Patent Description:
In the past, as methods for isolating a target biological sample from a specific biological sample, although the selection is suitably made according to the kind of the target biological sample, various methods are known, including a membrane separation method, a centrifugal separation method, an electrical separation method, a method in which biological samples other than the target biological sample are killed, a magnetic bead method in which the target biological sample is labeled with a magnetic bead and separated, flow cytometry, and the like.

As a method for isolating a target biological sample using the magnetic bead method, the method disclosed in NPL <NUM> is known.

According to this method, T cells, which are target biological samples, are labeled with magnetic beads, and the target biological samples are isolated on the basis of the magnetic beads.

In addition, as an isolation device utilizing the flow cytometry, the device shown in PTL <NUM> is known. PTL <NUM> discloses a microchip-type isolation device, which forms a sheath flow in a channel formed in a microchip made of plastic and glass, etc., to perform analysis.

In the isolation device disclosed in PTL <NUM>, a sample channel, through which a fluid containing microparticles passes, and an orifice, which discharges the fluid from the sample channel into the space outside the chip, are formed by attaching substrate layers together. The isolation device includes: a microchip formed of the lumen of a microtubule formed of the sample channel of the orifice unit embedded between the substrate layers; a vibration element for making the fluid into droplets and discharging the same in the orifice; a charging section that applies an electrical charge to the droplets discharged; an optical detection section that irradiates microparticles passing through the sample channel with light on the upstream side of the orifice in the fluid delivery direction and detects light emitted from the microparticles; a pair of electrodes arranged along the moving direction of droplets discharged into the space outside the chip and facing each other via the moving droplets; and at least two recovery sections that recover droplets that have passed between the pair of electrodes. In the sample channel between a light irradiation unit to be irradiated with light from the optical detection section and the orifice unit, a conversion channel whose cross-sectional shape changes from a square shape to a circular shape in the direction of fluid delivery is formed. <CIT> describes an apparatus for isolation of particles, which comprises a separation unit and which separates a given type of particles from a sample.

However, in the past isolation methods, such as a membrane separation method, a centrifugal separation method, an electrical separation method, a method in which biological samples other than the target biological sample are killed, and a magnetic bead method in which the target biological sample is labeled with a magnetic bead and separated, there has been a problem in that the degree of purification of the isolation of target biological samples is low. For example, in the case of the centrifugal separation method, when a target biological sample is isolated from a cell suspension, there is a possibility that samples other than the target biological sample may be incorporated.

In addition, for example, in the magnetic bead method, there has been a problem in that upon mixing of an isolation object sample with magnetic beads, when the magnetic beads are not sufficiently joined to the target biological sample, a certain amount of target biological sample is not isolated.

Further, in so-called flow cytometer as described in PTL <NUM>, the droplets fly in the space, leading to a problem in that the flow cytometer and the surrounding environment are contaminated with a mist containing the isolation object biological sample. In addition, because the isolation mechanism is in contact with the external atmosphere, there also has been a problem in that other substances in the external atmosphere are incorporated into the biological sample after isolation. Therefore, there has been a problem in that it is difficult to use a flow cytometer for immune cell therapy and the like. Thus, there is a need for providing a sample isolation kit that allows a target biological sample to be isolated and stored in an enclosed space, and also a sample isolation device.

According to an embodiment of the present technology, the isolation of a target biological sample and the storage of the target biological sample can be implemented in an enclosed space, whereby the degree of purification of the isolation of target biological samples can be improved. In addition, the contamination of a sample isolation device, etc., with a mist containing a target biological sample and/or the incorporation of other substances into the isolated target biological sample can be prevented.

Incidentally, the effects described herein are not necessarily limited, and may be any of the effects described in the present technology.

Hereinafter, best modes for carrying out the present technology will be described with reference to the drawings.

The embodiments described below show examples of typical embodiments of the present technology, and do not narrow the interpretation of the scope of the present technology.

Incidentally, the description will be given in the following order.

A first embodiment of the sample isolation kit according to an embodiment of the present technology will be described using <FIG>. The sample isolation kit <NUM> according to an embodiment of the present technology at least includes a housing unit <NUM>, an isolation unit <NUM>, and a storage unit <NUM>. The housing unit <NUM>, the isolation unit <NUM>, and the storage unit <NUM> are connected to each other through a hermetically sealing unit <NUM>. In addition, as necessary, the sample isolation kit <NUM> may also include a labeling unit <NUM>, a biological sample housing unit <NUM>, a separation unit <NUM>, a sheath container <NUM>, and a disposal unit <NUM>. Each unit will be described hereinafter.

The sample isolation kit <NUM> according to an embodiment of the present technology includes a housing unit <NUM>. In the housing unit <NUM>, an isolation object sample, which is the object of the isolation unit <NUM>, is housed. The housing unit <NUM> is composed of, for example, a cylindrical tubular body, which has an opening at one end, and a lid portion, which fits into the tubular body and blocks the opening. Then, the lid portion has formed therein a plurality of opening valves for housing the isolation object sample in the tubular body, and each opening valve employs the configuration of a check valve. Therefore, in the state where an isolation object sample is housed in the housing unit <NUM> through the opening valves, the isolation object sample does not come out from the housing unit <NUM>. In addition, because of the configuration of the opening valves, the isolation biological sample is hermetically sealed from the external atmosphere.

The isolation object sample is not particularly limited, any biological sample is acceptable as long as it contains a target biological sample to be isolated using the sample isolation kit according to an embodiment of the present technology. Specific examples of isolation object samples include whole blood, peripheral blood mononuclear cells contained in whole blood, a cell suspension containing only lymphocytes, and like cells from the patient.

The sample isolation kit according to an embodiment of the present technology includes an isolation unit <NUM> that isolates a target biological sample necessary for analysis from the isolation object sample. As in the case of past flow cytometers, the isolation unit <NUM> is configured such that a sheath flow is formed inside to perform isolation.

The specific configuration of the isolation unit <NUM> is not particularly limited. For example, the configuration of a microchip,.

in which the sheath flow is formed and a channel for the isolation object sample to flow is provided, etc., is possible.

The configuration and the isolation operation of a microchip-type isolation unit <NUM> will be described using <FIG> and <FIG>. Examples of suitable isolation units include a bubble-generating chip and a dielectric cytometry sorting chip.

The configuration of the isolation unit <NUM> will be described in detail with reference to <FIG>.

The isolation unit <NUM> roughly includes: a channel which is connected to the housing unit <NUM> through the hermetically sealing unit <NUM> and in which the isolation object sample flows; and a first pressure regulation unit that regulates the pressure in the channel to isolate.

That is, an isolation object sample is introduced from an isolation object sample inlet <NUM> into an isolation object sample channel <NUM>.

In addition, a sheath fluid is introduced from a sheath fluid inlet <NUM>. The sheath fluid introduced from the sheath fluid inlet <NUM> is divided and delivered to two sheath fluid channels <NUM>,<NUM>. The isolation object sample channel <NUM> and the sheath fluid channels <NUM>,<NUM> join together to form a main channel <NUM>. The isolation object sample laminar flow S, which is delivered through the isolation object sample channel <NUM>, and the sheath fluid laminar flows T, which are delivered through the sheath fluid channels <NUM>,<NUM>, join together in the main channel <NUM>, thereby forming a sheath flow having the isolation object sample laminar flow sandwiched between the sheath fluid laminar flows (see the below-described <FIG>).

In addition, the sheath fluid introduced from the sheath fluid inlet <NUM> is also delivered to a sheath fluid bypass channel <NUM> formed separately from the sheath fluid channels <NUM>. One end of the sheath fluid bypass channel <NUM> is connected to the sheath fluid inlet <NUM>, while the other end is connected near the communication port of the below-described isolation channel <NUM> to the main channel <NUM> (see <FIG>). The sheath fluid introduction end of the sheath fluid bypass channel <NUM> should be connected to one of the sheath fluid inlet <NUM> and sheath fluid passing parts including the sheath fluid channels <NUM>,<NUM>, but is preferably connected to the sheath fluid inlet <NUM>. When the sheath fluid bypass channel <NUM> is connected to the center position about which the two sheath fluid channels <NUM> are geometrically symmetric (i.e., the sheath fluid inlet <NUM> in this embodiment), the sheath fluid can be equally distributed into the two sheath fluid channels <NUM>. In <FIG>, the reference numeral <NUM> shows the communication port of the isolation channel <NUM> to the main channel <NUM>, and the reference numeral <NUM> shows the discharge port for the sheath fluid delivered through the sheath fluid bypass channel <NUM> into the isolation channel <NUM>.

In <FIG>, the reference numeral 115a shows a detection region to be irradiated with an excitation light, where the detection of fluorescence and scattered light emitted from an isolation object sample is performed. Isolation object samples are, while being arranged in a line in the sheath flow formed in the main channel <NUM>, delivered to the detection region 115a and irradiated with the excitation light.

The main channel <NUM> is branched into three channels downstream the detection region 115a. <FIG> show the configuration of the branch part of the main channel <NUM>. Downstream the detection region 115a, the main channel <NUM> communicates with three branch channels, that is, the isolation channel <NUM> and waste paths <NUM>,<NUM>. Among them, the isolation channel <NUM> is a channel into which a target biological sample is drawn. Samples other than the target biological sample contained in the isolation object sample (hereinafter sometimes referred to as "non-target biological samples") are not drawn into the isolation channel <NUM>, but flow into either of the two waste paths <NUM>. The sheath fluid bypass channel <NUM> is connected to a discharge port <NUM> provided near the communication port <NUM> of the isolation channel <NUM> to the main channel <NUM> (see <FIG>). The sheath fluid introduced from the sheath fluid inlet <NUM> is introduced into the isolation channel <NUM> from the discharge port <NUM>, and forms, at the communication port <NUM>, a flow of the sheath fluid from the isolation channel <NUM> side toward the main channel <NUM> side (detailed description of this flow will be given below).

The isolation unit <NUM> is composed of three substrate layers. The isolation object sample channel <NUM>, the sheath fluid channel <NUM>, the main channel <NUM>, the isolation channel <NUM>, and the waste path <NUM> are formed of a first substrate layer a<NUM> and a second substrate layer a<NUM> (see <FIG>). Meanwhile, the sheath fluid bypass channel <NUM> is formed of the second substrate layer a<NUM> and a third substrate layer a<NUM>. The sheath fluid bypass channel <NUM> formed in the substrate layers a<NUM> and a<NUM> is not connected to the isolation object sample channel <NUM>, the sheath fluid channel <NUM>, or the main channel <NUM> formed in the substrate layers a<NUM> and a<NUM>, and connects between the sheath fluid inlet <NUM> and the discharge port <NUM> of the isolation channel <NUM>. <FIG> show the configuration of the sheath fluid inlet <NUM>-side end and the discharge port <NUM>-side end of the sheath fluid bypass channel <NUM>, respectively.

Incidentally, the substrate layer structure of the isolation unit <NUM> is not limited to the three-layer structure. In addition, the configuration of the sheath fluid bypass channel <NUM> is not limited to the illustrated structure either, as long as it is capable of connecting between the sheath fluid inlet <NUM> and the discharge port <NUM> of the isolation channel <NUM> without intersecting the isolation object sample channel <NUM>, the sheath fluid channel <NUM>, and the main channel <NUM>.

For drawing a target biological sample into the isolation channel <NUM>, a negative pressure is generated in the isolation channel <NUM> by the first pressure regulation unit <NUM>, and then the target biological sample is sucked into the isolation channel <NUM> utilizing the negative pressure. The first pressure regulation unit <NUM> is a piezoelectric element. The first pressure regulation unit <NUM> is located at the position corresponding to the isolation channel <NUM>. More specifically, the first pressure regulation unit <NUM> is located at the position corresponding to a pressure chamber <NUM> that is provided as a region formed of an extended inner cavity in the isolation channel <NUM> (see.

<FIG> and <FIG>). The pressure chamber <NUM> is provided downstream the communication port <NUM> and the discharge port <NUM> in the isolation channel <NUM>.

The inner cavity of the pressure chamber <NUM> is extended in the plane direction (width direction of the isolation channel <NUM>) as shown in <FIG> and also extended in the cross-sectional direction (height direction of the isolation channel <NUM>) as shown in <FIG>. That is, the isolation channel <NUM> is extended in the width direction and the height direction in the pressure chamber <NUM>. In other words, the isolation channel <NUM> is formed such that its cross section perpendicular to the flow direction of the isolation object sample and the sheath fluid increases in the pressure chamber <NUM>.

The first pressure regulation unit <NUM> generates an expansion/contraction force with a change in the applied voltage, and causes a pressure change in the isolation channel <NUM> through the surface of the isolation unit <NUM> (contact surface). When a flow occurs in the isolation channel <NUM> as a result of a pressure change in the isolation channel <NUM>, the volume in the isolation channel <NUM> changes at the same time. The volume in the isolation channel <NUM> changes until the volume defined by the amount of displacement of the first pressure regulation unit <NUM> corresponding to the applied voltage is reached. More specifically, in the expanded state where a voltage is applied, the first pressure regulation unit <NUM> presses a displacement plate <NUM> (see <FIG>) that forms the pressure chamber <NUM> to keep the volume of the pressure chamber <NUM> small. Then, with a decrease in the applied voltage, the first pressure regulation unit <NUM> generates a force in the contracting direction and weakens the pressing of the displacement plate <NUM>, whereby a negative pressure is generated in the pressure chamber <NUM>.

In order for the expansion/contraction force of the first pressure regulation unit <NUM> to be efficiently transmitted to the pressure chamber <NUM>, as shown in <FIG>, it is preferable that the surface of the isolation unit <NUM> is depressed at the position corresponding to the pressure chamber <NUM>, and the first pressure regulation unit <NUM> is located in the depression. As a result, the displacement plate <NUM> to serve as the contact surface with the first pressure regulation unit <NUM> can be reduced in thickness, whereby the displacement plate <NUM> can be easily displaced with a change in the pressing force associated with the expansion/contraction of the first pressure regulation unit <NUM>, causing a volume change in the pressure chamber <NUM>.

In <FIG> and <FIG>, the reference numeral <NUM> shows the communication port of the isolation channel <NUM> to the main channel <NUM>. The target biological sample delivered in the sheath flow formed in the main channel <NUM> is drawn into the isolation channel <NUM> from the communication port <NUM>. In order to facilitate the drawing of the target biological sample from the main channel <NUM> into the isolation channel <NUM>, it is preferable that, as shown in <FIG>, the communication port <NUM> is opened at the position corresponding to the isolation object sample laminar flow S in the sheath flow formed in the main channel <NUM>. The shape of the communication port <NUM> is not particularly limited. However, for example, the shape shown in <FIG>, where the opening is formed in the plane, or the shape shown in <FIG>, where the channel walls of the two waste paths <NUM> are notched to form the opening, may be employed.

The isolation unit <NUM> may be formed by attaching substrate layers together, which have formed therein the main channel <NUM> and the like. The formation of the main channel <NUM> and the like in the substrate layers may be performed by injection-molding a thermoplastic resin using a mold. As the thermoplastic resin, a plastic known as a material for microchips so far may be employed, such as polycarbonate, polymethyl methacrylate resin (PMMA), cyclic polyolefin, polyethylene, polystyrene, polypropylene, and polydimethyl siloxane (PDMS).

Next, the isolation operation of the isolation unit <NUM> will be described using <FIG>.

A target biological sample sucked by the first pressure regulation unit <NUM> into the isolation channel <NUM> is drawn into the pressure chamber <NUM> as shown in <FIG>. In the figure, the reference numeral P shows a target biological sample drawn into the pressure chamber <NUM>, and the reference numeral <NUM> shows the drawing port for the target biological sample P into the pressure chamber <NUM>. When the flow of the isolation object sample containing the target biological sample P and the sheath fluid flows into the pressure chamber <NUM> formed of an extended inner cavity, the flow turns into a jet stream and separates from the channel wall surface (see the arrow in <FIG>). Accordingly, the target biological sample P comes away from the drawing port <NUM> and is drawn deep into the pressure chamber <NUM>.

In order to suck a target biological sample from the main channel <NUM> into the pressure chamber <NUM>, it is preferable that the amount of volume increase in the pressure chamber <NUM> is greater than the volume of the isolation channel <NUM> from the communication port <NUM> to a drawing port <NUM> (see <FIG>). In addition, it is preferable that the amount of volume increase in the pressure chamber <NUM> is such that a negative pressure sufficient to separate the flow of the isolation object sample containing the target biological sample P and the sheath fluid from the channel wall surface at the drawing port <NUM> is generated.

In this manner, when the target biological sample P is drawn deep into the pressure chamber <NUM> formed of an extended inner cavity of the isolation channel <NUM>, even in the case where the pressure in the isolation channel <NUM> is reversed and turns into a positive pressure, the target biological sample P can be prevented from reflowing out from the pressure chamber <NUM> toward the main channel <NUM> side. That is, as shown in <FIG>, even in the case where there is a positive pressure in the isolation channel <NUM>, because the isolation object sample and the sheath fluid flow out widely from near the drawing port <NUM>, the moving distance of the target biological sample P itself, which has been drawn to a position far from the drawing port <NUM>, is small. Accordingly, the target biological sample P does not reflow out and is maintained in the pressure chamber <NUM>.

In the pressure chamber <NUM>, it is preferable that the non-target biological samples or a sheath fluid containing the same is prevented from entering the isolation channel <NUM>. However, as shown in <FIG>, the flow of the isolation object sample and the sheath fluid (see the solid-line arrow in the figure) delivered through the main channel <NUM> has a large momentum, and thus may flow into the isolation channel <NUM> from the communication port <NUM>. The flow of the isolation object sample and the sheath fluid that has flowed into the isolation channel <NUM> from the communication port <NUM> changes its direction in the isolation channel <NUM> and flows toward the main channel <NUM> side along the channel wall of the isolation channel <NUM> (see the dotted-line arrow in the figure).

The flow of the isolation object sample and the sheath fluid flowing out from the isolation channel <NUM> along the channel wall toward the main channel <NUM> side is restrained by the channel wall and thus is slow, causing the stagnation of the non-target biological samples or an isolation object sample and sheath fluid containing the same at the communication port <NUM>. Such stagnation obstructs the operation for isolating a target biological sample and non-target biological samples from being performed at a high speed.

In contrast, in the sample isolation kit <NUM> according to an embodiment of the present technology, the sheath fluid introduced by the sheath fluid bypass channel <NUM> from the discharge port <NUM> into the isolation channel <NUM> functions to suppress the entry of non-target biological samples or an isolation object sample and sheath fluid containing the same into the isolation channel <NUM> during the non-isolation operation. That is, the sheath fluid introduced from the sheath fluid inlet <NUM> is introduced from the discharge port <NUM> into the isolation channel <NUM> and forms a sheath fluid flow from the isolation channel <NUM> side toward the main channel <NUM> side (hereinafter sometimes referred to as "reverse flow") at the communication port <NUM> (see <FIG>). Then, this reverse flow opposes the flow of the isolation object sample and the sheath fluid that is entering the isolation channel <NUM> from the main channel <NUM>, whereby the entry of the isolation object sample and the sheath fluid into the isolation channel <NUM> is prevented.

It is preferable that the reverse flow has a momentum corresponding to the momentum (strength) of the flow of the isolation object sample and the sheath fluid that is entering the isolation channel <NUM> from the main channel <NUM>. The momentum of the reverse flow can be controlled by regulating the amount of sheath fluid delivered to the sheath fluid bypass channel <NUM>, and the amount of fluid delivery can be controlled by regulating the channel diameter of the sheath fluid bypass channel <NUM>. In addition, the fluid delivery amount may also be regulated using a fluid delivery section such as a syringe pump, a valve provided in the sheath fluid bypass channel <NUM>, or the like.

The flow rate ratio between the flow rate of the sheath fluid introduced from the sheath fluid inlet <NUM> into the sheath fluid channel <NUM> and that into the sheath fluid bypass channel <NUM> is determined by the channel resistance ratio between the two channels. Accordingly, even when the pressure of introducing the sheath fluid into the sheath fluid inlet <NUM> changes, the above flow rate ratio does not change, allowing for a stable operation. In addition, also in the case where the sheath fluid flow rate has to be changed in order to change the speed of the isolation object sample passing in the detection region 115a, there is no need to separately control the flow rate of the sheath fluid channel <NUM> and the flow rate of the sheath fluid bypass channel <NUM>.

It is preferable that the momentum of the reverse flow is such that the entry of the isolation object sample and the sheath fluid from the main channel <NUM> into the isolation channel <NUM> can be completely suppressed. However, the reverse flow does not necessarily have to completely suppress the entry as long as the entry is reduced to some extent. As described above, when there is a flow of the isolation object sample and the sheath fluid flowing out from the isolation channel <NUM> along the channel wall toward the main channel <NUM> side, such a flow causes the stagnation of the non-target biological samples or an isolation object sample and sheath fluid containing the same at the communication port <NUM>. As shown in <FIG>, when the entry of the isolation object sample and the sheath fluid from the main channel <NUM> into the isolation channel <NUM> can be reduced to some extent, the flow of the isolation object sample and the sheath fluid flowing out from the isolation channel <NUM> along the channel wall toward the main channel <NUM> side, which causes stagnation, can be suppressed. Incidentally, by suppressing the stagnation of non-target biological samples or an isolation object sample and sheath fluid containing the same at the communication port <NUM>, the adhesion of the target biological sample and non-target biological samples to the channel wall can also be prevented.

A reverse flow is formed at the communication port <NUM> also at the time of sucking the target biological sample into the isolation channel <NUM> (see <FIG>). Accordingly, during the isolation operation, it is necessary to suck the target biological sample into the isolation channel <NUM> with a suction pressure that overcomes the reverse flow (see <FIG>). The amount of volume increase in the pressure chamber <NUM> should be large enough to generate a suction pressure that overcomes the reverse flow.

Further, as shown in <FIG>, it is necessary that the target biological sample is sucked to a position beyond the discharge port <NUM> in the isolation channel <NUM>. When the suction into the isolation channel <NUM> is insufficient, it may happen that the reverse flow formed by the sheath fluid introduced by the sheath fluid bypass channel <NUM> from the discharge port <NUM> into the isolation channel <NUM> causes the target biological sample to reflow out into the main channel <NUM>.

In order to suck the target biological sample sufficiently to a position beyond the discharge port <NUM>, the amount of volume increase in the pressure chamber <NUM> is set larger than the flow rate of the reverse flow, and the flow rate of the isolation object sample and the sheath fluid sucked by a negative pressure from the main channel <NUM> into the isolation channel <NUM> is set higher than the flow rate of the reverse flow.

After a desired amount of target biological sample can be introduced into the pressure chamber <NUM> by the isolation unit <NUM> formed in this manner, the target biological sample flows toward an isolation channel terminal <NUM> connected to the pressure chamber <NUM> and also to the storage unit <NUM> (see <FIG>).

Incidentally, considering a pressure change in the pressure chamber <NUM> caused by the first the pressure regulation unit <NUM>, it is preferable that the pressure chamber <NUM> and the isolation channel terminal <NUM> are connected through an opening/closing valve or the like.

Here, the isolation unit <NUM> shown in <FIG> is configured such that the sheath fluid inlet <NUM> is connected to the sheath fluid bypass channel <NUM>. However, in the isolation unit according to an embodiment of the present technology, it is also possible that the sheath fluid bypass channel <NUM> is not connected to the sheath fluid inlet <NUM>, and an introduction path 118A is separately provided as shown in <FIG>. In this case, the sheath fluid is introduced from the sheath fluid inlet <NUM>, while another solution different from the sheath fluid (e.g., culture solution, etc.) can be introduced from the introduction path 118A. Then, the solution introduced from the introduction path 118A passes through the isolation channel <NUM>, the pressure chamber <NUM>, and the isolation channel terminal <NUM>.

Accordingly, although it may happen that the sheath fluid is incorporated on the downstream side of the pressure chamber <NUM>, because the environment in the introduction path 118A is such that a larger amount of culture solution than the sheath fluid is present, an environment favorable for the target biological sample after isolation and recovery by the isolation unit <NUM> can be automatically created. Further, the below-described storage unit <NUM> has gas permeability. Thus, when the environment in the storage unit <NUM> is suited for culturing the target biological sample (e.g., CO2 concentration: <NUM>%, temperature: <NUM>, humidity: <NUM> to <NUM>%), even then the isolation step by the isolation unit <NUM> is performed for a long period of time, the quality loss of the target biological sample isolated and recovered can be avoided.

In addition, in the case of the configuration as shown in <FIG>, the flow rate of the sheath fluid bypass channel <NUM> can be individually controlled. Therefore, in exchangeable microchip-type isolation units <NUM>, even in the case where there are design differences among the isolation units <NUM> (e.g., in the case where there are great variations in the channel width and height, etc.), by controlling the flow rate of the sheath fluid bypass channel <NUM>, the isolation conditions can be optimized considering the design differences among the isolation units <NUM>.

The sample isolation kit <NUM> according to an embodiment of the present technology includes a storage unit <NUM> in which a target biological sample is housed.

This storage unit is formed in a bag-like shape in which a target biological sample is housed, for example, and includes an opening valve that is connected to the isolation channel terminal <NUM> of the isolation unit <NUM> through the hermetically sealing unit <NUM>.

The opening valve employs the configuration of a so-called check valve, such that in the state where a target biological sample is housed in the storage unit <NUM> through the opening valve, the target biological sample does not come out from the storage unit <NUM>.

In addition, because of the configuration of the opening valve, the target biological sample does not contact the external atmosphere.

The configuration of the storage unit <NUM> described above is merely an example, and a known configuration may be employed as long as the configuration does not allow for the contact between the target biological sample and the external atmosphere.

In the sample isolation kit <NUM> according to an embodiment of the present technology, a hermetically sealing unit <NUM> is provided between the housing unit <NUM> and the isolation unit <NUM> and also between the isolation unit <NUM> and the storage unit <NUM>, and the units are hermetically connected to each other. Hereinafter, an example of the configuration of the hermetically sealing unit <NUM> will be described using <FIG>.

The hermetically sealing unit <NUM> roughly includes a male member <NUM> connected to the opening valve of the housing unit <NUM> (or the channel of the isolation unit <NUM>) and a female member <NUM> hermetically connected to the male member <NUM> through a sealing member <NUM>.

The male member <NUM> has formed therein a through hole 141a, and the entire body is formed in an approximately cylindrical shape. Further, the male member <NUM> includes a projection 141b projecting along the axis of the through hole 141a and a connection tube 141c projecting in the direction perpendicular to the axis of the through hole 141a. The connection tube 141c also has a through hole formed therein, and the through hole communicates with the through hole 141a. That is, the inside of the male member <NUM> is formed in a hollow shape.

The male member <NUM> formed in this manner is configured such that, for example, the projection 141b is inserted into the opening valve of the housing unit <NUM> (or the channel of the isolation unit <NUM>), and the through hole formed in the male member <NUM> communicates with the inside of the housing unit <NUM> (or the channel of the isolation unit <NUM>).

Meanwhile, the female member <NUM> is formed in an approximately cylindrical shape having formed therein a through hole 142a. Then, one end of the female member <NUM> (in <FIG>, the back-side end on the plane of paper) is inserted into the channel of the isolation unit <NUM> (or the opening valve of the storage unit <NUM>), and the through hole 142a provided in the female member <NUM> communicates with the channel of the isolation unit <NUM> (or the inside of the storage unit <NUM>).

In addition, the sealing member <NUM> is formed in a ring-like shape having a circular hole 143a formed therein, and the inner diameter of the circular hole 143a is the same as or slightly smaller than the inner diameter of the through hole 141a of the male member <NUM> and the inner diameter of the through hole 142a of the female member <NUM>.

The female member <NUM> formed in this manner is connected to the male member <NUM> through the sealing member <NUM> and a connection member <NUM>. The connection member <NUM> is formed in a ring-like shape having a through hole 144a, and the inner diameter of the through hole 144a is the same as or slightly larger than the outer diameter of the connection tube 141c of the male member <NUM> and the outer diameter of the female member <NUM>.

When the male member <NUM> and the female member <NUM> are connected using this connection member <NUM>, the sealing member <NUM> is interposed between the male member <NUM> and the female member <NUM>, whereby the male member <NUM> and the female member <NUM> are hermetically connected. As a result, the units <NUM>, <NUM>, and <NUM> are hermetically connected through the hermetically sealing unit <NUM>.

Meanwhile, although the male member <NUM> and the female member <NUM> are hermetically connected through the connection member <NUM>, the connection member <NUM> is detachable by certain procedures. As a result, the male member <NUM> and the female member <NUM> are easily detachable from each other. That is, the housing unit <NUM>, the isolation unit <NUM>, and the storage unit <NUM> connected through the hermetically sealing unit <NUM> are easily detachable from each other.

Incidentally, the configuration shown in <FIG> is merely an example, and an ordinary hermetical structure (such as a known aseptic connector, etc.) used for sample isolation kits may be employed. Alternatively, it is also acceptable that a tubular member projects from each of the units <NUM>, <NUM>, and <NUM>, and the tubular members projecting from the units <NUM>, <NUM>, and <NUM> are welded together to form a hermetical structure. In some embodiments, one or more projecting tubular members are located on the outside of a channel of the isolation unit. A basement layer may be configured to allow projecting tubular members to be located on the outside of the channel of the isolation unit.

As necessary, the sample isolation kit <NUM> according to an embodiment of the present technology may also include a labeling unit <NUM> that labels the isolation object sample with a fluorescent dye.

Before the target biological sample is isolated from the isolation object sample in the isolation unit <NUM>, the labeling unit <NUM> labels the isolation object sample flowing into the isolation unit <NUM> with a fluorescent dye. In addition, it is also preferable that the labeling unit <NUM> is detachably connected to the isolation unit <NUM> through the hermetically sealing unit <NUM>.

Incidentally, although the labeling unit <NUM> is hermetically connected to the isolation unit <NUM> in <FIG>, any configuration is possible as long as the isolation object sample can be labeled by the labeling unit <NUM>. In some embodiments, the labeling unit <NUM> is hermetically connected to the housing unit <NUM> having housed therein the isolation object sample. In some embodiments, the fluorescent dye for labeling may be present in the isolation unit <NUM> or the housing unit <NUM>.

The kind or number of fluorescent dyes, which with the labeling unit <NUM> labels the isolation object sample, is not particularly limited, and known dyes such as FITC (fluorescein isothiocyanate: C<NUM>H<NUM>NO<NUM>S), PE (phycoerythrin), PerCP (peridinin chlorophyll protein), PE-Cy5, and PE-Cy7 can be suitably selected and used as necessary. Further, each isolation object sample may be modified with a plurality of fluorescent dyes.

Here, in the medical environment where the sample isolation kit <NUM> according to an embodiment of the present technology is used, the presence of any remaining fluorescent dye may be unacceptable. Accordingly, it is preferable that the fluorescent dye is eliminated as much as possible.

Therefore, in order to facilitate the elimination of a fluorescent dye from the target biological sample, it is preferable that the fluorescent dye is bound to the isolation object sample through a degradable linker. A degradable linker is a connector molecule that is degraded upon specific external stimulation. Examples thereof include linkers that are degraded by light at a specific wavelength, linkers that are degraded by an enzyme, and linkers that are degraded by the temperature.

The degradable linker is not particularly limited. However, in terms of not causing damage or the like to the target biological sample, it is preferable to use a photodegradable linker.

A photodegradable linker is a molecule having a structure that is degraded at a specific wavelength.

Examples thereof include a methoxy nitrobenzyl group, a nitrobenzyl group (<CIT>), a parahydroxyphenacyl group (<NPL>), a <NUM>-nitroindoline group (<NPL>), a <NUM>-(<NUM>-nitrophenyl)ethyl group (<NPL>), and a (coumarin-<NUM>-yl)methyl group (<NPL>).

In the case where the isolation object sample is a peripheral blood mononuclear cell, the peripheral blood mononuclear cells can be obtained by separation from whole blood as a biological sample. When the whole blood separation step and the step of collecting peripheral blood mononuclear cells, which are isolation object samples, can be performed consistently in an enclosed space, the problem of the incorporation of other substances into the isolation object samples can be solved more reliably.

Accordingly, as necessary, the sample isolation kit <NUM> according to an embodiment of the present technology may include a biological sample housing unit <NUM> for housing a biological sample.

The configuration of the biological sample housing unit <NUM> is not particularly limited. For example, it is formed in a bag-like in which a biological sample is housed, and includes an opening valve that is connected with the housing unit <NUM> through the hermetically sealing unit <NUM>. The opening valve employs the configuration of a so-called check valve, such that in the state where a biological sample is housed in the biological sample housing unit <NUM> through the opening valve, the biological sample does not contact the external atmosphere. This configuration is merely an example, and a known configuration may be employed as the configuration of the biological sample housing unit <NUM>. In the case where the biological sample is whole blood, the configuration of a so-called blood bag may be employed.

As necessary, the sample isolation kit <NUM> according to an embodiment of the present technology may include a separation unit <NUM> that separates an isolation object sample from the biological sample. The separation unit <NUM> is not indispensable in the sample isolation kit <NUM> according to an embodiment of the present technology, and it is also acceptable that the biological sample is separated using an external separation device, for example.

The configuration of the separation unit <NUM> is not particularly limited, and a known configuration may be employed. For example, it is possible that the configuration of a so-called spiral channel is employed, and the biological sample housed in the biological sample housing unit <NUM> flows into the spiral channel. As a result, the isolation object sample is separated from the biological sample.

Then, the separated isolation object sample flows into the housing unit <NUM>. Here, it is preferable that the separation unit <NUM> is also hermetically connected to the housing unit <NUM> through the hermetically sealing unit <NUM>.

Further, it is preferable that the biological sample housing unit <NUM> is also hermetically connected to the separation unit <NUM> through the hermetically sealing unit <NUM>.

In the sample isolation kit <NUM> according to an embodiment of the present technology, as described above, the isolation unit <NUM> forms a sheath flow and performs the isolation of a target biological sample from the isolation object sample.

As necessary, the sample isolation kit <NUM> according to an embodiment of the present technology may also include a sheath container <NUM> that houses the sheath fluid for use in the isolation unit <NUM>.

The sheath container <NUM> includes, for example, a tubular member into which the sheath fluid flows, and the tubular member communicates with the sheath fluid inlet <NUM> of the isolation unit <NUM>. As a result, the sheath fluid flows into the channel of the isolation unit <NUM>, whereby a sheath flow is formed.

It is preferable that the sheath container <NUM> is detachably connected to the isolation unit <NUM> as necessary, and it is more preferable that the sheath container <NUM> is hermetically connected to the isolation unit <NUM> through the hermetically sealing unit <NUM>.

Incidentally, the configuration of the sheath container <NUM> is not particularly limited, and a known configuration may be employed. In addition, the configuration for discharging a sheath fluid from the sheath container <NUM> is not particularly limited either, and it is also acceptable to use a driving source such as an actuator, for example. Further, in the sample isolation kit <NUM> according to an embodiment of the present technology, the sheath container <NUM> is not indispensable, and a configuration that is integrally formed in the housing unit <NUM> is also possible, for example.

In the sample isolation kit <NUM> according to an embodiment of the present technology, when a target biological sample is isolated from an isolation object sample in the isolation unit <NUM>, it is necessary to eliminate the non-target biological samples. In addition, because a sheath flow is formed and the target biological sample is isolated in the isolation unit <NUM>, it is necessary to eliminate the isolation object sample and sheath fluid containing non-target biological samples.

Accordingly, the sample isolation kit <NUM> according to an embodiment of the present technology may also include a disposal unit <NUM> for disposing biological samples and a sheath fluid other than the target biological sample (hereinafter sometimes referred to as "waste").

In addition, for example, in the case where the disposal unit <NUM> is filled with waste, it is necessary to eliminate the disposal unit <NUM> itself. Therefore, it is preferable that the disposal unit <NUM> is detachably connected to the isolation unit <NUM> through the hermetically sealing unit <NUM>.

Further, in the case where the sample isolation kit <NUM> according to an embodiment of the present technology includes the biological sample housing unit <NUM>, the biological sample in the biological sample housing unit <NUM> may contain samples other than the isolation object sample. In such a case, samples other than the isolation object sample may be separated by the separation unit <NUM>. In order to dispose such samples other than the isolation object sample, it is preferable that the disposal unit <NUM> is detachably hermetically connected to the separation unit <NUM> through the hermetically sealing unit <NUM> (see <FIG>).

Incidentally, with respect to a waste path provided in the disposal unit <NUM>, through which samples other than the isolation object sample flow, in the case where the path is connected to the channel through which the isolation object sample and the target biological sample flow (hereinafter sometimes referred to as "regular channel"), it is preferable that a reclosable valve is provided between each waste path and the regular channel.

In the sample isolation kit <NUM> according to an embodiment of the present technology described above, the housing unit <NUM>, the isolation unit <NUM>, and the storage unit <NUM> are hermetically connected through the hermetically sealing unit <NUM>. Accordingly, the isolation of a target biological sample and the storage of the target biological sample can be implemented in an enclosed space, whereby the degree of purification of the isolation of target biological samples can be improved. In addition, the contamination of a sample isolation kit itself with a mist containing a target biological sample and/or the incorporation of other substances into the isolated target biological sample can be prevented.

As a result, the sample isolation kit <NUM> according to an embodiment of the present technology can be used for clinical applications, such as immune cell therapy, where the purity of the target biological sample is necessary.

In the sample isolation kit <NUM> according to an embodiment of the present technology shown in <FIG> and so forth, the pressure regulation unit <NUM> forms a part of the isolation unit <NUM>.

Meanwhile, in a sample isolation kit <NUM> according to a second embodiment shown in <FIG>, a second pressure regulation unit <NUM> may regulate the pressure of the whole sample isolation kit <NUM>. In some embodiments, a sample isolation kit may have a first pressure regulation unit <NUM> configured to regulate sorting of a sample by isolation unit <NUM> in addition to a separate second pressure regulation unit <NUM>.

Hereinafter, the configuration different from the sample isolation kit <NUM> according to the first embodiment, that is, the configuration of the pressure regulation unit, will be mainly described. The configurations other than the pressure regulation unit, which are common to the sample isolation kit <NUM> according to the first embodiment, will be indicated with the same reference numerals, and the description thereof will be omitted.

In the sample isolation kit <NUM> according to an embodiment of the present technology, the housing unit <NUM>, the isolation unit <NUM>, and the storage unit <NUM> are hermetically connected to each other. Therefore, when the inside of the storage unit <NUM> is filled with a target biological sample, and the pressure in the storage unit <NUM> increases accordingly, the pressure in the isolation unit <NUM> may also increase. As a result, the isolation of a target biological sample by the isolation unit <NUM> may be affected.

Accordingly, the sample isolation kit <NUM> according to an embodiment of the present technology includes the pressure regulation unit <NUM> for regulating the pressure in the storage unit <NUM>. In the following description, for the convenience of description, the pressure regulation unit <NUM> is referred to as "second pressure regulation unit <NUM>" so as to distinguish from the first the pressure regulation unit <NUM>.

The configuration of the second pressure regulation unit <NUM> is not particularly limited, and a known configuration may be employed. For example, as in the case of the first pressure regulation unit <NUM>, the configuration may be such that a negative pressure is generated in the storage unit <NUM>. Specifically, a piezoelectric element, can be mentioned.

In addition, as described above, the housing unit <NUM>, the isolation unit <NUM>, and the storage unit <NUM> are hermetically connected to each other. Therefore, the pressure in the housing unit <NUM> may increase following a pressure increase in the storage unit <NUM> or the isolation unit <NUM>. As a result, the isolation object sample may be inhibited from flowing into the isolation unit <NUM>, for example.

While the flow rate of the isolation object sample flowing out from the housing unit <NUM> is fixed in a constant value, it is preferable that the second pressure regulation unit <NUM> is configured to regulate the pressure in the sheath container. In addition, it is preferable that the second pressure regulation unit <NUM> is configured to regulate the pressure in the housing unit <NUM> and/or the storage unit <NUM>. Incidentally, as in the case of the sample isolation kit <NUM> according to the first embodiment, the sample isolation kit <NUM> shown in <FIG> may be configured such that the isolation unit <NUM> includes the pressure regulation unit <NUM>, or that two pressure regulation units are provided.

Also in the sample isolation kit <NUM> according to the second embodiment described above, the housing unit <NUM>, the isolation unit <NUM>, and the storage unit <NUM> are hermetically connected through the hermetically sealing unit <NUM>. Accordingly, the isolation of a target biological sample and the storage of the target biological sample can be implemented in an enclosed space, whereby the degree of purification of the isolation of target biological samples can be improved. In addition, the contamination of a sample isolation kit itself with a mist containing a target biological sample and/or the incorporation of other substances into the isolated target biological sample can be prevented.

As a result, the sample isolation kit <NUM> according to an embodiment of the present technology can be used for clinical applications, where the purity of the target biological sample is necessary.

Further, because of the presence of the second pressure regulation unit <NUM>, even when the pressure in the storage unit <NUM> increases, the pressure in the isolation unit <NUM> and the housing unit <NUM> can be regulated. Therefore, the flowing in/out of the isolation object sample and the isolation of a target biological sample can be suitably performed.

The present technology also provides a sample isolation device using the sample isolation kit <NUM>. Hereinafter, a sample isolation device <NUM> according to an embodiment of the present technology will be described using <FIG>.

As shown in <FIG>, the sample isolation device <NUM> roughly includes a sample isolation kit <NUM>, a light irradiation unit <NUM>, a light detection unit <NUM>, and an arithmetic processing unit <NUM>, and, as necessary, may also include a position control unit <NUM>, a degradation light irradiation unit <NUM>, a drug loading control unit <NUM>, a culture unit <NUM>, and a pressure regulation unit <NUM>. Each unit will be described hereinafter.

The sample isolation device <NUM> according to an embodiment of the present technology includes a sample isolation kit <NUM> that performs the isolation and storage of a target biological sample.

Incidentally, the configuration of this sample isolation kit <NUM> is the same as the configuration of the sample isolation kit <NUM> shown in <FIG>, and thus the description thereof will be omitted.

The sample isolation device <NUM> according to an embodiment of the present technology includes a light irradiation unit <NUM> that irradiates the isolation object sample with light.

Specifically, the light irradiation unit <NUM> irradiates, with light (excitation light), the isolation object sample passing through the detection region 115a provided on the main channel <NUM> of the isolation unit <NUM>.

The light irradiation unit <NUM> is configured to include, for example, a light source that emits an excitation light, an objective lens that concentrates the excitation light on the isolation object sample passing through the main channel <NUM>, and the like. The light source is suitably selected according to the purpose of the analysis from laser diodes, SHG lasers, solid lasers, gas lasers, high-intensity LEDs, and the like. As necessary, the light irradiation unit <NUM> may also include an optical element other than the light source and the objective lens.

The sample isolation device <NUM> according to an embodiment of the present technology includes a light detection unit <NUM> that detects fluorescence and scattered light emitted from the isolation object sample irradiated with an excitation light.

Specifically, the light detection unit <NUM> detects fluorescence and scattered light emitted from the isolation object sample, and converts the same into an electrical signal. Then, the electrical signal is output to the arithmetic processing unit <NUM>.

The configuration of the light detection unit <NUM> is not particularly limited, and a known configuration may be employed. Further, the method for conversion into an electrical signal is not particularly limited either.

The sample isolation device <NUM> according to an embodiment of the present technology includes an arithmetic processing unit <NUM> into which an electrical signal obtained by conversion in the light detection unit <NUM> is input.

On the basis of the input electrical signal, the arithmetic processing unit <NUM> judges the optical properties of the isolation object sample and the target biological sample contained in the isolation object sample.

The arithmetic processing unit <NUM> further includes a gating circuit for computing a threshold for isolating the target biological sample from the isolation object sample, a threshold for determining whether a greater number of target biological samples than necessary have been isolated, a threshold for screening the target biological sample on the basis of the fluorescence intensity of the fluorescent dye used for labeling by the labeling unit <NUM>, and the like. Because of the configuration of the gating circuit, in the case where a threshold for isolating the target biological sample from the isolation object sample is computed, the threshold is converted into an electrical signal for isolation, and the isolation signal is output to the first pressure regulation unit <NUM> provided in the isolation unit <NUM>.

Incidentally, the configuration of the arithmetic processing unit <NUM> is not particularly limited, and a known configuration may be employed. Further, the method for arithmetic processing performed by the gating circuit of the arithmetic processing unit <NUM> may also be a known method.

As necessary, the sample isolation device <NUM> according to an embodiment of the present technology may also include a position control unit <NUM>.

In the case where the isolation unit <NUM> is configured as above, the excitation light has to irradiate the detection region 115a of the isolation unit <NUM>, and the position control unit <NUM> controls the relative positional relationship between the sample isolation kit <NUM> and the light irradiation unit <NUM>.

The configuration of the position control unit <NUM> is not particularly limited, and a known configuration may be employed. For example, an actuator to serve as a driving source can be mentioned.

As necessary, the sample isolation device <NUM> according to an embodiment of the present technology may also include a degradation light irradiation unit <NUM>.

In the case where the configuration is such that the sample isolation kit <NUM> includes the labeling unit <NUM>, and the isolation object sample is labeled with a fluorescent dye through a photodegradable linker, depending on the usage environment, it is necessary to eliminate the fluorescent dye from the isolation object sample.

The degradation light irradiation unit <NUM> irradiates the photodegradable linker with a predetermined light. As a result, the fluorescent dye can be eliminated from the isolation object sample. Here, the wavelength of the light to irradiate the degradable linker should be a wavelength corresponding to each photodegradable linker. For example, in the case of methoxy nitrobenzyl, the degradation efficiency is the highest at <NUM>. Taking this as <NUM>, the degradation efficiency is <NUM> at <NUM>, <NUM> at <NUM>, and <NUM> at <NUM>. A wavelength of <NUM> or less may cause damage to the isolation object sample, and thus is preferably not used. In addition, in order not to damage the isolation object sample, particularly the target biological sample, it is preferable that irradiation is performed at <NUM> mW/cm<NUM>, <NUM> sec → <NUM> J/cm<NUM>, for example. As the amount of irradiation, in the case where the target biological sample is a cell, although this depends on its kind, it is said that damage to DNA is caused at <NUM> J/cm<NUM>, resulting in the inhibition of cell growth (<NPL>)). In addition, it is also reported that cytotoxicity does not occur at <NUM> J/cm<NUM> (<NPL>.

As necessary, the sample isolation device <NUM> according to an embodiment of the present technology may also include a drug loading control unit <NUM>.

The target biological sample stored in the storage unit <NUM> of the sample isolation kit <NUM> has to be activated and subjected to gene introduction as necessary. The drug loading control unit <NUM> loads a drug for activating the target biological sample or a drug for introducing a gene into the target biological sample into the storage unit <NUM>. Alternatively, the unit controls the loading amount of each drug according to the state of the stored target biological sample.

As the drug, known drugs are usable, such as various cytokines (interleukin-<NUM> (IL-<NUM>), IL-<NUM>, IL-<NUM>, IL-<NUM>, etc.), various antibodies (anti-CD3 antibody, anti-CD28 antibody, etc.), and the like for activation, and various viral vectors into which a plasmid that expresses the target gene has been introduced (adeno-associated vector, adenovirus vector, retrovirus vector, lentivirus vector, etc.) for gene introduction. A suitable drug may be selected according to the kind and state of the target biological sample stored. Further, it is also possible to use several kinds of known drugs in combination.

As necessary, the sample isolation device <NUM> according to an embodiment of the present technology may also include a culture unit <NUM>.

According to the intended use of the sample isolation device <NUM>, it may be necessary to increase the number of target biological samples isolated by the sample isolation kit <NUM>. That is, in the culture unit <NUM>, the target biological sample stored in the storage unit <NUM> is cultured.

Specifically, the temperature in the storage unit <NUM> is controlled to increase the amount of the target biological sample housed in the storage unit <NUM>.

Incidentally, the method for temperature control in the culture unit <NUM> is not particularly limited, and a known method may be employed.

For example, it is possible that a heating element is provided in the storage unit <NUM>, and an electrical signal to control the temperature rise/fall is output from the culture unit <NUM> to the heating element.

As necessary, the sample isolation device <NUM> according to an embodiment of the present technology may also include the pressure regulation unit <NUM>.

As described above, the housing unit <NUM>, the isolation unit <NUM>, and the storage unit <NUM> in the sample isolation kit <NUM> are hermetically connected to each other. Therefore, a pressure change in the storage unit <NUM> may cause a pressure change in the housing unit <NUM> and/or the isolation unit <NUM>. The pressure regulation unit <NUM> regulates the pressure in the storage unit <NUM>.

Specifically, a piezoelectric element, which is a configuration that generates a negative pressure in the storage unit <NUM>, can be mentioned. Further, it is preferable that the pressure regulation unit <NUM> is configured to regulate the flow rate of the isolation object sample flowing out from the housing unit <NUM>, thereby regulating the pressure in the housing unit <NUM>. In addition, it is preferable that the pressure regulation unit <NUM> is configured to regulate the flow rate of the sheath fluid flowing out from the sheath container <NUM>, thereby regulating the pressure in the sheath container <NUM>.

That is, the pressure regulation unit <NUM> employs the same configuration as the second pressure regulation unit <NUM> provided in the sample isolation kit <NUM> shown in <FIG>.

In the sample isolation device <NUM> according to an embodiment of the present technology, the sample isolation kit <NUM> includes the separation unit <NUM> described above, and the isolation object sample is separated from the biological sample by such a sample isolation kit <NUM>.

However, the configuration does not have to be such that the sample isolation kit <NUM> performs the separation, and it is also possible that the sample isolation device <NUM> according to an embodiment of the present technology includes a separation unit (not shown) that performs the separation.

That is, for example, the separation unit may be a known centrifugal separator, and configured to centrifuge the entire sample isolation kit <NUM> or the biological sample housing unit <NUM> provided in the sample isolation kit <NUM>.

The operation of the sample isolation device <NUM> will be described using <FIG>.

First, in the sample isolation device <NUM> according to an embodiment of the present technology, an isolation object sample is separated from the biological sample by the sample isolation kit <NUM> or by the separation unit provided separately from the sample isolation kit <NUM>. The method for the separation step S1 is not particularly limited, and a centrifugal separation method may be used.

After the isolation object sample is separated in the separation step S1, a fluorescent antibody reagent is flowed by the labeling unit <NUM> into the housing unit <NUM> having housed therein the isolation object sample, and the reagent is bound to the isolation object sample (reagent binding step S2).

Incidentally, the kind or number of fluorescent dyes is not particularly limited, and known dyes such as FITC (fluorescein isothiocyanate: C<NUM>H<NUM>NO<NUM>S), PE (phycoerythrin), PerCP (peridinin chlorophyll protein), PE-Cy5, and PE-Cy7 can be suitably selected and used as necessary. Further, each isolation object sample may be modified with a plurality of fluorescent dyes.

In addition, as described above, it is preferable that the fluorescent dye is bound to the isolation object sample through a degradable linker, particularly a photodegradable linker.

After the isolation object sample is labeled with a fluorescent dye in the reagent binding step S2, an isolation step S3 of isolating a target biological sample from the isolation object sample on the basis of the optical properties is performed.

The details of the isolation step S3 will be described using <FIG>. Incidentally, the step shown in <FIG> is merely an example.

As shown in <FIG>, the isolation step S3 in the sample isolation device <NUM> according to an embodiment of the present technology includes a valve switching first step S31, a preliminary measurement step S32, a main measurement step S33, a valve switching second step S34, a target biological sample acquisition step S35, and a valve closing step S37. Each step will be described hereinafter.

In the sample isolation device <NUM> according to an embodiment of the present technology, in the case where the valve is provided between a regular channel provided in the isolation unit <NUM> and a waste path provided in the disposal unit <NUM>, the valve is opened such that the entire isolation object sample containing the target biological sample in the isolation unit <NUM> flows into the disposal unit <NUM> (valve switching first step S31).

The preliminary measurement step S32 will be described using <FIG>. As shown in <FIG>, the preliminary measurement step S32 includes at least a fluorescence intensity information acquisition step S322, a machine learning step S323, and a threshold setting step S324.

That is, in the state where the valve is opened through the valve switching first step S31, the isolation object sample is introduced into the channel in the isolation unit <NUM> from the housing unit <NUM> (S321). Then, the fluorescence intensity information of the isolation object sample is acquired by the light detection unit <NUM> (fluorescence intensity information acquisition step S322). Further, on the basis of the fluorescence intensity information, the arithmetic processing unit <NUM> performs machine learning using the information of the origin of the isolation object sample or the information of prior cases (machine learning step S323).

Then, in the arithmetic processing unit <NUM>, it is estimated into what kind of groups the isolation object sample is divided. Subsequently, with respect to the fluorescence intensity obtained through the fluorescence intensity information acquisition step S322, a threshold for isolating the target biological sample from the isolation object sample, a threshold for determining whether a greater number of target biological samples than necessary have been isolated, a threshold for screening the target biological sample on the basis of the fluorescence intensity of the fluorescent dye used for labeling by the labeling unit <NUM>, and the like are set (threshold setting step S324). Upon the completion of the threshold setting step S324 in this manner, the preliminary measurement step S32 is completed.

After the completion of the preliminary measurement step S32, the main measurement step S33 is performed.

In the main measurement step S33, as shown in <FIG>, in the state where the valve is opened through the valve switching first step S31, the isolation object sample is introduced into the channel in the isolation unit <NUM> from the housing unit <NUM> (S331). Then, the parameters of the first pressure regulation unit <NUM> included in the isolation unit <NUM> (piezo-amplitude, the time until the object biological sample reaches the branch part from the detection region 115a: delay time, etc.) are adjusted (S332). Upon the completion of the adjustment of each parameter, the main measurement step S33 is completed.

After the parameters for the first pressure regulation unit <NUM> are properly set through the main measurement step S33, the valve is switched (valve switching second step S34). This valve switching second step S34 makes it possible for the target biological sample to flow into the storage unit <NUM>.

After the valve switching second step S34, the isolation object sample is introduced into the channel in the isolation unit <NUM> from the housing unit <NUM>. Then, the target biological sample is isolated by the isolation unit <NUM>, the light irradiation unit <NUM>, the light detection unit <NUM>, and the arithmetic processing unit <NUM> and stored in the storage unit <NUM> (target biological sample acquisition step S35).

At that time, on the basis of the thresholds computed by the arithmetic processing unit <NUM>, it is determined whether the number of the target biological samples in the storage unit <NUM> has reached the necessary number.

In the determination step S36, in the case where the number of target biological samples is smaller than the necessary number (NO in S36), the target biological sample acquisition step S35 is performed again. This operation is repeated until the number of target biological samples reaches the necessary number.

Meanwhile, in the case where the number of target biological samples is greater than the necessary number (YES in S36), the process moves on to the next valve closing step S37.

In the case where the number of target biological samples reaches the necessary number, in order to prevent the incorporation of the isolation object sample from the isolation unit <NUM> into the storage unit <NUM>, the valve is closed (valve closing step S37). Upon the completion of the valve closing step S37, the isolation step S3 is completed.

After the isolation step S3 is completed, the internal processing step S4 is performed. In the internal processing step S4, the drug loading control unit <NUM> loads a predetermined drug into the storage unit <NUM>, for example, and the activation of the target biological sample, the gene introduction into the target biological sample, and the like are performed.

Further, after the internal processing step S4 is performed, the culture step S5 is performed. Specifically, the temperature of the storage unit <NUM> is controlled by the culture unit <NUM>, and the target biological sample is cultured.

After the culture step S5 is performed, the procedure of concentrating the target biological sample stored in the storage unit <NUM> and cultured is performed (concentration step S6). Incidentally, the method for concentrating a target biological sample is not particularly limited, and a known method may be employed.

After the concentration step S6 is performed, the step of preserving the concentrated target biological sample is performed (preservation step S7). Incidentally, the method for preserving a target biological sample is not particularly limited, and may be suitably selected according to the kind of the target biological sample, etc..

In the sample isolation devices <NUM> according to an embodiment of the present technology described above, the housing unit <NUM>, the isolation unit <NUM>, and the storage unit <NUM> are hermetically connected through the hermetically sealing unit <NUM>. Accordingly, the isolation of a target biological sample and the storage of the target biological sample can be implemented in an enclosed space, whereby the degree of purification of the isolation of target biological samples can be improved. In addition, the contamination of a sample isolation kit itself with a mist containing a target biological sample and/or the incorporation of other substances into the isolated target biological sample can be prevented.

Claim 1:
A sample isolation kit (<NUM>) comprising:
a separation unit (<NUM>) configured to separate an isolation object sample from a biological sample;
a biological sample housing unit (<NUM>) configured to detachably couple to the separation unit and provide the biological sample to the separation unit;
an isolation unit (<NUM>) having a sample fluid channel (<NUM>) and an isolation channel (<NUM>) and at least one sheath fluid channel, wherein
the isolation unit is configured to isolate a target biological sample from the isolation object sample; and
a sheath container (<NUM>) configured to detachably couple to the at least one sheath fluid channel of the isolation unit and provide the sheath fluid to the isolation unit;
wherein the separation unit and the biological sample housing unit are coupled using a hermetic seal (<NUM>); and wherein the isolation unit and
the sheath container are coupled using a further hermetic seal (<NUM>).