JP2012100625A - Cell enrichment method for cell separation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enrich a large size cell on the basis of difference in cell sizes by only the flow in a branched micro flow path without physically or chemically influencing cells.SOLUTION: When a cell solution introduced in one micro flow path is made to flow passing through the separation chamber and separated into two branch flow paths, large size cells go into one path and small size cells go into both paths. Therefore, in the solution which has passed one path, large size cells are enriched.

Description

  The present invention relates to cell separation / enrichment in the fields of biology, regenerative medical engineering, and medicine.

  Selecting and extracting cells having specific characteristics from many types of cells mixed in a solution is extremely important in areas such as cell biology research, pathological diagnosis / treatment, and regenerative medical engineering. The most widely used methods for this cell separation are the specific gravity method, filter method, or high-speed flow cytometer based on fluorescent staining of the cells to extract more specific cells. It has been. However, the above methods give strong physical / chemical manipulations to the target cells, causing damage to the cells, and problems such as increased energy consumption, high cost, and adverse environmental impact.

  In recent years, research and development of microchannels and microanalyzers have been actively conducted due to advances in semiconductor microfabrication technology and expansion of application areas. Among them, a cell separation system based on microfluidics (microfluidics) was also reported. For example, when a cell introduced into one micro flow channel is separated into two branch flow channels, the flow rate is controlled by an electroosmotic flow pump, and the cells are extracted from the subsequent flow channel (Patent Document 1). In addition, a large number of right-angled branch microchannels are connected to both sides of the inflow microchannel, and particles contained in the inflow solution are selected and extracted by particle size by controlling the flow rate of each branch channel (Non-patent Document 1). ) When the whole blood flows through the microchannel, the movement along the wall of the large white blood cell is used to enrich the leukocytes in the liquid flowing into the micro branch channel (Non-patent Document 2). .

 Each of the above examples is a small and inexpensive cell separation device based on micro branching channels by microfabrication. In particular, the latter two use only a micro flow to enrich large cells (particles). I can do it. However, the problem is that the enrichment rate is still low, and according to each paper, the enrichment rate is less than 50 times.

JP 2004-113223 A

MYamada and M Seki. Lab Chip, 2005, 5: 1233-1239 SS Shevkoplyas, et.al .. Anal Chem2005, 77: 933-939

In the conventional method, the cells are identified and separated by physical or biochemical means such as fluorescent staining or voltage application when the cells are separated. This damages cells, increases costs, and adversely affects the environment.

It is an object of the present invention to provide means for separating and enriching cells using an inexpensive disposable substrate without causing electrical or chemical stimulation or adverse effects on cells.

A cell solution containing a plurality of cell groups having different average sizes is circulated in a flow path structure in which a separation chamber is provided with an inflow channel for inflow of cell solution and an outflow channel for outflow of inflowed cell solution. A cell enrichment method that enriches a cell group having the maximum average size and has the following characteristics (1) to (4).
(1) The channel width of the inflow channel is set to 1 to 3 times the average diameter of the cell group having the maximum average size.
(2) The separation chamber has a structure in which the flow rate of the cell solution is slower than the flow rate in the inflow channel.
(3) The streamline distribution in the separation chamber is asymmetric with respect to the mainstream line (streamline with the highest flow velocity).
(4) The channel width of the outflow channel having a smaller outflow rate is set to be equal to or larger than the average diameter of the cell group having the maximum average size.

Unlike the prior art, the present invention makes it possible to separate and enrich large-sized cells using only the flow in the microchannel by utilizing the difference in cell size.

It is an example (schematic diagram) of the microchannel structure used in the present invention. It is a figure which shows the flow line figure of the hydrodynamic analysis in the microchannel structure of FIG. 1, and the flow locus | trajectory of a large cell. It is a pressure distribution map in the separation chamber containing a large cell. It is a figure which shows the total pressure which the big cell received, and the changed speed direction. It is a figure which shows an example of embodiment of this microchannel structure used by this invention. It is a figure which shows an example of the modification of this microchannel structure used by this invention. It is a figure which shows an example of the modification of this microchannel structure used by this invention. It is a figure which shows an example of the modification of this microchannel structure used by this invention.

The present invention is described below with reference to the accompanying drawings.

FIG. 1 is an example of a microchannel structure used in the cell enrichment method according to the present invention. In the microchannel structure, an inflow channel 100 through which a cell solution flows in and two outflow channels 102 and 103 through which the cell solution flows out are provided for the separation chamber 101.

FIG. 2 is a streamline diagram showing a flow state when a fluid is caused to flow through the microchannel shown in FIG. The streamline diagram can be created not only based on the measurement in the actual flow path but also by a known hydrodynamic analysis.

As shown in FIG. 2, in each cross section of the inflow channel 100, the largest flow velocity is observed near the center of the channel due to friction between the fluid and the channel wall. In the present invention, a line obtained by connecting points having the fastest flow velocity in each cross section of the inflow channel 100 is defined as a “mainstream line”. In the micro-channel structure shown in FIG. 1, as shown in FIG. 2, an inflow channel 100, a separation chamber 101, an outflow channel 102, and an outflow channel 103 into which a main stream line introduced from the inflow channel enters are configured. Has been.

In the present invention, when the cell solution containing a plurality of cell groups having different average sizes is introduced from the inflow channel 100 to the microchannel structure configured as described above, the channel width of the inflow channel 100 Is adjusted to about 1 to 3 times the diameter of a cell group having the maximum average size contained in the cell solution (hereinafter simply referred to as “maximum cell”). By using the microchannel configured as described above, the maximum cell flowing through the inflow channel 100 is limited to be positioned at approximately the center of the channel width of the inflow channel 100. The cells will move along the main stream line in the inflow channel 100. Then, the largest cell once along the main stream line moves away from the main stream line due to the pressure difference in the separation chamber 101 and flows into the outflow channel 102 different from the outflow channel 103 of the main stream line. A higher enrichment rate can be obtained by streamline crossing movement away from the mainstream line. At this time, by setting the flow channel width of the outflow channel 102 to a size that allows the maximum cells to flow, almost all of the maximum cells flow out through the outflow channel 102.

On the other hand, the relatively small cells contained in the cell solution are distributed almost uniformly in the width direction in the inflow channel 100, and therefore do not necessarily flow into the outflow channel 102, and a part of the cells flows into the outflow channel 103. And will be separated from the largest cell. Therefore, the largest cell in the cell solution flowing out from the outflow channel 102 is enriched.

In the microchannel structure shown in FIG. 1, a neck portion is provided in the middle of the outflow channel 103, and an extended portion is provided thereafter. In this way, by appropriately setting the shapes and positions of the separation chamber 101 and the outflow channels 102 and 103 with respect to the inflow channel 100 in which the channel width and the like are determined according to the cell solution to be introduced, The streamline distribution in the channel can be adjusted and the rate of enrichment of the largest cells in the cell solution can be determined.

FIG. 3 shows the pressure distribution experienced by the largest cell in the separation chamber 101.

A pressure gradient is created in the separation chamber, and as shown in FIG. 4, an angle is formed with the main stream line of the solution in the direction of the total pressure received by the large-sized cell, so that the cell has power over the lower stream line. The direction of speed changes from the original streamline direction to the bottom. Therefore, even if the main stream line enters the outflow channel 103, the largest cell can flow into the outflow channel 102. This further increases the enrichment rate of large size cells passing through the outflow channel 102.

A specific flow channel structure that realizes the present invention can be sufficiently produced by a conventional microfabrication technique. For example, a flow path / chamber on the order of several tens of μm is fabricated by using a technique such as photoresist or laser etching on a substrate such as glass, silicon, plastic, or polyimide resin.

FIG. 5 shows a specific example of this microchannel structure. The solution contains rat adipose tissue-derived stem cells: about 14 μm in diameter and erythrocytes: about 6 μm in diameter. The width of the inflow channel 100 is 25 μm, and the shape of the separation chamber 101 is as shown in FIG. The width of the outflow channel 102 is 40 μm, and the width of the outflow channel 103 is 14 μm at a minimum, and is expanded to a width of 100 μm through a neck portion having a length of 40 μm.

Through this structure once, the enrichment rate of the stem cells is about twice that of the inflow side. Furthermore, if this method is repeated ⁿ times, the enrichment rate becomes about ²ⁿ times.

FIG. 6 shows one modification of this microchannel structure. The shape of the micro inflow channel is curved, and it is possible to further increase the enrichment rate by using centrifugal force to further control the trajectory of large cells.

FIG. 7 shows one modification of this microchannel structure. The shape of the micro inflow channel is tapered, making the distribution of small cells in the channel width direction non-uniform, increasing the outflow rate to the outflow channel 103, and enriching large cells It is possible to increase the rate.

FIG. 8 shows one modification of the microchannel structure. By utilizing the fact that large-sized cells flow along the upper wall when flowing into the outflow channel 102, and further branching the 102 channel, large-sized cells flow out into the branch channel 104. The enrichment rate of large cells in the road is further increased.

As described above, based on the difference in cell size, the cells are not affected physically or chemically, and only by the flow in the branch microchannel, for example, in a mixed solution of rat stem cells and erythrocytes, It has been confirmed that stem cells can be enriched. Because this channel structure is small and inexpensive, it does not damage cells, saves energy, is low cost, and is environmentally friendly in the field where cell separation / enrichment is required in the fields of biology, regenerative medicine, and medicine. Easy cell separation and enrichment process is possible.

Claims (1)

A cell solution containing a plurality of cell groups having different average sizes is circulated in a flow path structure in which a separation chamber is provided with an inflow channel for inflow of cell solution and an outflow channel for outflow of inflowed cell solution. A cell enrichment method for enriching a cell population having a maximum average size,
(1) The channel width of the inflow channel is 1 to 3 times the average diameter of the cell group having the maximum average size,
(2) In the separation chamber, the flow rate of the cell solution is slower than the flow rate in the inflow channel,
(3) The streamline distribution in the separation chamber is asymmetric with respect to the mainstream line (streamline with the highest flow velocity),
(4) The width of the outflow channel having a smaller outflow rate is set to be equal to or larger than the average diameter of the cell group having the maximum average size.
A cell enrichment method having the characteristics of (1) to (4) above.