Separation and culture device for cells
The single-cell separation and culture device designed through microfluidic control technology uses microfluidic channels and parallel structure to solve the problems of low single-cell culture efficiency and susceptibility to the environment in the prior art, and realizes efficient single-cell separation and three-dimensional culture, simulating a complex microfluidic environment.
Patent Information
- Application Number
- CN202311475169.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-11-07
- Publication Date
- 2025-05-13
AI Technical Summary
The existing single-cell culture methods have problems such as low efficiency, easy environment and poor cell grasping effectiveness, making it difficult to achieve efficient and sensitive single-cell isolation and culture.
A separation and culture device is designed using microfluidic technology, including microfluidic channels, inlets, discharge ports and separation culture areas. Through the parallel structure of the main channel and the branch channel, the separation and three-dimensional culture of individual cells are achieved.
It realizes efficient isolation and culture of individual cells, provides a three-dimensional cell culture space, simulates a complex real microfluidic environment, and makes the cell culture results more guiding.
Smart Images
Figure CN119979324A_ABST
Abstract
Description
Technical Field
[0001] The present application relates to the field of medical technology, and more specifically, to a device for separating and culturing cells. Background Art
[0002] Even in the same environment, neighboring cells in the same population have heterogeneity, and this heterogeneity may exhibit different biophysical properties related to various mechanisms such as cell development, regeneration, and evolution. Therefore, the analysis of single cells is crucial to improve researchers' understanding of cell function at the cellular and subcellular levels, and facilitate subsequent clinical diagnosis and treatment. Single cell culture is one of the most important processes in single cell analysis, and its purpose is to culture single cells that are not in contact with each other, monitor the actual information of single cells, and provide sufficient single cell clones and cell-derived products for further analysis.
[0003] There are many methods for single-cell culture, such as through microgels or micro-patterned matrices, but these methods all have certain problems. For example, the former has low efficiency and is easily affected by the environment (temperature, pH, etc.), while the latter has problems with effective cell capture.
[0004] Microfluidics can achieve efficient and sensitive single-cell culture and real-time analysis of single cells. It can be operated inside or outside the cell culture device, and allows the capture, culture, operation and analysis of cells in a single device. It can be flexibly processed at the level of single cells, with low cost and high throughput. Cell culture devices using microfluidics can highly reproduce the complex three-dimensional microenvironment in the body (where there are factors such as soluble factors, cell-cell contact, cell-extracellular matrix interaction, and shear stress caused by fluid flow), and can restore the natural cell growth environment in the body by integrating liquid flow controllers, gas temperature controllers, chip culture plates, etc., thereby realizing the combination of cell culture and analysis. Summary of the invention
[0005] The purpose of the present application is to provide a separation and culture device that can separate single cells from a cell group and provide a three-dimensional structured culture environment for them.
[0006] To this end, the present invention provides a device for separating and culturing cells, the device comprising a microfluidic channel, with reference to the input flow direction of a fluid sample, the microfluidic channel comprising an inlet for inputting the fluid sample, a discharge port downstream of the inlet, and a separation and culture region between the inlet and the discharge port, the separation and culture region comprising a main channel and a plurality of branch channels, each branch channel being connected in parallel with a main channel section of the main channel as a bypass,
[0007] Each branch channel includes a cell niche chamber, which is an expanded part of the branch channel relative to the rest of the branch channel, and the size of the cell niche chamber is set to accommodate cells in the terminal state after culture, the cross-sectional size of the first part of the branch channel upstream of the cell niche chamber is set to enable cells in the initial state to enter the cell niche chamber through the first part, and the cross-sectional size of the second part of the branch channel downstream of the cell niche chamber is set to prevent cells in the initial state from entering the second part from the cell niche chamber.
[0008] The cell separation and culture device according to the present invention can separate and capture a single cell from a fluid sample containing multiple cells and culture it separately, and can provide a three-dimensional cell culture space for the cells, thereby enabling monitoring and analysis of single cells, and helps to simulate a complex and realistic three-dimensional microfluidic environment, making the cell culture results more instructive.
[0009] In some embodiments, the length of the first portion of the branch channel is less than or equal to half the length of a section of the main channel connected in parallel to the branch channel.
[0010] In some embodiments, the length of the first portion of the branch channel is less than or equal to two-thirds of the maximum linear dimension of the outer contour of the cell in the initial state.
[0011] In some embodiments, the cross-sectional size of the main channel is configured to allow only a single cell in the initial state to pass through.
[0012] In some embodiments, the cell niche chamber includes a cell culture area and a cell capture area, wherein the cell capture area is located downstream of the cell culture area and at least partially overlaps with the cell culture area, the volume of the cell capture area is approximately equal to or smaller than the volume of the cells in the initial state, and the volume of the cell culture area is greater than or equal to the volume of the cells in the terminal state.
[0013] In some embodiments, the outer contour of the cell culture region smoothly transitions to the outer contour of the cell capture region.
[0014] In some embodiments, the main flow channel includes a plurality of main flow channel sections connected to each other, and each main flow channel section is equipped with one or more branch flow channels.
[0015] In some embodiments, two main flow channel sections that follow each other are configured such that the fluid from the preceding main flow channel section can flow into the succeeding main flow channel section substantially in a straight line.
[0016] In some embodiments, the microfluidic channel includes at least one group of multiple segments connected to each other, each segment includes a main channel segment of the main channel and an associated branch channel, the main channel segments of the multiple segments extend in a wavy manner, and the branch channels of the multiple segments extend in a straight line.
[0017] In some embodiments, the main channel includes one or more turning parts, and the main channel turns at the turning parts so that the sections upstream and downstream of each turning part of the microfluidic channel are generally parallel to each other.
[0018] In some embodiments, the direction of any one of each main channel segment and each branch channel is substantially straight, arc-shaped, or zigzag.
[0019] In some embodiments, the cross-section of the microfluidic channel is substantially rectangular, and the ratio between the width and the height of the cross-section at any position of the microfluidic channel is between 1.0 and 2.0.
[0020] In some embodiments, the microfluidic channel includes an auxiliary inlet, which is fluidically connected to the separation and culture area in parallel with the injection port.
[0021] In some embodiments, the microfluidic channel includes a mixing valve, which is positioned downstream of the injection port and upstream of the separation and culture area in the input flow direction of the fluid sample.
[0022] In some embodiments, the cell separation and culture device includes an elongated sheet-shaped body, and the microfluidic channel is formed in the body.
[0023] In some embodiments, the cell separation and culture device further comprises a transparent or translucent bottom plate, and the bottom plate can be positioned below the main body and fit with the main body to close the microfluidic channel.
[0024] In some embodiments, the thickness of the bottom plate is less than or equal to 0.17 mm.
[0025] The various technical features mentioned above and the various technical features to be mentioned below as well as the technical features that can be derived from the drawings can be combined with each other arbitrarily, as long as the individual technical features combined with each other are not contradictory to each other. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] The present invention is described in more detail below with reference to the accompanying drawings by means of exemplary embodiments, but the present invention is not limited thereto.
[0027] Figure 1is a schematic circuit diagram of a device for separating and culturing cells according to some embodiments of the present invention.
[0028] Figure 2 yes Figure 1 Schematic plan views of some embodiments of devices for separation and culture of cells.
[0029] Figure 3 yes Figure 2 An enlarged schematic plan view of a branch channel and a corresponding main channel section shown in FIG. DETAILED DESCRIPTION
[0030] In the context of the present invention, the "initial state" of cells refers to a state in which the cells are uncultured and have a small size, and the "terminal state" of cells refers to a state in which the cells are cultured in a separation and culture device for a predetermined period of time and have increased in size.
[0031] Figure 1 FIG. 1 is a simplified circuit diagram of a cell separation and culture device 1 according to an embodiment of the present invention. Figure 2 yes Figure 1 Schematic plan views of some embodiments of the device 1 for separating and culturing cells. Figure 1 and Figure 2 As shown, the separation and culture device 1 may include a microfluidic channel 2. The microfluidic channel 2 may include an injection port 3 and an auxiliary inlet 4. The injection port 3 may be used to input a fluid sample containing cells, and the auxiliary inlet 4 may be used to input auxiliary substances such as oxygen, carbon dioxide, etc. required in the cell culture process. It should be understood that the microfluidic channel 2 may be configured to have no auxiliary inlet 4 but only an injection port 3, and both the fluid sample and the auxiliary substance may be input through the injection port 3.
[0032] Optionally, the injection port 3 and the auxiliary inlet 4 can be fluidically connected to the mixing valve 5, respectively, so that the substances input from the injection port 3 and / or the auxiliary inlet 4 can be mixed at the mixing valve 5. The mixing valve 5 can be configured to have an S-shaped bend, for example, to achieve effective material mixing by changing the flow path of the fluid. With reference to the input flow direction of the fluid sample, downstream of the mixing valve 5, the microfluidic channel 2 may include a separation culture area 6 for separating and culturing the input cells. The separation culture area 6 can be configured to separate and capture single cells, and culture the single cells therein, so as to achieve separate culture and analysis for single cells. Further details about the separation culture area 6 will be described in detail below. Finally, the microfluidic channel 2 may include a discharge port 7 downstream of the separation culture area 6 for discharging waste liquid in the fluid sample.
[0033] like Figure 2As shown, the cell separation and culture device 1 may include a generally elongated sheet-like body 8, and the main structure of the microfluidic channel 2 may be formed in the body 8. The cell separation and culture device 1 may also include a bottom plate (not shown), which may be positioned to fit the body 8 so that the microfluidic channel 2 is formed between the body 8 and the bottom plate. The bottom plate may be constructed to be transparent or translucent, and may be positioned below the body 8 during use to facilitate observation of the growth state of cells in the microfluidic channel 2 from below. The thickness of the bottom plate may be set to be less than or equal to 0.17 mm to facilitate adaptation to a large-aperture microscopic system (e.g., a confocal microscope, a super-resolution microscope, etc.). The cross-section of the microfluidic channel 2 may be generally rectangular, and the ratio between the width and height of the cross-section at any position of the microfluidic channel 2 may be set to be between 1.0 and 2.0 to facilitate smooth flow of the fluid sample in the microfluidic channel 2.
[0034] like Figure 2 As shown, the separation and culture area 6 of the microfluidic channel 2 may include a main channel 11 and a plurality of branch channels 12, and each branch channel 12 may be configured to be connected in parallel with a main channel section 13 of the main channel 11 as a bypass. The main channel 11 is configured to allow cells in a fluid sample to pass through, and the branch channels 12 are configured to capture and culture single cells. Advantageously, the cross-sectional size of the main channel 11 may be configured to allow only a single cell in an initial state to pass through. Thus, at the position where the main channel 11 does not intersect with the branch channel 12, the individual cells can be arranged more neatly along the direction of the main channel 11, which helps to deliver the cells to the various branch channels 12 in a more orderly manner. In addition, although Figure 2 It is shown that each main channel segment 13 is connected in parallel with one branch channel 12, and it should be understood that in other embodiments not shown, one main channel segment 13 may also be connected in parallel with multiple branch channels 12. For example, in some embodiments, the main channel 11 may include multiple main channel segments 13 connected to each other, and each main channel segment 13 may be equipped with one or more branch channels 12.
[0035] Figure 3 yes Figure 2 FIG. 1 is an enlarged schematic plan view of a branch channel 12 and a corresponding main channel section 13 shown in FIG. Figure 3It is more clearly shown in the figure that the branch channel 12 can include a cell niche 14 for capturing and culturing single cells therein. The cell niche 14 can be configured as an expanded portion of the branch channel 12 relative to the rest of the branch channel 12, so as to accommodate cells in the terminal state after culture. The cross-sectional size of the first portion 18 of the branch channel 12 upstream of the cell niche 14 can be set so that cells in the initial state can enter the cell niche 14 through the first portion 18, and the cross-sectional size of the second portion 19 of the branch channel 12 downstream of the cell niche 14 can be set so that cells in the initial state cannot enter the second portion 19 from the cell niche 14, thereby preventing cells from escaping from the cell niche 14 to the downstream. After a cell enters the cell niche 14, since the cell cannot escape from the cell niche 14 to the downstream, under the pressure of the fluid flow, the cell can block the flow from the cell niche 14 to the downstream, so that the flow resistance in the corresponding branch channel 12 becomes larger, so that subsequent cells tend not to flow into the branch channel 12, but tend to flow along the main channel 11.
[0036] like Figure 3 As further shown, the length of the first portion 18 of the branch channel 12 can be set to be less than or equal to one-half of the length of the main channel section 13 connected in parallel with the branch channel 12, so as to avoid or reduce the possibility of multiple cells being blocked in the branch channel 12. More advantageously, the length of the first portion 18 of the branch channel 12 can be set to be less than or equal to two-thirds of the maximum linear dimension of the outer contour of the cells in the initial state, so as to further help avoid the blockage of cells in the branch channel 12, and help to carry away the excess cells entering the branch channel 12 from the branch channel 12 and return to the main channel 11 through the flow of the subsequent fluid sample.
[0037] like Figure 3 As further shown, with reference to the input flow direction of the fluid sample, the cell niche chamber 14 may include a cell culture area 15 and a cell capture area 16 downstream of the cell culture area 15, and the cell culture area 15 and the cell capture area 16 are configured to overlap at least partially. The volume of the cell capture area 16 may be configured to be approximately equal to or less than the volume of the cells in the initial state to capture the cells in the initial state, and the volume of the cell culture area 16 may be configured to be greater than or equal to the volume of the cells in the final state to accommodate the cells cultured to the final state. Such a structure allows the cells to be roughly positioned at the cell capture area 16 at the initial stage of culture, and gradually move to the cell culture area 15 as the cells grow, so that the cells can be more reliably positioned during the entire culture process. Advantageously, the outer contour of the cell culture area 15 can be smoothly transitioned to the outer contour of the cell capture area 16 to reduce the risk of damage to the cells.
[0038] like Figure 2 and Figure 3 As shown, in some embodiments, the main channel sections 13 connected in parallel with the branch channels 12 can each have a substantially zigzag orientation, and the plurality of branch channels 12 can be connected end to end in the input flow direction of the fluid sample to form a substantially straight orientation. Figure 2 As further shown, two main channel segments 13 connected to each other can be respectively positioned on both sides of the straight-line direction formed by the connected branch channels 12. In other words, the microfluidic channel 2 may include at least one group of multiple segments connected to each other, each segment including a main channel segment 13 of the main channel 11 and an associated branch channel 12, the main channel segments 13 of the multiple segments extend in a wavy manner, and the branch channels 12 of the multiple segments extend in a straight line. However, the present invention is not limited to such a structure. Each main channel segment 13 can be formed to have a roughly straight-line direction, or an arc-shaped or other zigzag direction. Each branch channel 12 can also be formed to have an arc-shaped, zigzag, or other zigzag directions. Advantageously, two main channel segments 13 connected one after another can be configured so that the fluid from the previous main channel segment 13 can flow substantially in a straight line into the next main channel segment 13, which helps to reduce the flow resistance between the two connected main channel segments 13, so that the flow at the junction between the two main channel segments 13 can be smoother than the flow toward the branch channel 12. This helps to remove excess cells that have entered the branch channel 12 from the branch channel 12.
[0039] like Figure 2 As further shown, the main channel 11 may include one or more return portions 17, and the main channel 11 may be configured to return at the return portion 17 so that the sections of the microfluidic channel 2 located upstream and downstream of each return portion 17 are parallel to each other as a whole. This helps to avoid the overall length of the microfluidic channel 2 being too long, and the overall size of the microfluidic channel 2 can be flexibly adjusted according to the size of the main body 2 or other requirements.
[0040] The cell separation and culture device 1 according to the present invention can separate and capture a single cell from a fluid sample containing multiple cells and culture it separately, so that the single cell can be monitored and analyzed. In addition, the cell separation and culture device 1 causes less damage to the cells during the cell separation process, and can provide a three-dimensional cell culture space for the cells, thereby facilitating the simulation of a complex and real three-dimensional microfluidic environment, making the cell culture results more instructive.
[0041] It should be noted that the terms used herein are for the purpose of illustrating specific aspects only and are not intended to limit the disclosure. As used herein, the singular forms "a" and "the one" shall include the plural forms unless the context clearly states otherwise. It is understood that the terms "include" and "comprises" and other similar terms, when used in the application documents, specify the existence of the stated operations, elements and / or parts, without excluding the existence or addition of one or more other operations, elements, parts and / or combinations thereof. As used herein, the term "and / or" includes all arbitrary combinations of one or more related enumerated items. In the description of the drawings, similar reference numerals always represent similar elements.
[0042] Finally, it should be pointed out that the above embodiments are only used to understand the present invention, and do not limit the protection scope of the present invention. For those skilled in the art, modifications can be made based on the above embodiments, and these modifications do not depart from the protection scope of the present invention.
Claims
1. A device for separating and culturing cells, characterized in that: The device comprises a microfluidic channel. With reference to the input flow direction of the fluid sample, the microfluidic channel comprises an inlet for inputting the fluid sample, a discharge port downstream of the inlet, and a separation and culture region between the inlet and the discharge port. The separation and culture region comprises a main channel and a plurality of branch channels, each branch channel being connected in parallel with a main channel section of the main channel as a bypass. Each branch channel includes a cell niche chamber, which is an expanded part of the branch channel relative to the rest of the branch channel. The size of the cell niche chamber is set to accommodate cells in the terminal state after culture. The cross-sectional size of the first part of the branch channel upstream of the cell niche chamber is set to enable cells in the initial state to enter the cell niche chamber through the first part, and the cross-sectional size of the second part of the branch channel downstream of the cell niche chamber is set to prevent cells in the initial state from entering the second part from the cell niche chamber.
2. The cell separation and culture device according to claim 1, characterized in that: The length of the first part of the branch channel is less than or equal to one half of the length of the main channel section connected in parallel with the branch channel. Preferably, the length of the first part of the branch channel is less than or equal to two thirds of the maximum linear dimension of the outer contour of the cell in the initial state; and / or The cross-sectional size of the main channel is set to allow only a single cell in the initial state to pass through; and / or The cell niche chamber includes a cell culture area and a cell capture area, wherein the cell capture area is located downstream of the cell culture area and at least partially overlaps with the cell culture area, the volume of the cell capture area is approximately equal to or smaller than the volume of the cells in the initial state, and the volume of the cell culture area is greater than or equal to the volume of the cells in the terminal state.
3. The cell separation and culture device according to claim 2, characterized in that: The outer contour of the cell culture area transitions smoothly into the outer contour of the cell capture area.
4. The device for separating and culturing cells according to any one of claims 1 to 3, characterized in that: The main flow channel comprises a plurality of main flow channel sections connected to each other, each main flow channel section is provided with one or more branch flow channels; and / or Two main flow channel sections that follow each other are configured so that the fluid from the previous main flow channel section can flow into the next main flow channel section substantially along a straight line; and / or The microfluidic channel comprises at least one group of multiple segments connected to each other, each segment comprises a main channel section of the main channel and an associated branch channel, the main channel sections of the multiple segments extend in a wavy shape, and the branch channels of the multiple segments extend in a straight line; and / or The main flow channel includes one or more return portions, and the main flow channel returns at the return portions so that the sections upstream and downstream of each return portion of the microfluidic channel are parallel to each other as a whole; and / or The direction of each main channel section and each branch channel is generally straight, arc-shaped, or zigzag.
5. The device for separating and culturing cells according to any one of claims 1 to 3, characterized in that: The cross section of the microfluidic channel is substantially rectangular, and the ratio between the width and the height of the cross section at any position of the microfluidic channel is between 1.0 and 2.
0.
6. The device for separating and culturing cells according to any one of claims 1 to 3, characterized in that: The microfluidic channel includes an auxiliary inlet, which is fluidically connected to the separation and culture area in parallel with the injection port.
7. The device for separating and culturing cells according to any one of claims 1 to 3, characterized in that: The microfluidic channel includes a mixing valve, which is located downstream of the injection port and upstream of the separation and culture area in the input flow direction of the fluid sample.
8. The device for separating and culturing cells according to any one of claims 1 to 3, characterized in that: The cell separation and culture device includes a long sheet-shaped body, and the microfluidic channel is formed in the body.
9. The cell separation and culture device according to claim 8, characterized in that: The cell separation and culture device further comprises a transparent or translucent bottom plate, which can be positioned below the main body and fit with the main body to close the microfluidic channel.
10. The cell separation and culture device according to claim 9, characterized in that: The thickness of the bottom plate is less than or equal to 0.17 mm.