Controllable cell lysis method in emulsion

Abstract

The invention relates to the field of single cell analysis of droplet microfluidics, in particular to a controllable cell lysis method in an emulsion. The invention provides a method for cell lysis in a liquid drop, which comprises the following steps: 1) liquid drop single cell wrapping: wrapping a single cell into the liquid drop through a micro-fluidic chip in the presence of a first oil phase to obtain a first mixture containing the first oil phase and the liquid drop; and 2) cell lysis: replacing the first oil phase in the first mixture with a second oil phase containing a cell lysis solution, and incubating to realize cell lysis. For realizing cell lysis in the liquid drop, the second oil phase containing the cell lysis solution can realize cell lysis of the liquid drop outside the micro-fluidic chip, so that the operation difficulty is low, the cell lysis method is simple and efficient, low-cost, high-throughput and controllable cell lysis can be realized, and the applicability is wide.

Description

Technical Field

[0001] The present application relates to the field of single-cell analysis by droplet microfluidics, and in particular to a controllable cell lysis method in an emulsion. Background Art

[0002] Cells are the basic unit of all living organisms. Similar to the human body, cells also have a group of components called organelles, which are responsible for performing various cellular functions. In addition, the genetic information for the development and function of any organism is encoded in the DNA or RNA sequences within the cell. Cells have an external boundary called the cell membrane, which encloses all the cell contents. The cell membrane acts as a barrier and regulates the transport of substances inside and outside the cell. Cell lysis is a method of destroying the external boundaries of the cell, allowing DNA, RNA, proteins, etc. to be released from the cell. Cell lysis is an important unit of operation in technologies such as molecular diagnosis of pathogens, point-of-care diagnostic immunoassays, cancer diagnosis, drug screening, and mRNA transcriptome determination.

[0003] According to the lysis effect, cell lysis can be divided into complete lysis and partial lysis. Partial cell lysis is performed using techniques such as patch clamping, which is used for drug testing and studying intracellular ion currents. Complete cell lysis is the complete disintegration of the cell membrane, which is used to analyze DNA, RNA and subcellular components.

[0004] Microfluidics is one of the emerging platforms for cell lysis. Microfluidic manipulation is mainly about manipulating and processing small volumes (nano to picoliter) of liquids in microchannels. Due to the microscale operation mechanism, microfluidics is very suitable for applications with small samples or sample volumes. Microfluidic lysis techniques can be roughly divided into six types. They include mechanical lysis, thermal lysis, chemical lysis, optical lysis, acoustic lysis, and electrical lysis.

[0005] The chemical lysis method is easy to operate and has controllable effects, so it is the main technology in microfluidic droplet lysis. The chemical lysis method is a method that uses chemical reagents to dissolve lipids and proteins in the cell membrane to create pores and lyse cells. It is mainly divided into three types: alkaline lysis, detergent lysis and enzyme lysis. ① Alkaline lysis method: In alkaline lysis, hydroxide ions are the main component used to lyse cell membranes. The lysis buffer consists of sodium hydroxide and sodium dodecyl sulfate (SDS). Hydroxyl ions react with cell membranes and destroy fatty acid-glyceride bonds, which then make the cell membrane permeable, and SDS dissolves proteins and membranes. ② Detergent lysis method: Detergents, also known as surfactants, have the ability to destroy hydrophobic-hydrophilic interactions. Since the cell membrane is a bilayer composed of hydrophobic and hydrophilic molecules, detergents can be used to break them. Detergents are able to destroy lipid-lipid, lipid-protein and protein-protein interactions. According to their charge carrying capacity, they can be divided into cationic, anionic and non-ionic detergents. Detergents are most widely used to lyse mammalian cells. Among them, detergents are used to chemically destroy the membrane. Detergents react with cell membranes to form pores on the membrane surface, leading to the release of intracellular components (such as DNA, RNA, proteins, etc.). ③ Enzymatic lysis: It is a biological cell lysis method in which enzymes such as lysozyme, lysozyme, zymolyase, cellulase, protease or glycine are used. Most of these enzymes are commercially available and can be used for large-scale lysis. One advantage of enzymatic lysis is its specificity. For example, lysozyme is used for bacterial cell lysis, while chitinase can be used for yeast cell lysis and pectinase is used for plant cell lysis.

[0006] The current mainstream method of cell lysis in droplets is to encapsulate detergent (cell lysate) and cells into droplets together, and in small volume droplets, detergent acts on cells, thereby realizing the lysis of cells in droplets. However, in this case, it is necessary to ensure that detergent and cells cannot be mixed in advance, and only when encapsulated into droplets can both be contacted to cause cell lysis, so the method of cell lysis in most droplets is to separate lysate and cell solution, and by the method of additionally increasing the lysate channel, cells and lysate are encapsulated into droplets together, thereby realizing cell lysis in droplets. In addition, for cells that have been encapsulated into droplets, only by the method of droplet injection or droplet fusion, the method of adding lysate to the droplet to add lysate and realize cell lysis in droplets, but these methods require more complicated microfluidic devices, and at the same time, all have high requirements for operation, equipment, etc. Therefore, a simple, efficient and generally applicable method of controllable cell lysis in droplets is urgently needed to be developed. Summary of the invention

[0007] In view of the shortcomings of the prior art mentioned above, in order to solve the technical problems that the methods for cell lysis in droplets in the prior art are complicated and difficult to operate, the purpose of the present application is to provide a method for controllable cell lysis in an emulsion, by replacing the oil phase with a surfactant in the droplets, thereby successfully achieving controllable cell lysis in the droplets.

[0008] To achieve the above-mentioned object and other related objects, the present application provides a method for cell lysis in a droplet in a first aspect, comprising the following steps:

[0009] 1) Single cell encapsulation in droplets: encapsulating single cells into droplets through a microfluidic chip in the presence of a first oil phase, thereby obtaining a first mixture comprising the first oil phase and droplets;

[0010] 2) Cell lysis: replacing the first oil phase in the first mixture with a second oil phase containing a cell lysis solution, and incubating to achieve cell lysis.

[0011] Compared with the prior art, the beneficial effects of this application are:

[0012] 1. For cell lysis in droplets, the present application uses a second oil phase containing a cell lysis solution to achieve cell lysis in droplets outside the microfluidic chip. Not only is the operation easy, but the cell lysis method is simple and efficient, and can achieve low-cost, high-throughput, controllable cell lysis, with wide applicability.

[0013] 2. The spiral channel of the microfluidic chip of the present application utilizes fluid mechanics to achieve full dispersion of cells. The inertial effect increases the single-cell encapsulation rate of cells in droplets, which is beneficial for the subsequent efficient lysis of single cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a diagram of the controllable cell lysis pattern in droplets. First, the cells are encapsulated into the droplets, and then the oil phase is replaced with an oil phase containing cell lysis solution, and incubated for a period of time to achieve controllable cell lysis.

[0015] Figure 2 Schematic diagram of the design of a microfluidic chip for encapsulating cells into droplets. After the cells are introduced, they will first pass through the spiral channel to fully disperse the cells, and at the same time, the inertial effect can increase the cell encapsulation rate.

[0016] Figure 3 The lysis of cells in the droplets after adding different proportions of igpal to the oil phase. a, b, c, d represent the lysis of cells in the droplets after 60 minutes of incubation in the droplets under the conditions of adding 0%, 0.5%, 1%, and 2% igpal to the oil phase, respectively. Scalebar is 200um.

[0017] Figure 4To optimize the lysis time, a, b, and c are the cell lysis conditions after incubation for 1 h, 2 h, and 3 h, respectively. The scalebar is 200 μm.

[0018] Figure 5 Comparison of the lysis effects of igpal and Triton X-100, a is the cell lysis when 2% igpal is added to the oil phase, b is the cell lysis when 2% igpal is added to the oil phase.

[0019] Component number description:

[0020] 1 Cell fluid compartment

[0021] 11. Cell fluid inlet

[0022] 12 Cytosolic Fluid Channel

[0023] 121 Spiral Channel

[0024] 122 straight channels

[0025] 2 Droplet collection port

[0026] 3 Oil chamber

[0027] 31 Oil chamber

[0028] 32 Oil chamber channel

[0029] 4 Main Channel

[0030] 41 Cut DETAILED DESCRIPTION

[0031] In order to make the invention purpose, technical scheme and beneficial effect of the present application clearer, the present application is further described below in conjunction with the examples. It should be understood that the examples are only used to explain the present application and are not used to limit the scope of the application. The test methods used in the following examples are conventional methods unless otherwise specified, and those familiar with the technology can easily understand other advantages and effects of the present application from the contents disclosed in this description.

[0032] After extensive exploration and research, the inventors of this application discovered a method for controllable cell lysis in emulsion. By replacing the oil phase with surfactant in the droplets, they successfully achieved controllable cell lysis in droplets, and completed this application on this basis.

[0033] On the one hand, the present application provides a method for cell lysis in a droplet, comprising the following steps:

[0034] 1) Single cell encapsulation in droplets: encapsulating single cells into droplets through a microfluidic chip in the presence of a first oil phase, thereby obtaining a first mixture comprising the first oil phase and droplets;

[0035] 2) Cell lysis: replacing the first oil phase in the first mixture with a second oil phase containing a cell lysis solution, and incubating to achieve cell lysis.

[0036] In the method provided in the present application, step 1) refers to single cell encapsulation in droplets: encapsulating single cells into droplets through a microfluidic chip in the presence of a first oil phase to obtain a first mixture comprising the first oil phase and droplets. In some embodiments, Figure 2 As shown, the microfluidic chip in step 1) of the present application includes a substrate, on which an oil chamber 3, a cell fluid chamber 1, and a droplet collection port 2 connected by a main channel 4 are provided. The oil chamber 3 includes a connected oil cavity 31 and an oil chamber channel 32, the cell fluid chamber 1 includes a connected cell fluid inlet 11 and a cell fluid channel 12, and a shearing opening 41 is provided on the main channel 4 for generating droplets, which flow into the droplet collection port 2 after being generated; the oil chamber channel 32 and the cell fluid channel 12 merge at the shearing opening 41, and the cell fluid channel 12 includes a connected spiral channel 121 and a straight channel 122.

[0037] In the microfluidic chip provided in this application, Figure 2 As shown, the spiral channel 121 is connected to the cell fluid inlet 11, and the straight channel 122 is connected to the shear opening 41. The spiral channel 121 takes the cell fluid inlet 11 as the starting point, and the width of the spiral channel 121 is 50-150 μm; specifically, it can be 50-100 μm, 100-120 μm, 120-130 μm, or 130-150 μm, etc. The height of the spiral channel 121 is 50-150 μm; specifically, it can be 50-80 μm, 80-100 μm, 100-110 μm, 110-120 μm, or 120-150 μm, etc. The size of the spiral channel 121 of the actual microfluidic chip can be adjusted according to the specific required droplet size. The spiral channel of the microfluidic chip of the present application utilizes fluid mechanics to achieve full dispersion of cells, and utilizes the inertial effect of the liquid to achieve effective arrangement of single cells, thereby increasing the encapsulation rate of cells in droplets, which is beneficial for subsequent efficient lysis of single cells.

[0038] In the microfluidic chip provided in the present application, the oil chamber 31 is used to contain the first oil phase. In some embodiments, the first oil phase includes fluorinated oil. In a specific embodiment of the present application, the first oil phase can be, for example, HFE-7500 containing 2% (w / w) PEG-PFPE, or FC-40 oil containing 5% PEG-PFPE surfactant. Unless otherwise specified, the fluorinated oils of the present application are all conventional commercially available products.

[0039] In the microfluidic chip provided in the present application, the cell fluid inlet 11 is used to accommodate a single cell.

[0040] In step 1), the first oil phase includes fluorinated oil. In a specific embodiment of the present application, the first oil phase can be, for example, HFE-7500 containing 2% (w / w) PEG-PFPE, or FC-40 oil containing 5% PEG-PFPE surfactant. Unless otherwise specified, the fluorinated oils of the present application are all commercially available conventional products.

[0041] In step 1), single cells are obtained by centrifuging suspended cells and then precipitating them, and then resuspending them with a cell resuspension solution. Single cells may be conventional cells such as MCF7, HEK293, and 3T3. Optionally, single cells may also be stained, and the staining agent may be, for example, CellTracker Orange, DAPI, TRITC-Phalloidin, Trypan Blue, Hoechst 33342, and other common staining agents. In a specific embodiment of the present application, the condition for centrifugation of suspended cells is 1500rpm / min centrifugation for 1min. The cell resuspension solution is selected from conventional culture media such as PBS and DMEM culture medium; further, the cell resuspension solution includes an iodixanol aqueous solution. Based on the volume of the cell resuspension solution, the concentration of the iodixanol aqueous solution is 15-25% (v / v); specifically, it may be 15-18% (v / v), 18-20% (v / v), or 20-25% (v / v), etc. The density of iodixanol is 1.3 g / mL, which is greater than that of water. It is biologically inert and will not react with cells. It can balance the cells in the cell resuspension, making it difficult for them to sink to the bottom, which is more conducive to the efficient encapsulation of cells in droplets.

[0042] In step 1), in the microfluidic chip, the flow rate of the first oil phase is 500-10000 μL / h; specifically, it can be 500-2000 μL / h, 2000-3000 μL / h, 3000-4000 μL / h, or 4000-10000 μL / h, etc. The flow rate of a single cell is ≥500 μL / h. The single cell of the present application is set to a high flow rate in order to utilize the effect of fluid mechanics to achieve its orderly arrangement in the first oil phase and improve the efficient encapsulation in the droplets.

[0043] In the method provided in the present application, step 2) refers to cell lysis: the first oil phase in the first mixture is replaced with a second oil phase containing a cell lysate, and incubated with the droplets to achieve cell lysis. Step 2) can be performed in a centrifuge tube, for example. Due to the density difference, after the first mixture is allowed to stand, the first oil phase and the droplets are clearly stratified, the droplets are located in the upper layer of the centrifuge tube, and the first oil phase is located in the lower layer of the centrifuge tube. The first oil phase at the bottom of the centrifuge tube is carefully sucked off with a pipette, and the second oil phase containing a cell lysate is added; then the centrifuge tube is laid flat, and the centrifuge tube is slowly rotated to allow the second oil phase to fully contact the droplets.

[0044] In step 2), the second oil phase includes fluorinated oil; further, the fluorinated oil includes HFE-7500 and / or PEG-PFPE. In a specific embodiment of the present application, the second oil phase can be, for example, HFE-7500 containing 2% (w / w) PEG-PFPE. Unless otherwise specified, the fluorinated oils of the present application are all commercially available conventional products.

[0045] In step 2), the cell lysate in the second oil phase is selected from CA-630、Triton TM X-100, or NP-40. Based on the volume of the second oil phase, the concentration of the surfactant is 0.5-2% (w / w); specifically, it can be 0.5-1% (w / w), 1-1.5% (w / w), or 1.5-2% (w / w).

[0046] In step 2), the incubation time is 30 min to 3 h; specifically, it can be 30 min to 1 h, 1 h to 2 h, or 2 h to 3 h, etc. The incubation temperature is room temperature.

[0047] Another aspect of the present application provides use of the aforementioned method in cell lysis.

[0048] As for achieving cell lysis in droplets, the present application can achieve cell lysis in droplets outside the microfluidic chip by using a second oil phase containing cell lysis solution. Not only is the operation easy, but the cell lysis method is simple and efficient, and can achieve low-cost, high-throughput, controllable cell lysis, with wide applicability.

[0049] The present application is further described below by way of examples, but the scope of the present application is not limited thereby.

[0050] The material information for this application is:

[0051] Fluorinated oil: 3M Company

[0052] Iodixanol: Merck; CAS: 92339-11-2

[0053] PEG-PFPE surfactant: Dapu Biotech Co., Ltd.

[0054] Dye: Thermo Fisher Scientific Inc.

[0055] Cells: Cell Bank of Chinese Academy of Sciences

[0056] Example 1

[0057] (1) Droplet single cell encapsulation

[0058] ① Cell staining: The MCF-7 cells to be encapsulated into the droplets were stained with CellTracker Orange to obtain cells labeled with red dye; and the number of cells was quantified using a cell counter;

[0059] ② Design and processing of single-cell encapsulated microfluidic chips: Design of inertial microfluidic chips with spiral channels (such as Figure 2 ), used to efficiently encapsulate single cells into droplets;

[0060] ③ Droplet single cell encapsulation: The suspended cell solution was centrifuged at 1500 rpm / min for 1 min, and then the supernatant was removed; 20% (v / v) OptiPrep TM The cells were resuspended in iodixanol PBS solution, and the resuspended cell solution was loaded into a 1 ml syringe and injected from the cell solution inlet 11 of the microfluidic chip. HFE7500 fluorinated oil containing 2% PEG-PFPE was placed in the oil chamber 31 of the microfluidic chip as the first oil phase, and the resuspended cell solution flowed through the spiral channel 121 and the straight channel 122, and merged with the first oil phase at the shear port 41. The flow rate of the first oil phase was set to 3000 μL / h, and the single cell flow rate was set to 500 μL / h. After the two merged, the first oil phase wrapped the single cell into the droplet, and the droplet containing the single cell, i.e., the first mixture, was obtained.

[0061] (2) In-droplet cell lysis: preparation of lysis oil phase; replacement of oil phase after cell encapsulation; incubation time to allow complete cell lysis;

[0062] ① Preparation of the oil phase containing the lysate: Add a certain proportion of the lysate igpal or Triton X-100 to the oil phase (HFE7500 fluorinated oil containing 2% PEG-PFPE) to prepare the second oil phase, and mix thoroughly with a mixer to allow the lysate to fully dissolve in the oil phase;

[0063] ② If Figure 1 As shown, the first mixture in step (1), i.e., the droplets containing single cells, is first transferred to a centrifuge tube. Due to the density difference, the first oil phase and the droplets are clearly separated. The droplets are located in the upper layer of the centrifuge tube, and the first oil phase is located in the lower layer of the centrifuge tube. The first oil phase (shown in orange) at the bottom of the centrifuge tube is carefully sucked out with a pipette, and the second oil phase (shown in purple) containing the cell lysate is added; then the centrifuge tube is laid flat, and the centrifuge tube is slowly rotated to allow the second oil phase to fully contact the droplets;

[0064] ③ Incubate at room temperature to allow the cells in the droplets to completely lyse.

[0065] The results are as follows Figures 3 to 5 As shown, Figure 3 The lysis of cells in the droplets after adding different concentrations of igpal to the oil phase is shown. Figure 3c~3d show that adding 1% and 2% igpal to the second oil phase can achieve complete cell lysis. Considering the actual cost, adding 1% igpal to the second oil phase is the optimal concentration. Figure 4 It shows that when incubated for 1h, 2h, and 3h, cell lysis reaches an ideal state. Figure 5 It is shown that if the cell lysate is treated with igpal ( Figure 5 a) Replaced with Triton X-100 ( Figure 5 b), the same cracking effect can be achieved.

[0066] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present application. Anyone familiar with the technology may modify or change the above embodiments without violating the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by a person of ordinary skill in the art without departing from the spirit and technical ideas disclosed by the present invention shall still be covered by the claims of the present application.

Claims

1. A method for cell lysis in droplets, characterized in that: The following steps are involved: 1) Single cell encapsulation in droplets: encapsulating single cells into droplets through a microfluidic chip in the presence of a first oil phase, thereby obtaining a first mixture comprising the first oil phase and droplets; 2) Cell lysis: replacing the first oil phase in the first mixture with a second oil phase containing a cell lysis solution, and incubating to achieve cell lysis.

2. The method according to claim 1, characterized in that In step 1), the microfluidic chip comprises a substrate, and the substrate is provided with an oil chamber (3), a cell fluid chamber (1), and a droplet collection port (2) connected via a main channel (4); the oil chamber (3) comprises a connected oil cavity (31) and an oil chamber channel (32); the cell fluid chamber (1) comprises a connected cell fluid inlet (11) and a cell fluid channel (12); the main channel (4) is provided with a shearing opening (41) for generating droplets; the oil chamber channel (32) and the cell fluid channel (12) merge at the shearing opening (41); and the cell fluid channel (12) comprises a connected spiral channel (121) and a straight channel (122).

3. The method according to claim 2, characterized in that Includes one or more of the following characteristics: a1) In step 1), the spiral channel (121) is connected to the cell fluid inlet (11), and the straight channel (122) is connected to the shear opening (41); a2) In step 1), the oil chamber (31) is used to contain the first oil phase; a3) In step 1), the cell fluid inlet (11) is used to accommodate a single cell.

4. The method according to claim 3, characterized in that In step 1), the spiral channel (121) takes the cell fluid inlet (11) as a starting point, and the width of the spiral channel (121) is 50 to 150 μm, and the height is 50 to 150 μm.

5. The method according to claim 1, characterized in that In step 1), the first oil phase comprises fluorinated oil; further, the fluorinated oil is HFE-7500 fluorinated oil containing 2% PEG-PFPE surfactant, or FC-40 oil containing 5% PEG-PFPE surfactant.

6. The method according to claim 1, characterized in that In step 1), the single cell is obtained by centrifuging the suspended cells and then resuspending the precipitate with a cell resuspension solution.

7. The method according to claim 5, characterized in that In step 1), the cell resuspension solution includes an iodixanol aqueous solution.

8. The method according to claim 6, characterized in that The concentration of the iodixanol aqueous solution is 15-25% (v / v).

9. The method according to claim 1, characterized in that Includes one or more of the following characteristics: b1) Step 2) is carried out in a centrifuge tube; b2) In step 2), replacement means that the first mixture is allowed to stand, and after separation, the upper layer is the liquid droplets, and the lower layer is the first oil phase, and after the first oil phase is sucked off, the second oil phase is added to make the second oil phase and the liquid droplets fully contact; b3) In step 2), the second oil phase comprises fluorinated oil; further, the fluorinated oil comprises HFE-7500 and / or PEG-PFPE; b4) In step 2), the cell lysate is selected from CA-630、Triton TM X-100, or NP-40; b5) in step 2), the concentration of the cell lysate is 0.5-2% (v / v) based on the volume of the second oil phase; b6) In step 2), the incubation time is 30 min to 3 h; b7) In step 2), the incubation temperature is room temperature.

10. Use of the method according to any one of claims 1 to 9 in cell lysis.

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