CN117247826A - Cell separation device and method of operating the same - Google Patents
Cell separation device and method of operating the same Download PDFInfo
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- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
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Abstract
A cell separation device and method of operation thereof, the cell separation device comprising: a channel through which a liquid to be separated flows, wherein the liquid to be separated comprises cells to be separated, basic cells and free liquid, and the ratio of the effective diameter of the cells to be separated to the effective diameter of the channel is greater than or equal to a first diameter ratio; a flow rate control part for controlling a flow rate of the liquid to be separated flowing through the channel; a plurality of liquid flow channels coupled to the channels, at least one separation liquid flow channel for flow out of the channels of cells to be separated; the ratio of the total volume of the basic cells to the total volume of the liquid to be separated is greater than or equal to a first volume ratio, and the ratio of the volume of free liquid in the liquid to be separated to the total volume of the liquid to be separated is less than or equal to a second volume ratio. The invention can effectively separate cells, and is beneficial to improving the physiological cleanliness of the residual liquid.
Description
Technical Field
The invention relates to the technical field of biotechnology detection, in particular to a cell separation device and an operation method thereof.
Background
Nucleated cells in blood have important clinical value. Nucleated cells in blood may include, for example, white blood cells, nucleated red blood cells, circulating tumor cells, and the like. However, since nucleated cells belong to rare cells, a large amount of blood is required to obtain a certain number of nucleated cells, which may cause a certain physical burden for individuals with a small blood amount (such as infants or patients with poor physical conditions) and may not even be separable for non-human race (such as laboratory dogs or mice).
However, existing cell separation techniques mainly include erythrocyte lysis, centrifugation and microfluidic methods. The above methods often require destruction of cells in the original blood during the separation process, or introduction of foreign fluids, resulting in the separated residual blood not being reused, for example, resulting in the blood not being recovered and returned to the organism.
There is a need for a cell separation device that can effectively separate cells without destroying the cells in the fluid to be separated and without introducing extraneous fluids, thereby allowing the opportunity for reuse of the remaining fluid after separation.
Disclosure of Invention
The invention solves the technical problem of providing a cell separation device and an operation method thereof, which can effectively separate cells without damaging the cells in liquid to be separated and introducing external liquid, and the original components are not biochemically influenced, thereby being beneficial to improving the cleanliness of the residual liquid.
To solve the above technical problems, an embodiment of the present invention provides a cell separation device, including: a channel through which a liquid to be separated flows, wherein the liquid to be separated comprises cells to be separated, basic cells and free liquid, and the ratio of the effective diameter of the cells to be separated to the effective diameter of the channel is greater than or equal to a first diameter ratio; a flow rate control part for controlling a flow rate of the liquid to be separated flowing through the channel; a plurality of liquid flow channels coupled to the channel, wherein the liquid flow channels comprise a separation liquid flow channel for flowing out the cells to be separated and a residual liquid flow channel for flowing out residual liquid, at least one separation liquid flow channel being used for flowing out the cells to be separated of the channel; the ratio of the total volume of the basic cells to the total volume of the liquid to be separated is greater than or equal to a first volume ratio, and the ratio of the volume of free liquid in the liquid to be separated to the total volume of the liquid to be separated is less than or equal to a second volume ratio.
Optionally, the basal cell is a red blood cell; wherein the first volume ratio is selected from 30% to 60%; and/or, the second volume ratio is selected from 40% to 70%.
Optionally, the channel satisfies one or more of: the cross-sectional shape of the channel is selected from: round, oval, rounded polygon, and polygon; when the cross section of the channel is elliptical, rounded polygonal or polygonal, the effective diameter of the channel is the hydraulic diameter of the channel.
Alternatively, the effective diameter of the channel is selected from 25 μm to 200 μm.
Optionally, the effective diameter of the passageway does not vary by more than a predetermined percentage of variation under a pressure variation of a predetermined pressure.
To solve the above technical problems, an embodiment of the present invention provides an operation method based on the above cell separation device, including: and controlling the liquid to be separated to flow through the channel at a first flow rate so that the Reynolds number of the liquid to be separated is in a preset range.
Optionally, the reynolds number of the liquid to be separated is located in a first subinterval, so that the cells to be separated are gathered in a central area of the channel; wherein the separated liquid flow channel is one or more liquid flow channels corresponding to the central area, and the rest liquid flow channels are other liquid flow channels except the separated liquid flow channel.
Optionally, the reynolds number of the liquid to be separated is located in a second subinterval, so that the cells to be separated are gathered in a preset area of the channel; the separation liquid flow channel is one or more liquid flow channels corresponding to the preset area, and the residual liquid flow channels are other liquid flow channels except the separation liquid flow channel.
Optionally, the first flow rate is determined using the following formula:
Re＝ρvd/μ
where v is used to denote the first flow rate, re is used to denote the reynolds number, ρ is used to denote the density of the free liquid, μ is used to denote the viscosity of the free liquid, and d is used to denote the effective diameter of the channel.
Optionally, the lower limit of the reynolds number is selected from: 0.8 to 1.2; and/or, the upper limit of the Reynolds number is selected from: 20 to 28.
Optionally, the operation method of the cell separation device further comprises: and collecting the liquid flowing out of the separation liquid flow channel and serving as a diversion liquid for bearing the cells to be separated.
Optionally, the liquid to be separated is blood, and the method further comprises: collecting liquid flowing out of the residual liquid flow channel; wherein the liquid flowing out of the residual liquid flow passage can be returned to the living body after being treated.
Compared with the prior art, the technical scheme of the embodiment of the invention has the following beneficial effects:
in the embodiment of the invention, the flow position of the cells to be separated in the liquid to be separated is more close to the area of the separation liquid flow passage of the channel, the flow position of the cells to be separated in the liquid to be separated is more close to the area of the residual liquid flow passage of the channel, and then at least one separation liquid flow passage is arranged for flowing out of the cells to be separated of the channel, so that the partial flow liquid for bearing the cells to be separated is obtained, and the cells are effectively separated without damaging the cells in the liquid to be separated and introducing external liquid. Furthermore, as the basic cells in the separated residual liquid are not destroyed or doped with external liquid, the original components are not biochemically influenced, which is beneficial to improving the physiological cleanliness of the residual liquid.
Further, by setting the basal cells as erythrocytes; wherein the first volume ratio is selected from 30% to 60%; and/or the second volume ratio is selected from 40% to 70%, so that the liquid to be separated is closer to blood liquid, and separation of cells to be separated (such as nucleated cells) in blood is realized by adopting the scheme of the embodiment of the invention, and the separated residual blood is recovered and returned to the organism.
Further, when the cross-sectional shape of the channel is an ellipse, a rounded polygon, or a polygon, the effective diameter of the channel is a hydraulic diameter of the channel, such as 4 times the ratio of the cross-sectional area to the cross-sectional perimeter of the channel, so that the size of the channel can be effectively restricted when forming other channels than a circular channel. Further, the cross section of the channel adopts an arc line (such as a circle, an ellipse and a round corner polygon), so that the flow stability can be improved, and the flow position of cells to be separated in the liquid to be separated is further promoted to be more close to the area where the separation liquid flow channel of the channel is located.
Further, by setting the change of the effective diameter of the channel at the pressure change of the preset pressure not exceeding the preset change percentage, better balance between the viscosity and the smoothness of the liquid flow can be achieved, and the problem that the flow position of the cells to be separated is difficult to change due to too smooth liquid flow caused by too large flow inertia force is solved; and the problem that the flow of the liquid is too stagnant due to excessive flow viscosity is solved, and the flow position of the cells to be separated approaches to the side wall of the channel is solved.
Further, the first flow rate is controlled to flow through the channel, so that the Reynolds number of the liquid to be separated is in a preset range, the Reynolds number is influenced by the first flow rate to change on the premise that the effective diameters of the liquid to be separated and the channel meet the scheme of the embodiment of the invention, and the Reynolds number of the liquid to be separated can meet the requirement by controlling the first flow rate, so that the flowing position of the cell to be separated in the liquid to be separated is further promoted to be more close to the region where the separating liquid flow channel of the channel is located, and the flowing position of the basic cell is more close to the region where the residual liquid flow channel of the channel is located.
Drawings
FIG. 1 is a schematic diagram showing the structure of a cell separation device according to an embodiment of the present invention;
FIG. 2 is a schematic illustration of a planar spiral curved microtube configuration;
FIG. 3 is a schematic illustration of a three-dimensional helical curved microtube morphology;
FIG. 4 is a schematic illustration of a planar periodically curved microtube configuration;
FIG. 5 is a schematic representation of a three-dimensional self-twisting double-helical curved microtube morphology;
FIG. 6 is a schematic diagram showing the structure of another cell separation device according to an embodiment of the present invention;
FIG. 7 is a flow chart of a method of operating a cell separation device in accordance with an embodiment of the present invention;
FIG. 8 is a schematic diagram showing a state in which cells to be separated flow in a central region of a channel according to an embodiment of the present invention;
FIG. 9 is a schematic diagram showing the distribution of discrete positions of cells to be separated in a channel according to an embodiment of the present invention;
FIG. 10 is a graph showing the recovery of nucleated cells and hard particles at various outlets according to an embodiment of the present invention;
FIG. 11 is a schematic diagram showing the position distribution of cells to be separated according to the change of Reynolds number under the condition that the effective diameters of channels are the same and the basal cell pressure volumes are different in the embodiment of the invention;
FIG. 12 is a schematic diagram showing the position distribution of cells to be separated according to the change of Reynolds number under the condition that the effective diameters of channels are different and the basal cell pressure volumes are the same in the embodiment of the invention.
Detailed Description
Nucleated cells in blood play an important role in diagnosis and therapy. Nucleated cells in blood include white blood cells, nucleated red blood cells, circulating tumor cells, and the like. The type and the number of the leucocytes have important roles in clinical diagnosis, the fetal-derived nucleated red blood cells in the maternal can be used for prenatal diagnosis, the circulating tumor cells can be used for diagnosis and prognosis evaluation of cancers, and the leucocytes can also be used for diagnosis and treatment of diseases and the like. These diagnostic methods all require the acquisition of nucleated cells from the blood.
The existing cell separation technology mainly comprises a red blood cell lysis method, a centrifugation method and a microfluidic method.
In the erythrocyte lysis method, it is necessary to lyse erythrocytes by adding an erythrocyte lysate to blood, which damages erythrocytes in blood and cannot be used for in vivo blood treatment.
In the centrifugation method, nucleated cells or white blood cells in blood can be separated by using centrifugal force, however, the work flow is intermittent, a certain amount of blood must be extracted and then the blood is centrifuged, and more blood is extracted each time, so that the donor is in a state of losing part of the blood in the treatment process, and the separation cannot be performed or a certain physical burden is caused for individuals with a small blood amount such as infants or patients with poor physical conditions; while for non-human race, such a regimen is almost impossible for animals like experimental dogs or mice.
In microfluidic methods, a large amount of external liquid is introduced to dilute the blood, so that the cell-cell interaction of the blood is reduced, and its non-newtonian character is converted into newtonian fluid for further leukocyte separation.
The present inventors have studied and found that in the existing cell separation technique, it is often necessary to destroy cells in original blood or to introduce an external liquid, so that the remaining blood after separation cannot be reused, for example, so that the blood cannot be recovered and returned to the organism.
In the embodiment of the invention, the flow position of the cells to be separated in the liquid to be separated is more close to the area of the separation liquid flow passage of the channel, the flow position of the cells to be separated in the liquid to be separated is more close to the area of the residual liquid flow passage of the channel, and then at least one separation liquid flow passage is arranged for flowing out of the cells to be separated of the channel, so that the partial flow liquid for bearing the cells to be separated is obtained, and the cells are effectively separated without damaging the cells in the liquid to be separated and introducing external liquid. Furthermore, as the basic cells in the separated residual liquid are not destroyed or doped with external liquid, the original components are not biochemically influenced, which is beneficial to improving the cleanliness of the residual liquid.
In order to make the above objects, features and advantages of the present invention more comprehensible, embodiments accompanied with figures are described in detail below.
Referring to fig. 1, fig. 1 is a schematic diagram showing the structure of a cell separation device according to an embodiment of the present invention.
The cell separation device may include a channel 11, a flow rate control member 12, and a liquid flow channel 13, may further include a bottom plate 10, and may further include a fixing member 14.
Specifically, the channel 11 is used for flowing a liquid to be separated, which may include cells to be separated, basal cells, and free liquid.
Wherein the basal cells can be used to represent the most numerous cells, to distinguish them from a small number of cells to be separated, and the free liquid can be used to represent the liquid carrying the cells to be separated and the basal cells. It is noted that the liquid to be separated may also comprise other suitable cells.
Taking the liquid to be separated as blood as an example, the blood can contain red blood cells, nucleated cells, platelets and plasma. Wherein, red blood cells can be used as basic cells, plasma can be used as free liquid, nucleated cells can be used as cells to be separated, and platelets can be used as other cells.
Specifically, the ratio of the effective diameter of the cells to be separated to the effective diameter of the channel 11 may be equal to or greater than the first diameter ratio.
The effective diameter of the cells to be separated can be determined by a method of measuring a sample, and a plurality of measurement results are obtained after mathematical treatment.
Taking nucleated cells in blood as an example, the cell diameters can be measured by optical or electron microscopy, or diameter data can be obtained from literature studying similar cell populations.
As a non-limiting example, after obtaining a plurality of measurements, the center value of the gaussian distribution may be determined and used as the effective diameter of the cells to be isolated.
In a specific implementation, the effective diameter of the channel 11 can be obtained by optical or electron microscopy measurements.
Further, the channel 11 may satisfy one or more of the following: the cross-sectional shape of the channel 11 is selected from: round, oval, rounded polygon, and polygon; wherein, when the cross-sectional shape of the passage 11 is elliptical, rounded polygonal or polygonal, the effective diameter of the passage 11 is the hydraulic diameter of the passage 11.
Wherein the hydraulic diameter (hydraulic diameter) may be a comparison of the cross-sectional diameter of the non-circular channel, for example 4 times the ratio of the cross-sectional area to the cross-sectional perimeter.
In the embodiment of the present invention, when the cross-sectional shape of the channel 11 is an ellipse, a rounded polygon, or a polygon, the effective diameter of the channel 11 is the hydraulic diameter of the channel 11, such as 4 times the ratio of the cross-sectional area to the cross-sectional perimeter of the channel, so that the size of the channel 11 can be effectively limited when forming other channels than a circular channel. Further, the cross section of the channel 11 adopts an arc (such as a circle, an ellipse, and a rounded polygon), which can improve the flow stability, and further promote the flow position of the cells to be separated in the liquid to be separated to be more close to the area of the channel 11 where the flow channel of the liquid to be separated is located.
It should be noted that the effective diameter of the channel 11 should not be too large, otherwise it is difficult to meet the requirement that the ratio of the effective diameter of the cells to be separated to the effective diameter of the channel 11 is large; the effective diameter of the channel 11 should not be too small, otherwise larger cells to be separated may have difficulty passing through the channel 11.
As a non-limiting example, the liquid to be separated is blood, and when the cells to be separated are nucleated cells, the effective diameter of the channel 11 may be selected from 25 μm to 200 μm, for example, from 60 μm to 140 μm, for example, 100 μm.
As a non-limiting example, the first diameter ratio may be selected from: 0.06 to 0.6.
In the embodiment of the present invention, the ratio of the effective diameter of the cell to be separated to the effective diameter of the channel 11 is set to be larger, so that the flow position of the cell to be separated in the liquid to be separated is further promoted to be closer to the area where the separation liquid flow channel of the channel 11 is located, and the flow position of the basic cell is further promoted to be closer to the area where the rest liquid flow channel of the channel 11 is located.
In a specific implementation, the ratio of the total volume of the basal cells to the total volume of the liquid to be separated may be greater than or equal to a first volume ratio, and the ratio of the volume of free liquid in the liquid to be separated to the total volume of the liquid to be separated is less than or equal to a second volume ratio.
In a specific implementation, the first volume ratio is the total volume ratio of the basal cells, which may also be referred to as basal cell volume, which may be the hematocrit when the liquid to be separated is blood, as may be measured using centrifugation or a hemocytometer.
The centrifugation method can be that after liquid is filled into a glass microtube for centrifugal treatment, the liquid to be separated can be divided into three layers: the volume ratio of different components can be obtained by measuring the length ratio of the three layers of the plasma layer, the white membrane layer and the erythrocyte layer. The first volume ratio may be a ratio of a length of the red blood cell layer to a total length of the liquid.
The blood cell analyzer method may be to add a liquid to be measured to the blood cell analyzer to obtain said first volume ratio.
In a specific implementation, the second volume ratio is the volume of extracellular free liquid in the liquid to be separated, and may be determined using centrifugation or filtration.
The centrifugation method can be that after liquid is filled into a glass microtube for centrifugal treatment, the liquid to be separated can be divided into three layers: the volume ratio of different components can be obtained by measuring the length ratio of the three layers of the plasma layer, the white membrane layer and the erythrocyte layer. The second volume ratio may be a ratio of a length of plasma to a total length of liquid.
The filtration method may be to filter the liquid to be separated using a filter having a pore diameter smaller than all cell diameters to obtain the extracellular free liquid in the liquid to be separated, and the second volume ratio may be a ratio of a volume thereof to a total volume of the liquid to be separated.
As a non-limiting example, the base cells may be red blood cells and the first volume ratio may be selected from 30% to 60%, for example, may be 40% to 50%, for example, 44%; and/or the second volume ratio may be selected from 40% to 70%, for example may be 50% to 60%, for example 54%.
In the embodiment of the invention, the volume occupied by the basic cells in the liquid to be separated is relatively large, the volume occupied by the free liquid in the liquid to be separated is relatively small, the basic cells and the cells to be separated can generate cell-cell interaction, and the cells to be separated flow in an aggregation mode at a certain flow rate, so that the flowing position of the cells to be separated in the liquid to be separated is further promoted to be more close to the area of the separating liquid flow passage of the channel 11, and the flowing position of the basic cells is more close to the area of the remaining liquid flow passage of the channel 11.
It is noted that when there are a plurality of cells to be separated and having different effective diameters, the smallest effective diameter may be employed to achieve a larger ratio of the effective diameter of the cells to be separated to the effective diameter of the channel 11.
In the embodiment of the invention, the basic cells are erythrocytes; wherein the first volume ratio is selected from 30% to 60%; and/or the second volume ratio is selected from 40% to 70%, so that the liquid to be separated is closer to blood liquid, and separation of cells to be separated (such as nucleated cells) in blood is realized by adopting the scheme of the embodiment of the invention, and the separated residual blood is recovered and returned to the organism.
Further, the effective diameter of the channel 11 does not vary by more than a preset percentage of variation under a pressure variation of a preset pressure.
In a non-limiting example, taking the preset pressure as 1MPa as an example, the effective diameter of the channel 11 may be set to vary by no more than 4% to 6%, for example no more than 5%, under a pressure variation of 1 MPa.
In one embodiment, the channels 11 may be formed using quartz microtubes reinforced with an external polyimide coating, and the channels 11 may also be formed using other suitable flexible materials (e.g., polydimethylsiloxane, PDMS).
Further, the microtubes forming the channel 11 may be straight pipes or curved pipes.
Still further, the morphology of the microtubes may be selected from: planar wave bending, three-dimensional wave bending, planar helical bending, three-dimensional self-twisting double helix, and planar periodic bending.
Referring to fig. 2-5 in combination, fig. 2-5 are schematic views of four alternative microtube configurations in accordance with embodiments of the present invention.
Referring to fig. 2, fig. 2 is a schematic view of a planar spiral curved microtube configuration.
In particular, in the microtube with the spiral bending plane, the adoption of the circular interface channel eliminates the flow stagnation areas of the near-wall areas at four corners in the rectangular fluid channel, so that the flow in the section is more uniform, and coagulation is not easy to form; the effective flow area wrapped by the perimeter of the boundary of the same cross section is larger, the flow resistance is smaller, and the separation is more effective; meanwhile, the axisymmetric wall surface boundary enables the internal flow field to be uniform, stable dean vortex under lower Reynolds number is easier to form in a bent pipeline, and the separation of cells with target diameter can be realized in a larger flow range by combining with proper pipeline diameter, so that the separation robustness is greatly enhanced; and the PDMS pipeline does not need to be assisted by other surfaces, and forms a closed passage, so that the overall size of a chip manufactured by adopting the PDMS microtube is smaller and compact.
As a non-limiting example, the parameters of the planar spiral curved microtube may be selected from: the initial bending radius is 0.5-2 mm and the length is 20-100 mm.
It should be noted that fig. 2 shows only one alternative planar spiral-curved microtube configuration, and in practice, a convex spiral-curved microtube configuration or a concave spiral-curved microtube configuration may be used, for example, with the spiral central region being lower or higher than the spiral edge region.
Referring to fig. 3, fig. 3 is a schematic view of a three-dimensional helical curved microtube morphology.
Specifically, the bending radius of the three-dimensional spiral bent microtube is consistent along the advancing direction of the screw thread, and is not limited by the photoetching technology, and the PDMS pipeline can form a closed passage by itself without the assistance of other surfaces, so that a three-dimensional structure like a spiral shape is formed. The three-dimensional spiral structure can also generate larger secondary flow Dien vortex, so that separation under small flow can be effective, the area in the plane of the separation device can be reduced, the efficiency of particle separation is improved by efficient secondary flow, long pipelines are not needed, and the whole separation device is improved in structure and efficiency, so that the separation device can be made into compact micro devices.
Referring to fig. 4, fig. 4 is a schematic view of a planar periodically curved microtube configuration.
In particular, a planar periodically curved microtube is a rule that particles gather into a streamline in continuously bending flow by utilizing inertia during movement of the particles with fluid. The two-dimensional plane rectangular pipeline microfluidic chip can be used for realizing different density particle aggregation lines, and the PDMS microtubes can be adopted at present to realize the purpose more simply, conveniently and efficiently.
As a non-limiting example, the parameters of the planar periodically curved microtubes may be selected from: the length is 20-200 mm, and the bending radius is 0.5-2 mm.
It should be noted that fig. 4 shows only one alternative planar periodically curved microtube configuration, and in particular implementations, a three-dimensional periodically curved microtube configuration may be used, for example, with one end of the periodic curve being higher or lower than the other end.
Referring to fig. 5, fig. 5 is a schematic view of a three-dimensional self-twisting double-spiral bent microtube.
The three-dimensional self-twisting double-helix bent microtube is based on the robustness of a three-dimensional helix bent microtube chip, in an emergency, PDMS microtubes are simply folded in half and then twisted, so that the two microtubes form a double helix by taking the microtubes as an axis, and the purpose of inducing secondary flow Dien vortex and realizing separation is achieved.
As a non-limiting example, the parameters of the three-dimensional self-twisting double-helical curved microtube may be selected from: the twist radius is 100-500 microns.
It is noted that in practice other suitable curved microtubes may be used, such as planar wavy curves, three-dimensional wavy curves, wherein the three-dimensional wavy curved microtubes may be, for example, wavy curved with one end higher or lower than the other.
Wherein the bending mode of the planar wave bending and the three-dimensional wave bending can be periodic or aperiodic.
Wherein the microtubes may be placed on the bottom plate 10, the liquid flow channels 13 are fixed on the bottom plate 10, for example, using an adhesive, the microtubes are fixed on the bottom plate 10 using a fixing member 14, such as a microtube fixing block made of Polydimethylsiloxane (PDMS), and linearity of the microtubes is maintained.
In the embodiment of the invention, the channel 11 is formed by adopting the quartz microtube reinforced by the external polyimide coating, so that better balance between viscosity and smoothness of liquid flow can be achieved, and the problem that the flow position of cells to be separated is difficult to change due to too smooth liquid flow caused by too large flow inertia force is solved; and reduces the problem that the flow of the liquid is too stagnant due to excessive flow viscosity, and the flow position of the cells to be separated approaches the side wall of the channel 11.
In a specific implementation, the flow rate control means 12 may be used to control the flow rate of the liquid to be separated through the channel 11.
In one embodiment, the flow rate control unit 12 may be a syringe pump, and the first flow rate may be effectively controlled by pumping the liquid to be separated in the sample injector into the channel 11 by using the syringe pump.
In another embodiment, the flow rate control unit 12 may also be a suction pump, so as to effectively control the first flow rate by pumping the liquid to be separated in the channel 11.
In a specific implementation, a plurality of liquid flow channels 13 may be coupled to the channel 11, wherein the liquid flow channels 13 may comprise separate liquid flow channels 131 and remaining liquid flow channels 132, at least one separate liquid flow channel 131 being for liquid flowing out of a central region of the channel 11.
Further, the separation liquid channel 131 may be one or more channels, and since the flow position of the cells to be separated in the liquid to be separated is closer to the area of the separation liquid channel of the channel 11, the liquid flowing out from the separation liquid channel 131 can more support the cells to be separated.
The residual liquid flow channel 132 may be one or more flow channels, and the multiple residual liquid flow channels 132 may be located on the same plane as the separation liquid flow channel 131, or may be a three-dimensional structure with different planes. Since the flow position of the basal cell is closer to the area of the channel 11 where the residual liquid flow channel is located, the liquid flowing out from the residual liquid flow channel 132 can bear more basal cells.
In the embodiment of the invention, by arranging the liquid to be separated, the channel 11 through which the liquid to be separated flows, the plurality of liquid channels 13 coupled with the channel 11, and the ratio of the effective diameter of the cell to be separated to the effective diameter of the channel 11 is large, the volume occupied by the basic cell in the liquid to be separated is large, the volume occupied by the free liquid in the liquid to be separated is small, the flow position of the cell to be separated in the liquid to be separated is more close to the area of the separating liquid channel of the channel 11, the flow position of the basic cell is more close to the area of the remaining liquid channel of the channel 11, and then by arranging at least one separating liquid channel 131 for flowing out of the cell to be separated of the channel 11, the part of the split liquid bearing the cell to be separated is opportunely obtained, so that the cell to be separated is effectively separated without damaging the cell in the liquid to be separated and without introducing external liquid. Furthermore, as the basic cells in the separated residual liquid are not destroyed or doped with external liquid, the original components are not biochemically influenced, which is beneficial to improving the cleanliness of the residual liquid.
Referring to fig. 6, fig. 6 is a schematic diagram showing the structure of another cell separation device according to an embodiment of the present invention. The other cell separation device may include a base plate 10, a channel 11, a flow rate control part 12, and a liquid flow channel 13 shown in fig. 1, and may further include an optical device, an image sensor, and a data processing module.
Wherein, a channel 11 through which the liquid to be separated flows can be arranged on the separated bottom plate 10, a flow rate control component 12 can be used for controlling the flow rate of the liquid to be separated flowing through the channel 11, and the cells to be separated and the carrier liquid thereof in the channel 11 can flow out through the channel of the liquid to be separated, for example, a collecting device (such as a test tube) can be used for collecting.
It should be noted that, since the cells to be separated may be located in the central region of the channel 11 or may be located in the middle region of the channel 11 (e.g., adjacent to the central region and on one side), the types of cells collected by each collecting device may not be limited in advance, but it may be determined which separating liquid flow channel and its collecting device to collect after collecting experimental data or after determining the collecting position of the cells to be separated based on historical empirical data.
The optical device may comprise, for example, an optical microscope or an electron microscope, and may also employ a Light Emitting Diode (LED) to observe the effective diameter of the channel 11 and the morphology of the liquid to be separated flowing within the channel 11. The image sensor and the data processing module can acquire images and conduct data analysis.
Referring to fig. 7, fig. 7 is a flowchart of a method of operating a cell separation device in accordance with an embodiment of the present invention. The operation method of the cell separation device may include step S31, may further include step S32, and may further include step S33:
step S31: controlling the liquid to be separated to flow through the channel at a first flow rate so that the Reynolds number of the liquid to be separated is in a preset range;
step S32: collecting the liquid flowing out of the separation liquid flow channel and taking the liquid as a diversion liquid for bearing the cells to be separated;
step S33: and collecting the liquid flowing out of the residual liquid flow channel.
In a specific implementation of step S31, the flow rate control means shown in fig. 1 or 6 may be used to control the flow of the liquid to be separated through the channel at the first flow rate.
Further, the first flow rate may be determined using the following formula:
Re＝ρvd/μ
where v is used to denote the first flow rate, re is used to denote the reynolds number, ρ is used to denote the density of the free liquid, μ is used to denote the viscosity of the free liquid, and d is used to denote the effective diameter of the channel.
From the above, it can be seen that the reynolds number is changed by the influence of the first flow rate on the premise that the effective diameter of the liquid to be separated and the channel meet the scheme of the embodiment of the invention. When the Reynolds number exceeds the upper limit value, the cells to be separated are more likely to be located on a ring centered on the axis of the channel than to be gathered in the central region of the channel, resulting in difficulty in separation from the separation liquid flow channel; when the reynolds number is lower than the lower limit value, the cells to be separated are more likely to be located at the side wall of the channel than to be gathered in the central region of the channel, resulting in difficulty in separation from the separation liquid flow channel.
Further, the reynolds number of the liquid to be separated may be located within a first subinterval so that the cells to be separated are concentrated in a central region of the channel; wherein the separated liquid flow channel is one or more liquid flow channels corresponding to the central area, and the rest liquid flow channels are other liquid flow channels except the separated liquid flow channel.
The first subinterval may be determined according to historical empirical data, or may be determined through experimental data.
Further, the reynolds number of the liquid to be separated may be located in a second subinterval, so that the cells to be separated are gathered in a preset area of the channel; the separation liquid flow channel is one or more liquid flow channels corresponding to the preset area, and the residual liquid flow channels are other liquid flow channels except the separation liquid flow channel.
The first subinterval may be determined according to historical empirical data, or may be determined through experimental data.
It will be appreciated that by adjusting the first flow rate, the density of the free liquid, the viscosity of the free liquid and the effective diameter of the channel, the reynolds number can be adjusted and by having the reynolds numbers lie in different preset ranges (e.g. in the first sub-zone or the second sub-zone) an aggregated flow of cells to be separated is achieved and out through the separation liquid flow channel.
It should be noted that for cells of different hardness to be isolated, having different cell aspect ratios in the flow, a preset range of respective reynolds numbers may be provided.
For example, when the aspect ratio of the cells to be separated in the flow is equal to or greater than a preset threshold, the degree of deformation may be considered to be large, and the upper limit value of the reynolds number may be high; when the aspect ratio of the cells to be separated in the flow is smaller than the preset threshold, the deformation degree can be considered to be smaller, and the upper limit value of the Reynolds number can be lower.
Further, when the aspect ratio of the cells to be separated in the flow is large (for example, nucleated cells in blood), the lower limit of the reynolds number may be selected from: 0.8 to 1.2, for example 1; and/or, the upper limit of the reynolds number may be selected from: 20 to 28, for example 24.
Where the aspect ratio of the cells to be separated in the flow is small (e.g., organic polymer microspheres), the lower limit of the Reynolds number may be selected from: 0.8 to 1.2, for example 1; and/or, the upper limit of the reynolds number may be selected from: 4 to 8, for example 6.
In the embodiment of the invention, the reynolds number of the liquid to be separated flows through the channel by controlling the first flow rate so that the reynolds number of the liquid to be separated is in a preset range, and on the premise that the effective diameter of the liquid to be separated and the channel meet the scheme of the embodiment of the invention, the reynolds number is influenced by the first flow rate to change, and the reynolds number of the liquid to be separated can meet the requirement by controlling the first flow rate, so that the flowing position of the cell to be separated in the liquid to be separated is further promoted to be more close to the region where the separating liquid flow channel of the channel is located, and the flowing position of the basic cell is more close to the region where the residual liquid flow channel of the channel is located.
In practice, if the fluid to be separated is blood, it may also be sterilized by rinsing with 75% ethanol, then rinsing with Phosphate Buffer (PBS) to remove ethanol, and then wetting with 5% Bovine Serum Albumin (BSA) containing 10ug/ml tirofiban to prevent cell and platelet adhesion in the fluid to be separated, before flowing through the fluid to be separated using the channel.
Referring to fig. 8, fig. 8 is a schematic view showing a state in which cells to be separated flow in a central region of a channel according to an embodiment of the present invention.
As can be seen from fig. 8, when the ratio of the total volume of the base cells to the total volume of the liquid 41 to be separated is equal to or greater than the first volume ratio, and the ratio of the volume of the free liquid in the liquid 41 to the total volume of the liquid 41 to be separated is equal to or less than the second volume ratio, the ratio of the effective diameter D of the channel 42 to the effective diameter D of the channel 42 is equal to or greater than the first diameter ratio, and the reynolds number of the liquid 41 to be separated is within the preset range, the flowing position of the liquid 41 to be separated in the channel 42 is more similar to the central region of the channel 42, and the flowing position of the base cells is more similar to the edge region of the channel 42.
Referring to FIG. 9 in combination, FIG. 9 is a schematic diagram showing the distribution of discrete positions of cells to be separated in a channel according to an embodiment of the present invention.
It can be seen that the flow position of the cells to be separated is more approximate to the central region of the channel, which approximates to a gaussian distribution, and the central value of the gaussian distribution can be used as the position of the cells to be separated in the channel, so that the flow channel of the separation liquid is provided with better theoretical support.
Referring to fig. 10, fig. 10 is a graph showing the recovery rate of nucleated cells and hard particles at various outlets according to an embodiment of the present invention.
From the graph, the particle hardness of the cells has an influence on the equilibrium position of the particles, and different particle hardness have the optimal Reynolds number interval.
In a specific application of the embodiment of the invention, blood is used as the liquid to be separated, a channel with an effective diameter of 100 μm is used, and under the condition of retaining about 4/5 of blood, the hard fluorescent particles obtain the optimal collecting position with the Reynolds number Re=2 and the flow rate Q=0.67 ml/h, and the recovery efficiency is as high as 92%.
In addition, taking the cell to be separated as nucleated cell MCF7 and hardened MCF7 as an example, when Re=12 and Q=4ml/h, the position aggregation of the cell to be separated in the channel is higher and is positioned in the central area, so that a better recovery effect is obtained, and the recovery efficiency reaches 94.8%.
Referring to fig. 11, fig. 11 is a schematic diagram showing a position distribution of cells to be separated according to a change of reynolds number under the condition that effective diameters of channels are the same and basal cell pressure volumes are different in an embodiment of the present invention.
As a non-limiting example, erythrocytes in blood are used as the basal cells, and the effective diameter of the channel is 100 μm, and the hematocrit is 0, 0.15, 0.3, 0.45, respectively.
In a specific application of the embodiment of the present invention, blood (for example, ht=45%) is used as the liquid to be separated, and the liquid is subjected to non-newton effect and cell-cell interaction so that the liquid can better perform in separating nucleated cells (for example, circulating tumor cells CTCs), and at a specific flow rate, the circulating tumor cells have a narrower cell distribution width in the blood (ht=45%) which is diluted in the blood (Ht < 45%) and are more beneficial to separating from basic cells.
Referring to fig. 12, fig. 12 is a schematic diagram showing a position distribution of cells to be separated according to a change of reynolds number under the condition that effective diameters of channels are different and a basal cell pressure product is the same in the embodiment of the present invention.
As a non-limiting example, erythrocytes in blood are used as the basal cells, and the effective diameter of the channel is 100 μm, 150 μm, 200 μm, and the hematocrit is 0.45.
In the embodiment of the invention, the flow position of the cells to be separated in the liquid to be separated can be promoted to be more approximate to the central area of the channel and the flow position of the basic cells is more approximate to the edge area of the channel by setting the ratio of the effective diameter of the cells to be separated to the effective diameter of the channel to be larger, the occupied volume of the basic cells in the liquid to be separated to be larger, the occupied volume of the free liquid in the liquid to be separated to be smaller and adopting the proper first flow rate to realize the preset range of the Reynolds number.
Then, by providing at least one separation liquid flow channel for outflow of the cells to be separated, there is an opportunity to obtain a fraction of the split liquid carrying the cells to be separated, thereby efficiently separating the cells without damaging the cells in the liquid to be separated and without introducing extraneous liquid.
Further, there is also an opportunity for the separated residual liquid to be reused since the basal cells in the separated residual liquid are not destroyed or doped with foreign liquid.
It should be understood that the term "and/or" is merely an association relationship describing the associated object, and means that three relationships may exist, for example, a and/or B may mean: a exists alone, A and B exist together, and B exists alone. In this context, the character "/" indicates that the front and rear associated objects are an "or" relationship.
The term "plurality" as used in the embodiments herein refers to two or more.
The first, second, etc. descriptions in the embodiments of the present application are only used for illustrating and distinguishing the description objects, and no order division is used, nor does it indicate that the number of the devices in the embodiments of the present application is particularly limited, and no limitation on the embodiments of the present application should be construed.
Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the invention, and the scope of the invention should be assessed accordingly to that of the appended claims.
Claims (12)
1. A cell separation device, comprising:
a channel through which a liquid to be separated flows, wherein the liquid to be separated comprises cells to be separated, basic cells and free liquid, and the ratio of the effective diameter of the cells to be separated to the effective diameter of the channel is greater than or equal to a first diameter ratio;
a flow rate control part for controlling a flow rate of the liquid to be separated flowing through the channel;
a plurality of liquid flow channels coupled to the channel, wherein the liquid flow channels comprise a separation liquid flow channel for flowing out the cells to be separated and a residual liquid flow channel for flowing out residual liquid, at least one separation liquid flow channel being used for flowing out the cells to be separated of the channel;
The ratio of the total volume of the basic cells to the total volume of the liquid to be separated is greater than or equal to a first volume ratio, and the ratio of the volume of free liquid in the liquid to be separated to the total volume of the liquid to be separated is less than or equal to a second volume ratio.
2. The cell separation device of claim 1, wherein the basal cell is a red blood cell;
wherein the first volume ratio is selected from 30% to 60%;
and/or the number of the groups of groups,
the second volume ratio is selected from 40% to 70%.
3. The cell separation device of claim 1, wherein the channel satisfies one or more of:
the cross-sectional shape of the channel is selected from: round, oval, rounded polygon, and polygon;
when the cross section of the channel is elliptical, rounded polygonal or polygonal, the effective diameter of the channel is the hydraulic diameter of the channel.
4. A cell separation device according to claim 1 or 3, wherein the effective diameter of the channel is selected from 25 μm to 200 μm.
5. The cell separation device according to claim 1, wherein,
the effective diameter of the passageway does not vary by more than a predetermined percentage of variation under a pressure variation of a predetermined pressure.
6. A method of operating a cell separation device according to any one of claims 1 to 5, comprising:
and controlling the liquid to be separated to flow through the channel at a first flow rate so that the Reynolds number of the liquid to be separated is in a preset range.
7. The method of claim 6, wherein the reynolds number of the liquid to be separated is located in a first subinterval so that the cells to be separated are concentrated in a central region of the channel;
wherein the separated liquid flow channel is one or more liquid flow channels corresponding to the central area, and the rest liquid flow channels are other liquid flow channels except the separated liquid flow channel.
8. The method of claim 6, wherein the reynolds number of the liquid to be separated is located in a second subinterval so that the cells to be separated are collected in a predetermined region of the channel;
the separation liquid flow channel is one or more liquid flow channels corresponding to the preset area, and the residual liquid flow channels are other liquid flow channels except the separation liquid flow channel.
9. The method of operating a cell separation device of claim 6, wherein the first flow rate is determined using the formula:
Re＝ρvd/μ
where v is used to denote the first flow rate, re is used to denote the reynolds number, ρ is used to denote the density of the free liquid, μ is used to denote the viscosity of the free liquid, and d is used to denote the effective diameter of the channel.
10. The method of claim 6, wherein the lower limit of the reynolds number is selected from the group consisting of: 0.8 to 1.2;
and/or the number of the groups of groups,
the upper limit of the Reynolds number is selected from: 20 to 28.
11. The method of operating a cell separation device of claim 6, further comprising:
and collecting the liquid flowing out of the separation liquid flow channel and serving as a diversion liquid for bearing the cells to be separated.
12. The method of operating a cell separation device according to claim 6, wherein the liquid to be separated is blood, the method further comprising:
collecting liquid flowing out of the residual liquid flow channel;
wherein the liquid flowing out of the residual liquid flow passage can be returned to the living body after being treated.
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