JP6686768B2 - Cell separation and collection method - Google Patents

Description

  The present invention relates to a method for efficiently separating and recovering cells contained in a sample. In particular, the present invention relates to a method capable of efficiently separating and collecting the cells even when the number of cells contained in the sample is very small.

  In recent years, cells have been selectively separated and collected from body fluids such as blood and tissues or tissue culture samples obtained by suspending or dispersing tissues such as organs in a solution. Research is being conducted to apply it to clinical diagnosis and treatment. For example, a tumor cell (Circulating Tumor Cell, hereinafter CTC) is collected from blood collected from a cancer patient, and morphological analysis, tissue type analysis, and gene analysis are performed on the cell, and based on the findings obtained by the analysis. Research to determine treatment policy is ongoing.

  As a method for recovering cells contained in a sample, a method utilizing centrifugation has been conventionally used, and an efficient separation / recovery method for recovering more cells is required. However, when the sample is a sample containing blood components such as whole blood, by centrifugation, an aggregate is formed by the aggregating factors such as fibrinogen, fibrin, and platelets contained in the sample, and the aggregate is contained in the sample. By incorporating the cells contained therein, it has been difficult to separate and collect the cells.

  Patent Document 1 discloses a method of separating and collecting rare cells contained in a sample by adding heparin to a sample containing cells and suppressing blood coagulation by a fibrin polymer contained in the sample. There is. However, it was difficult to efficiently separate and collect a sample containing platelets even if the method described in Reference 1 was used.

International Publication No. 2004/029208

  An object of the present invention is to provide a method for efficiently separating and recovering cells contained in a sample by using centrifugation, without being affected by platelets contained in the sample. Especially.

  In order to solve the above problems, the inventors of the present invention have earnestly studied, and as a result, arrived at the present invention.

  That is, the first aspect of the present invention is a method of separating and collecting cells contained in a sample containing platelets by using centrifugation, wherein the sample containing platelets further contains an inhibitor for the platelet GPIIb / IIIa receptor. The above method.

  A second aspect of the present invention is the method according to the first aspect, wherein the sample containing platelets further contains a protein to which a hydrophilic polymer is bound.

  A third aspect of the present invention is the method according to the first or second aspect, wherein the sample containing platelets further contains sugar.

  A fourth aspect of the present invention is the method according to any one of the first to third aspects, wherein the inhibitor for the platelet GPIIb / IIIa receptor is tirofiban at 0.5 μg / mL or more. is there.

  Hereinafter, the present invention will be described in detail.

  Examples of the sample containing platelets in the present invention include biological samples such as blood, diluted blood, serum, plasma, cord blood, component blood, bone marrow fluid, lymph fluid, tissue fluid, ascites, and liver, lung, spleen, kidney, tumor. , A suspension of a piece of blood-containing tissue such as a lymph node suspended in an appropriate buffer solution. Further, a fraction containing target cells obtained by separating and collecting these samples and suspensions by centrifugation etc. is also included in the sample containing platelets of the present invention.

  The method for separating and recovering cells of the present invention is characterized in that a sample containing platelets is preliminarily impregnated with an inhibitor against the platelet GPIIb / IIIa receptor and then centrifuged to recover cells contained in the sample. There is. As described in the background art, when platelets contained in a sample form aggregates, it becomes difficult to separate and collect cells contained in the sample due to the aggregates, so it is necessary to inhibit the aggregation of platelets by some method. is there. As an example of the method of inhibiting, there is a method of incorporating a platelet aggregation inhibitor into a sample containing platelets.

  Platelet aggregation inhibitors include, for example, blocking the platelet ADP receptor, blocking the platelet serotonin 2 receptor, inhibiting the platelet cyclooxygenase, or blocking the platelet GPIIb / IIIa receptor to directly affect platelet function. There are substances that affect and inhibit platelet aggregation. An inhibitor that blocks the platelet ADP receptor, blocks the platelet serotonin 2 receptor, and inhibits platelet cyclooxygenase, mainly inhibits the receptor on the surface of the platelet membrane, or acts as an inhibitor of the signal pathway from the receptor, It is applied to suppress an increase in calcium ion concentration in platelets that contributes to the expression of platelet GPIIb / IIIa receptors involved in platelet aggregation. On the other hand, an inhibitor against the platelet GPIIb / IIIa receptor has a function of suppressing aggregation due to binding of platelet GPIIb / IIIa receptors expressed on the platelet membrane surface between platelets via von Willebrand factor, fibrinogen and the like. .

  As a result of diligent studies on the platelet aggregation inhibitor contained in the sample containing platelets, the present inventors have found that platelet inhibitors are more effective than the inhibitors that block platelet ADP receptor, block platelet serotonin 2 receptor, and inhibit platelet cyclooxygenase. It has been found that inhibitors for the GPIIb / IIIa receptor are preferred. In the operation of separating and recovering cells contained in a sample containing platelets by using centrifugal separation, it is considered that the calcium ion concentration in the platelets increases due to shear stress on the platelets due to centrifugal force. Therefore, it can be said that an inhibitor against the receptor which directly acts on the platelet GPIIb / IIIa receptor is preferable as the platelet aggregation inhibitor used in the method of the present invention.

  Examples of inhibitors for the platelet GPIIb / IIIa receptor include tirofiban, abciximab, and eptifibatide. When tirofiban is used as a platelet GPIIb / IIIa inhibitor, the sample containing platelets may be contained at 0.5 μg / mL or more, more preferably at 0.6 μg / mL or more, and 0.7 μg / mL to 5 μg / mL. Is more preferable, and it is even more preferable that it is contained in the range of 0.75 μg / mL to 3 μg / mL.

  In the present invention, it is preferable that the sample containing platelets further contains a protein bound to a hydrophilic polymer in addition to the inhibitor for the platelet GPIIb / IIIa receptor, since the cells contained in the sample can be efficiently recovered. . The hydrophilic polymer may be any hydrophilic polymer having no electric charge, and examples thereof include polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, poly (hydroxyalkyl) methacrylate, polyacrylamide, polymers having a phosphorylcholine group in a side chain, and polysaccharides. , And polypeptides. The protein may be water-soluble, and examples thereof include blood-derived proteins such as serum-derived proteins and plasma-derived proteins, and milk-derived proteins such as casein, and more specific examples include serum-derived proteins commonly used by those skilled in the art. The protein is bovine serum albumin (BSA). The protein to which the hydrophilic polymer is bound is a protein in which the hydrophilic polymer and the protein are bound to each other at a fixed ratio, and, for example, is provided with a functional group capable of binding to the protein (eg, N-hydroxysuccinimide group). It can be obtained by reacting the hydrophilic polymer and protein at a constant molar ratio. The reaction ratio between the hydrophilic polymer and the protein may be such that the hydrophilic polymer reacts with the protein in a molar ratio of 0.01 or more, more preferably 0.5 or more. It is most preferable to react at a molar ratio of 2 or more (for example, when a hydrophilic polymer is reacted with blood-derived protein or milk protein at a molar ratio of 2 or more, hydrophilic polymer is not reacted with blood-derived protein. The hydrophilic polymer binds at a measured molar ratio of 1 or more, and the hydrophilic polymer binds to the milk-derived protein at a measured molar ratio of 0.2 or more) (see JP-A-2016-106622).

  Further, in the present invention, it is preferable that the sample containing platelets further contains sugar in addition to the inhibitor for the platelet GPIIb / IIIa receptor, since damage to cells contained in the sample is reduced. Examples of sugars include mannitol, glucose and sucrose. The concentration of the sugar contained in the sample containing platelets is preferably such that it becomes an isotonic solution, and when mannitol is used as the sugar, a final concentration between 250 mM and 350 mM can be said to be a preferable range.

  Further, in the present invention, the sample containing platelets may further contain an electrolyte such as calcium chloride or magnesium chloride, or a protein such as BSA, in addition to the inhibitor for the platelet GPIIb / IIIa receptor.

The cells separated and collected by the method of the present invention may be applied to a slide, observed with a microscope or an optical detector, or measured using flow cytometry. When performing cell measurement by observing with a microscope or an optical detector, the suspension containing the cells is introduced into a cell holding means having a holding part capable of holding the cells, and After holding the cells, it may be observed with a microscope or an optical detector. Examples of the holding unit include a hole capable of accommodating the cells and a surface covered with a material capable of fixing the cells (for example, poly-L-lysine). It should be noted that it is preferable to set the size of the holding portion to a size capable of holding only one of the cells, because collection and analysis of specific cells (morphological analysis, tissue type analysis, gene analysis, etc.) can be easily performed. Further, when the cells are held in the holder, it is preferable to use the dielectrophoretic force because the cells can be efficiently held in the holder. When the dielectrophoretic force is used, specifically, an alternating voltage may be applied to generate dielectrophoresis and the cells may be introduced into the holding unit. The AC voltage to be applied is preferably an AC voltage having a waveform in which charging / discharging of cells in the holding section is periodically repeated, the frequency is between 100 kHz and 3 MHz, and the electric field strength is 1 × 10 ⁵ to 5 × 10 5. ^It is particularly preferable to set it to ⁵ V / m (see WO 2011/149032 and JP 2012-013549 A).

Hereinafter, a method for separating and collecting tumor cells (CTC) contained in blood will be described as an example of the separating and collecting method of the present invention, but the present invention is not limited to the contents of the present description.
(1) Blood is collected from a patient suspected of having cancer. An anticoagulant such as citric acid, heparin, or ethylenediaminetetraacetic acid (EDTA) may be added when collecting blood. If necessary, the collected blood may be diluted with physiological saline or the like.
(2) A solution containing an inhibitor (eg, tirofiban) for the platelet GPIIb / IIIa receptor is added to the blood (or diluted blood) collected in (1), and CTC is separated using a density gradient centrifugation method. . The concentration of tirofiban may be 0.5 μg / mL or more as the final concentration for the blood (or the diluted blood), more preferably 0.6 μg / mL or more, and 0.7 μg / mL to 5 μg / mL. Is more preferable, and 0.75 μg / mL to 3 μg / mL is even more preferable. The density gradient centrifugation method is a method of separating cells based on their specific gravities, and the collected blood (or diluted blood) is layered on a density gradient forming medium (density gradient forming medium) and then centrifuged. By collecting the layer containing CTC of interest (upper layer), a fraction containing CTC from which unnecessary cells have been removed is obtained. Before performing the density gradient centrifugation, a binding agent (for example, RosetteSep (manufactured by StemCell Technologies)) capable of binding unnecessary cells such as red blood cells and white blood cells may be added to blood or diluted blood. The binding agent forms cell aggregates by binding to surface antigens of erythrocytes, leukocytes, and / or these cells, and can increase the density of these cells, so that CTC separation by density gradient centrifugation is performed. make it easier. On the other hand, it is not preferable to add a binder that removes platelets by density gradient centrifugation (for example, Rosette Sep Human Circulating Epithelial Tumor Cell Enrichment Cocktail (manufactured by StemCell Technologies)). A part of CTC has acquired the property of collecting platelets in order to avoid recognition from phagocytes and acquire high metastatic ability to distant sites. Therefore, the addition of the above-mentioned binding agent that removes platelets is effective in suppressing the formation of aggregates by platelets, but it becomes difficult to separate and collect CTCs that have acquired high metastatic potential.
(3) The blood component is removed by centrifuging the CTC-containing fraction obtained in (2), and the CTC is pelletized, and then a solution containing an inhibitor for the platelet GPIIb / IIIa receptor is added. And suspend the CTCs. At this time, a solution containing a protein bound with a hydrophilic polymer (for example, BSA bound with polyethylene glycol) may be added. The concentration of the protein to which the hydrophilic polymer is bound may be 0.01 to 25% (w / v) as the final concentration of the protein in the suspension, and 0.02 to 5% (w / v). v) is more preferable, and 0.05 to 2% (w / v) is even more preferable.
(4) The suspension containing CTC prepared in (3) is centrifuged again to collect the pellet containing CTC. If necessary, a step of resuspending the recovered pellet in a solution containing an inhibitor against the platelet GPIIb / IIIa receptor and centrifuging may be added.

  The present invention is a method for recovering cells contained in a sample containing platelets by centrifugation, which is characterized in that the sample is impregnated with an inhibitor for the platelet GPIIb / IIIa receptor and then centrifuged. . According to the present invention, cells contained in a sample containing platelets can be separated and collected with high efficiency. This method is particularly useful when the amount of cells contained in the sample is very small.

  As an example, by applying the present invention to the separation and collection of tumor cells (CTC) contained in blood, the amount of blood collected can be reduced and the burden on the patient can be reduced. Further, when the cancer is diagnosed by the presence of the CTC, the reliability of the determination result of the presence or absence of the CTC is improved, so that the cancer can be diagnosed with high accuracy.

  Hereinafter, the present invention will be described in more detail using examples and comparative examples, but the present invention is not limited to the examples.

Example 1
(1) Human breast cancer cells (SKBR3) were cultured in RPMI-1640 medium containing 10% FBS in a 5% CO ₂ environment at 37 ° C. for 24 to 96 hours, and then 0.25% trypsin / 1 mM EDTA was used. The cells were detached from the culture medium and labeled with a fluorescent dye (CFSE, Dojindo Laboratories). Fluorescently labeled SKBR3 cells were used as the target cells.
(2) After collecting 3 mL of blood from a healthy person who obtained informed consent into an EDTA-2K blood collection tube (VP-DK050K, manufactured by Terumo), 3 mL of physiological saline and 75 μL of white blood cell / red blood cell binding agent in the blood collection tube A diluted blood sample was prepared by adding 180 μL of a 300 mM mannitol solution containing RosetteSep and StemCell Technologies, and about 50 SKBR3 cells fluorescently labeled in (1) and tirofiban.
(3) The prepared diluted blood sample was overlaid on a density gradient solution having a density of 1.091 g / mL, centrifuged at 2000 × g for 10 minutes at 25 ° C., and the supernatant was recovered.
(4) The supernatant collected in (3) was lysed with 0.9% (w / v) ammonium chloride and 0.1% (w / v) potassium hydrogen carbonate to 30 mL, and then 300 mL. Centrifuged at 25 g for 10 minutes at xg. By this operation, the erythrocytes mixed in the supernatant are destroyed, and the SKBR3 cells that have been separated and collected are well observed.
(5) After removing the supernatant, the pellet containing SKBR3 cells was resuspended in 30 mL of a 300 mM mannitol solution containing tirofiban.
(6) The resuspension was centrifuged at 300 × g for 5 minutes at 25 ° C., the supernatant was removed, and the pellet containing SKBR3 cells was suspended again in 30 mL of 300 mM mannitol solution containing tirofiban. This operation is an operation for removing blood components and concentrating the desired SKBR3 cells.
(7) The SKBR3 cell suspension obtained in (6) was centrifuged at 300 × g for 5 minutes at 25 ° C., and the supernatant was removed.
(8) The suspension containing the SKBR3 cells from which the supernatant had been removed in (7) was introduced into a cytodiagnostic chip, and an AC voltage was applied for 3 minutes to hold the SKBR3 cells in the holding part of the chip. The cytodiagnostic chip used in this example is composed of an insulator having a plurality of fine holes each having a diameter of 30 μm and a depth of 30 μm, and a light-shielding chromium film provided between the insulator and the lower electrode substrate. The holding part is sandwiched between a spacer having a thickness of 1 mm and a lower electrode substrate, and the spacer is sandwiched between an upper electrode substrate and a lower electrode substrate.
(9) The recovery rate was calculated by counting the number of SKBR3 cells retained on the cytodiagnostic chip and dividing by the number of SKBR3 cells added in (2).

Comparative Example 1
As a solution to be suspended in SKBR3 cells in Example 1 (2), (5) and (6),
300 mM mannitol solution (liquid temperature 25 ° C or 0 ° C),
300 mM mannitol solution containing heparin (solution temperature 25 ° C) or 300 mM mannitol solution containing ticlopidine (solution temperature 25 ° C),
In the same manner as in Example 1 except that the above was used, the SKBR3 cells were separated and collected and the recovery rate was calculated.

  The results of the recovery rates in Example 1 and Comparative Example 1 are summarized in Table 1. In Table 1, the additive concentration other than mannitol indicates the final concentration when the volume of the hemolyzed blood containing the ammonium chloride and potassium hydrogen carbonate or the 300 mM mannitol solution was adjusted to 30 mL.

  In the step of separating and recovering cancer cells from a blood sample using centrifugation, the sample may contain 0.5 μg / mL or more of tirofiban, which is an inhibitor for the platelet GPIIb / IIIa receptor. In comparison with the case where heparin was included and the case where ticlopidine, which is an inhibitor that blocks the platelet ADP receptor, was included, it was found that the platelet aggregation was suppressed. When 300 μm mannitol solution at a liquid temperature of 0 ° C was used, in which the aggregation of platelets was not confirmed by including 0.5 μg / mL or more of tirofiban and separating and collecting cancer cells (recovery rate 70%). It can be seen that the recovery rate is improved as compared to (recovery rate about 90%). In addition, in the step of adding hemolyzed blood containing ammonium chloride and potassium hydrogen carbonate (Example 1 (4)), when tirofiban was included, no precipitate was formed, but when ticlopidine was included, no precipitate was formed. When the concentration was 100 μg / mL or more, a white precipitate was formed, and at 1000 μg / mL, the white precipitate formed made it difficult to evaluate the presence or absence of platelet aggregation.

  When the concentration of tirofiban to be added was examined, the ability to suppress platelet aggregation was improved with the increase in the concentration of tirofiban, and at a concentration of 0.75 μg / mL or more of tirofiban, cancer cells (SKBR3 cells) contained in the sample had a high recovery rate. It was possible to recover at a recovery rate of about 90%.

実施例２
実施例１（１）での目的とする細胞として、ヒト小細胞肺がん細胞（Ｈ６９）を用い、実施例１（２）、（５）および（６）の細胞を懸濁させる溶液として、０．７５μｇ／ｍＬのチロフィバンを含む３００ｍＭマンニトール溶液を用いた他は、実施例１と同様な方法で細胞の分離回収および回収率の算出を行なった。

Example 2
Human small cell lung cancer cells (H69) were used as the target cells in Example 1 (1), and a solution for suspending the cells of Examples 1 (2), (5) and (6) was used as a solution. Separation and recovery of cells and calculation of recovery rate were performed in the same manner as in Example 1 except that a 300 mM mannitol solution containing 75 μg / mL tirofiban was used.

Comparative example 2
As a leukocyte / erythrocyte binding agent added to blood in Example 1 (2), Rosette Sep Human Circulating Epithelial Tumor Cell Enrichment Cocktail (manufactured by StemCell Technologies, Inc. 2) capable of binding leukocytes, erythrocytes and platelets was used (Example 1). , (5) and (6) were used as the solution for suspending the cells, and the separation and recovery of the cells and calculation of the recovery rate were performed in the same manner as in Example 1 except that the 300 mM mannitol solution was used.

  The results of the recovery rates of Example 2 and Comparative Example 2 are summarized in Table 2. Aggregation of platelets was not confirmed in both Example 2 and Comparative Example 2. On the other hand, as for the cell recovery rate, when a binding agent that also binds platelets is used as the leukocyte / erythrocyte binding agent (Comparative Example 2), a binding agent that does not bind platelets is used (Example 2, recovery rate 84). It was significantly decreased (0.7%) compared with 0.1%).

  From the above results, it can be seen that in the case of adding a binding agent that binds platelets as in Comparative Example 2, cells having the property of collecting platelets cannot be recovered, and thus the cell recovery rate is significantly reduced. On the other hand, in Example 2, since the binder that does not bind to platelets was used, the cell recovery rate was significantly higher than in Comparative Example 2. That is, since some cells have a property of collecting platelets, it may be possible to further recover the cells by using a leukocyte / erythrocyte binding agent that does not bind to platelets. It could be confirmed.

実施例３
（１）一方の末端がメトキシ基であり、もう一方の末端がＮ−ヒドロオキシスクシンイミドエステル基である、分子量５０００のポリエチレングリコール（ｍＰＥＧ−ＮＨＳ）と、ウシ血清アルブミン（ＢＳＡ）（３００ｍｇ、０．３ｍｍｏｌ／Ｌ）とを、炭酸水素ナトリウム緩衝液（０．１Ｍ、１５ｍＬ）に溶解させ、当該溶液を２５℃で３時間撹拌することでポリエチレングリコールを結合したＢＳＡ（ＰＥＧ−ＢＳＡ）を調製した。なお調製する際、ｍＰＥＧ−ＮＨＳとＢＳＡとのモル比（ｍＰＥＧ−ＮＨＳ／ＢＳＡ）を２となるようにした。調製後、分画分子量１００００の透析膜を用いて、純水への溶液置換を３日間行なった。
（２）実施例１（５）および（６）のＳＫＢＲ３細胞に懸濁させる溶液として、チロフィバンおよび（１）で調製したＰＥＧ−ＢＳＡ（ＢＳＡとして１％（ｗ／ｖ））を含む３００ｍＭマンニトール溶液、を用いた他は、実施例１と同様な方法でＳＫＢＲ３細胞の分離回収および回収率の算出を行なった。

Example 3
(1) Polyethylene glycol (mPEG-NHS) having a molecular weight of 5,000, one end of which is a methoxy group and the other end of which is an N-hydroxysuccinimide ester group, and bovine serum albumin (BSA) (300 mg, 0. 3 mmol / L) was dissolved in a sodium hydrogen carbonate buffer solution (0.1 M, 15 mL), and the solution was stirred at 25 ° C. for 3 hours to prepare polyethylene glycol-bonded BSA (PEG-BSA). During the preparation, the molar ratio of mPEG-NHS and BSA (mPEG-NHS / BSA) was set to 2. After the preparation, the solution was replaced with pure water for 3 days using a dialysis membrane having a molecular weight cut off of 10,000.
(2) A 300 mM mannitol solution containing tirofiban and PEG-BSA (1% (w / v) as BSA) prepared in (1) as a solution to be suspended in SKBR3 cells of Example 1 (5) and (6). Separation and recovery of SKBR3 cells and calculation of recovery rate were performed in the same manner as in Example 1 except that

Comparative Example 3
As the combination of the solution suspended in SKBR3 cells of Example 1 (2) and the solution suspended in SKBR3 cells of Examples 1 (5) and (6), the combinations shown in Table 3 were used, The SKBR3 cells were separated and collected in the same manner as in 1 and the recovery rate was calculated. The “PEG-BSA” described in Table 3 is the PEG-BSA prepared in Example 3 (1), and is a solution containing 1% (w / v) as BSA.

実施例３および比較例３での回収率の結果をまとめて表４に示す。なお表４において、マンニトール以外の添加物濃度は、塩化アンモニウムと炭酸水素カリウムとを含む溶血液、またはマンニトール溶液で３０ｍＬにメスアップしたときの終濃度を示す。

The results of the recovery rates in Example 3 and Comparative Example 3 are summarized in Table 4. In Table 4, the concentration of the additives other than mannitol indicates the final concentration when the volume of the lysed blood containing ammonium chloride and potassium hydrogen carbonate or the mannitol solution was adjusted to 30 mL.

  In the step of separating and recovering cancer cells using centrifugation, by containing 0.5 μg / mL or more of tirofiban, which is an inhibitor against the platelet GPIIb / IIIa receptor, heparin was included when tirofiban was not included. It can be seen that the platelet aggregation is suppressed as compared with the case where ticlopidine was added.

  In addition, when comparing the solutions containing tirofiban with each other, as the concentration of tirofiban was increased, the ability to suppress platelet aggregation was improved, and at a concentration of tirofiban of 0.75 μg / mL or more, the cancer cells (SKBR3 cells) contained in the sample were It was possible to recover with a high recovery rate (recovery rate of about 95%). Further, as compared with Example 1, the recovery rate is improved by adding PEG-BSA, which is a protein to which a hydrophilic polymer is bound, to the sample (without PEG-BSA (Example 1): about 90%). , With PEG-BSA (Example 3): about 95%).

Claims (4)

A method for separating and recovering cells contained in a sample containing platelets by using centrifugation, wherein the sample containing platelets further comprises an inhibitor for platelet GPIIb / IIIa receptors. The method according to claim 1, wherein the sample containing platelets further comprises a protein to which a hydrophilic polymer is bound. The method according to claim 1 or 2, wherein the sample containing platelets further comprises sugar. The method according to any one of claims 1 to 3, wherein the inhibitor for the platelet GPIIb / IIIa receptor is 0.5 μg / mL or more of tirofiban.