞接收装置包括孔板。
  11. 根据权利要求1所述的单细胞分选与鉴定方法，其特征在于，还包括细胞状态监测步骤，所述细胞状态监测步骤包括：在所述细胞接收装置内打印预设数量的单细胞之后，采用所述光学模组对所述细胞接收装置进行扫描拍照以监测细胞状态。
  12. 根据权利要求1所述的单细胞分选与鉴定方法，其特征在于，还包括分泌物检测步骤，所述分泌物检测步骤包括：在所述细胞接收装置内打印预设数量的单细胞之后，采用所述光学模组拍摄所述细胞接收装置内细胞分泌物的荧光图像以筛选目标细胞。
  13. 一种单细胞分选与鉴定装置，包括热泡打印芯片、光学模组及控制组件，其特征在于：所述单细胞分选与鉴定装置用于执行如权利要求1-12任意一项所述的单细胞分选与鉴定方法。
PCT/CN2022/103658 2021-09-02 2022-07-04 一种单细胞分选与鉴定方法及装置 WO2023029730A1 (zh)

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