JP2015047285A - Glass filter and method to separate cells - Google Patents
Glass filter and method to separate cells Download PDFInfo
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- JP2015047285A JP2015047285A JP2013180416A JP2013180416A JP2015047285A JP 2015047285 A JP2015047285 A JP 2015047285A JP 2013180416 A JP2013180416 A JP 2013180416A JP 2013180416 A JP2013180416 A JP 2013180416A JP 2015047285 A JP2015047285 A JP 2015047285A
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- 238000000034 method Methods 0.000 title abstract description 7
- 239000011324 bead Substances 0.000 claims abstract description 65
- 210000004027 cell Anatomy 0.000 claims abstract description 41
- 210000004369 blood Anatomy 0.000 claims abstract description 22
- 239000008280 blood Substances 0.000 claims abstract description 22
- 239000002245 particle Substances 0.000 claims abstract description 20
- 239000007788 liquid Substances 0.000 claims description 13
- 238000003320 cell separation method Methods 0.000 claims description 11
- 238000000926 separation method Methods 0.000 claims description 11
- 210000002751 lymph Anatomy 0.000 claims description 6
- 210000004880 lymph fluid Anatomy 0.000 abstract description 2
- 239000004793 Polystyrene Substances 0.000 description 27
- 229920002223 polystyrene Polymers 0.000 description 27
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- 210000003743 erythrocyte Anatomy 0.000 description 12
- 238000010304 firing Methods 0.000 description 10
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- 239000011148 porous material Substances 0.000 description 6
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- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 5
- 239000000463 material Substances 0.000 description 4
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- 108010039918 Polylysine Proteins 0.000 description 3
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- 239000000126 substance Substances 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 239000011800 void material Substances 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 239000006087 Silane Coupling Agent Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
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- 210000004204 blood vessel Anatomy 0.000 description 1
- 239000005385 borate glass Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
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Abstract
Description
本発明は、ガラスフィルタ及び細胞分離方法に関する。 The present invention relates to a glass filter and a cell separation method.
病気の予防やサンプル採取等の観点で、体液をフィルタに通過させて体液中の細胞や血球等の成分を分離する技術が注目されている。 From the viewpoint of disease prevention, sample collection, and the like, a technique for separating components such as cells and blood cells in a body fluid by passing the body fluid through a filter has attracted attention.
例えば、血球を除去分離するために、表面がポリリジンでコーティングされたガラスビーズを充填したフィルタが使用されている（特許文献１を参照）。このフィルタは、ガラスビーズの表面をポリリジンでコーティングすることによってガラスビーズ表面を正電荷に帯電させ、ガラスビーズ表面と負電荷を有する血球とを相互作用させ、ガラスビーズ表面に補足することにより分離するものである。 For example, in order to remove and separate blood cells, a filter filled with glass beads whose surface is coated with polylysine is used (see Patent Document 1). This filter separates the glass bead surface by coating the surface of the glass bead with polylysine to positively charge the glass bead surface, interacting the glass bead surface with negatively charged blood cells, and capturing the glass bead surface. Is.
しかしながら、上記フィルタは、正電荷と負電荷との相互作用を利用して血球を特異的に補足するものであるため、血球を分離することはできるが、その他の細胞を分離することはできない。また、ガラスビーズの表面をポリリジンでコーティングする必要があるため、手間がかかる。 However, since the filter specifically captures blood cells by utilizing the interaction between positive charges and negative charges, it can separate blood cells but cannot separate other cells. Moreover, since it is necessary to coat the surface of a glass bead with polylysine, it takes time.
本発明は、以上の実情に鑑みてなされたものであり、細胞を分離するのに適し、かつ簡便に製造可能な新しいガラスフィルタ及びこのガラスフィルタを用いた細胞分離方法を提供することを目的とする。 The present invention has been made in view of the above circumstances, and an object thereof is to provide a new glass filter that is suitable for separating cells and can be easily produced, and a cell separation method using the glass filter. To do.
本発明者らは、ガラスビーズを相互に融着させることで、細胞を分離できるガラスフィルタを簡便に得られることを見出し、本発明を完成するに至った。すなわち、本発明は以下の通りである。 The present inventors have found that a glass filter capable of separating cells can be easily obtained by fusing glass beads together, and have completed the present invention. That is, the present invention is as follows.
（１）相互に融着された複数のガラスビーズからなるガラスフィルタ。 (1) A glass filter composed of a plurality of glass beads fused to each other.
（２）前記ガラスビーズの平均粒径が１００〜２００μｍである（１）記載のガラスフィルタ。 (2) The glass filter according to (1), wherein the glass beads have an average particle size of 100 to 200 μm.
（３）細胞分離用である（１）又は（２）記載のガラスフィルタ。 (3) The glass filter according to (1) or (2), which is for cell separation.
（４）細胞を含む液体を（３）記載のガラスフィルタに通し、前記液体中の細胞を分離する工程を有する、細胞分離方法。 (4) A cell separation method comprising a step of passing a liquid containing cells through the glass filter according to (3) and separating the cells in the liquid.
（５）前記液体は血液又はリンパ液である（４）記載の細胞分離方法。 (5) The cell separation method according to (4), wherein the liquid is blood or lymph.
本発明によれば、細胞を分離するのに適し、かつ簡便に製造可能な新しいガラスフィルタ及びこのガラスフィルタを用いた細胞分離方法を提供することができる。 ADVANTAGE OF THE INVENTION According to this invention, it is suitable for isolate | separating a cell, and can provide the new glass filter which can be manufactured simply, and the cell separation method using this glass filter.
以下、本発明の実施形態について詳細に説明するが、本発明は以下の実施形態になんら限定されるものではなく、本発明の目的の範囲内において、適宜変更を加えて実施することができる。 Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments, and can be implemented with appropriate modifications within the scope of the object of the present invention.
＜ガラスフィルタ＞
本発明のガラスフィルタは、相互に融着された複数のガラスビーズからなる。かかる構成を有することにより、ガラスフィルタがガラスビーズ間に空隙を備えるので、ガラスフィルタとしての機能を発揮する。また、本発明のガラスフィルタによれば、ガラスビーズの表面に化学的性質を付加することを要さずに、物理的な分離が可能である。かかる性質を備える本発明のガラスフィルタによれば、細胞の大きさに応じた分離が可能であるため、細胞の分離に適している。しかし、本発明のガラスフィルタの用途は、特にこれに限定されない。さらに、本発明のガラスフィルタは無機材料であるガラスにより構成されることにより、化学的、熱的耐久性、耐熱性、放射線耐性が高い。
<Glass filter>
The glass filter of the present invention comprises a plurality of glass beads fused to each other. By having such a configuration, the glass filter has a gap between the glass beads, and thus functions as a glass filter. In addition, according to the glass filter of the present invention, physical separation is possible without the need to add chemical properties to the surface of the glass beads. According to the glass filter of the present invention having such properties, separation according to the size of the cells is possible, which is suitable for cell separation. However, the use of the glass filter of the present invention is not particularly limited to this. Furthermore, the glass filter of the present invention is made of glass that is an inorganic material, and thus has high chemical, thermal durability, heat resistance, and radiation resistance.
本発明で用いるガラスビーズの材料は、特に限定されないが、例えば、ソーダ石灰ガラス、鉛ガラス、バリウムガラス、ホウケイ酸ガラス、アルミノケイ酸ガラス、リチウムアルミノケイ酸ガラス等のケイ酸ガラス、鉛ホウ酸ガラス、アルミノホウ酸ガラス等のホウ酸ガラス、アルミノリン酸ガラス等のリン酸ガラス等が挙げられる。 The material of the glass beads used in the present invention is not particularly limited. Examples thereof include borate glasses such as aluminoborate glass and phosphate glasses such as aluminophosphate glass.
ガラスビーズの粒径は、特に限定されない。ガラスビーズの平均粒径を変更することによって、フィルタ内の空隙の径（後述する平均細孔径）が変更される。よって、ガラスビーズの平均粒径は、分離対象に応じて適宜変更可能である。 The particle size of the glass beads is not particularly limited. By changing the average particle diameter of the glass beads, the diameter of the voids in the filter (average pore diameter described later) is changed. Therefore, the average particle diameter of the glass beads can be appropriately changed according to the separation target.
従来より、がん細胞が転移する際に、がん細胞は血管やリンパ管を通ることが知られている。つまり、血液やリンパ液をフィルタに通して、血液中のがん細胞を除去することができれば、がん転移を予防することができる。本発明のガラスフィルタは、分離対象の大きさに応じた物理的な分離が可能であるため、がん細胞を血液やリンパ液中から分離することが可能である。特に本フィルタでは、少量〜大量まで様々な量や性質の血液又はリンパ液を濾過するために、その形状や大きさを制御して調整することができる。さらに、フィルタ内に捕捉したがん細胞をそのまま培養して増殖することが可能であり、がん細胞の詳細な形態学的及び遺伝子的解析に基づく診断と治療に有効である。 Conventionally, when cancer cells metastasize, it is known that cancer cells pass through blood vessels and lymph vessels. That is, cancer metastasis can be prevented if blood or lymph can be passed through a filter to remove cancer cells in the blood. Since the glass filter of the present invention can be physically separated according to the size of the separation target, it is possible to separate cancer cells from blood and lymph. In particular, in this filter, in order to filter blood or lymph fluid of various amounts and properties from a small amount to a large amount, the shape and size can be controlled and adjusted. Furthermore, cancer cells captured in the filter can be cultured and propagated as they are, which is effective for diagnosis and treatment based on detailed morphological and genetic analysis of cancer cells.
がん細胞の大きさは、一般に、約１０〜約２０μｍである。本発明のガラスフィルタが、かかる大きさを有するがん細胞の分離に適した空隙を有するためには、本発明のガラスビーズの平均粒径は、１００〜２００μｍが好ましく、１２０〜１８０μｍがより好ましく、１４０〜１６０μｍが最も好ましい。また、効率よくがん細胞の分離を行うためには、ガラスビーズの粒径の分散度は低い方が好ましいが、特に限定されず、例えば、５０％径に対する９０％径の比が、１．１以下であり、１０％径に対する５０％径の比が１．１以下であってもよい。 The size of the cancer cell is generally about 10 to about 20 μm. In order for the glass filter of the present invention to have voids suitable for separation of cancer cells having such a size, the average particle size of the glass beads of the present invention is preferably 100 to 200 μm, more preferably 120 to 180 μm. 140 to 160 μm is most preferable. Further, in order to efficiently separate cancer cells, it is preferable that the dispersion degree of the particle size of the glass beads is low, but there is no particular limitation. For example, the ratio of 90% diameter to 50% diameter is 1. The ratio of 50% diameter to 10% diameter may be 1.1 or less.
＜ガラスフィルタの製造方法＞
本発明のガラスフィルタの製造方法を図１に沿って説明するが、特にこれに限定されるものではない。
<Glass filter manufacturing method>
Although the manufacturing method of the glass filter of this invention is demonstrated along FIG. 1, it does not specifically limit to this.
図１に示すように、管３０を板４０に立てる。次いで、図１における矢印で示すようにガラスビーズ１０を管３０内に入れ、棒２０で棒を押し、ガラスビーズ１０を管３０内に密に充填する。その後、ガラスビーズ１０を焼成することにより、ガラスビーズ１０は相互に融着する。 As shown in FIG. 1, the tube 30 stands on the plate 40. Next, as shown by the arrows in FIG. 1, the glass beads 10 are put into the tube 30, and the rods 20 are pushed by the rod 20, so that the glass beads 10 are closely packed in the tube 30. Thereafter, the glass beads 10 are fused to each other by firing the glass beads 10.
本発明においては、かかる製造方法により、簡便にガラスフィルタを作製できる。特に、上述の製造方法によると、ガラスビーズ１０の表面処理を要さずに、ガラスフィルタを作製できるという点において簡便である。また、この製造方法によると、ガラスビーズ１０を充填する管３０の形状に合わせて、フィルタの構造を適宜設定することが容易である。 In the present invention, a glass filter can be easily produced by such a production method. In particular, according to the above-described manufacturing method, it is simple in that a glass filter can be produced without requiring surface treatment of the glass beads 10. Further, according to this manufacturing method, it is easy to appropriately set the structure of the filter in accordance with the shape of the tube 30 filled with the glass beads 10.
棒２０、管３０、板４０の材質は、後述する焼成に対して耐熱性を有するものであれば特に限定されない。例えば、これらの材質として、アルミナや、ステンレス等を使用することができる。 The material of the rod 20, the tube 30, and the plate 40 is not particularly limited as long as it has heat resistance against firing, which will be described later. For example, alumina or stainless steel can be used as these materials.
棒２０の形状は、管３０内でガラスビーズ１０を押し込めるものであれば、特に限定されない。管３０の形状は、図１では円柱として示されているが、特にこれに限定されず、四角柱や五角柱等の形状であってもよい。 The shape of the rod 20 is not particularly limited as long as the glass beads 10 can be pushed into the tube 30. The shape of the tube 30 is shown as a cylinder in FIG. 1, but is not particularly limited thereto, and may be a shape such as a quadrangular column or a pentagonal column.
焼成する温度及び時間は、ガラスビーズ１０を相互に融着できる温度、時間であれば特に限定されない。例えば、ガラスの材料としてソーダ石灰ガラスを用いた場合、５００〜８００℃の温度で１０分〜２時間焼成することでガラスビーズ１０を相互に融着することができる。 The firing temperature and time are not particularly limited as long as the glass beads 10 can be fused to each other. For example, when soda lime glass is used as the glass material, the glass beads 10 can be fused to each other by firing at a temperature of 500 to 800 ° C. for 10 minutes to 2 hours.
ガラスビーズ１０を焼成することによって、ガラスビーズ１０が融着し、ガラスビーズ１０間に空隙が発生する。この空隙における平均細孔径（ある空隙における、それぞれのガラスビーズの表面に内接する円の直径）の大きさは、特に限定されず、分離対象の大きさに応じて適宜設定することができる。がん細胞を分離する場合の平均細孔径は、好ましくは、８〜２０μｍであり、最も好ましくは８〜１２μｍである。平均細孔径は、ガラスビーズ１０の焼成温度及び焼成時間を調整することで、調整可能である。 By firing the glass beads 10, the glass beads 10 are fused and voids are generated between the glass beads 10. The size of the average pore diameter in this void (the diameter of a circle inscribed in the surface of each glass bead in a certain void) is not particularly limited, and can be appropriately set according to the size of the separation target. The average pore size when separating cancer cells is preferably 8 to 20 μm, and most preferably 8 to 12 μm. The average pore diameter can be adjusted by adjusting the firing temperature and firing time of the glass beads 10.
ガラスフィルタの高さは、管３０の高さの範囲内で、適宜設定することができる。ガラスフィルタの高さは特に限定されないが、ガラスフィルタの高さを高くすればするほど、ガラスフィルタの分離能が向上する。 The height of the glass filter can be appropriately set within the range of the height of the tube 30. The height of the glass filter is not particularly limited, but as the height of the glass filter is increased, the separation performance of the glass filter is improved.
また、本発明は、上述の通り、ガラスビーズ１０の表面処理を行わずにガラスフィルタを作製することができる。しかし、本発明は、特にこれに限定されず、上記焼成後に、シランカップリング剤等を用いて、ガラスビーズ１０の表面処理を行うことでその表面を疎水化してもよく、或いは、酸処理又はアルカリ処理等の表面処理をガラスビーズ１０に施すことで、その表面を親水化してもよい。その他、タンパクや抗体及び低分子薬剤やリガンドによる修飾も可能である。これらの表面処理は、従来の公知のいずれの方法によっても行うことができる。 Moreover, the present invention can produce a glass filter without performing the surface treatment of the glass beads 10 as described above. However, the present invention is not particularly limited to this, and the surface of the glass beads 10 may be hydrophobized by performing a surface treatment of the glass beads 10 using a silane coupling agent or the like after the firing, The surface of the glass beads 10 may be hydrophilized by subjecting the glass beads 10 to surface treatment such as alkali treatment. In addition, modification with proteins, antibodies, low molecular weight drugs and ligands is also possible. These surface treatments can be performed by any conventionally known method.
上記焼成後又は表面処理後に、ガラスフィルタを洗浄してもよいが、洗浄しなくてもよい。例えば、超音波洗浄によりガラスフィルタを洗浄することができる。 Although the glass filter may be washed after the firing or surface treatment, it may not be washed. For example, the glass filter can be cleaned by ultrasonic cleaning.
＜細胞分離方法＞
本発明は、上記ガラスフィルタを用いることによる、細胞分離方法を包含する。
<Cell separation method>
The present invention includes a cell separation method using the above glass filter.
本発明のガラスフィルタを用いた細胞分離方法は、特に限定されないが、例えば、細胞を含む液体を本発明のガラスフィルタに通し、液体中の細胞を分離することにより行ってもよい。また、本発明の細胞分離方法において、細胞を含む液体は特に限定されないが、例えば、血液やリンパ液を分離するのに適している。なお、細胞を含む液体として血液を用いた場合、分離を行う際にガラスフィルタに血液が付着しにくく、また、細胞中の血球も破壊されにくい。 Although the cell separation method using the glass filter of this invention is not specifically limited, For example, you may carry out by passing the liquid containing a cell through the glass filter of this invention, and isolate | separating the cell in a liquid. In the cell separation method of the present invention, the liquid containing cells is not particularly limited, but is suitable for separating blood and lymph, for example. In addition, when blood is used as the liquid containing cells, blood does not easily adhere to the glass filter during separation, and blood cells in the cells are not easily destroyed.
以下、実施例等に基づき本発明を詳細に説明するが、本発明は、かかる実施例等になんら限定されるものではない。 EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example etc., this invention is not limited to this Example etc. at all.
＜ガラスフィルタの作製＞
（実施例１）
内径２４ｍｍ、高さ３０ｍｍのアルミナ管を準備し、アルミナ板の上に立てた。ガラスビーズとして、平均粒径約１５０μｍであり、分散度が９０％径：１４５.２μｍ、５０％径：１４８.６μｍ、１０％径：１５２．７μｍであるソーダ石灰ガラスのビーズ（ユニオン社製高精度ユニビーズＳＰＬ−１５０）を準備した。ガラスビーズの高さが１ｍｍとなるように、ガラスビーズをアルミナ管に充填し、ステンレス棒で押して、ガラスビーズをアルミナ管内に密に充填した。その後、ガラスビーズを６６０〜７１０℃で、１時間焼成した。焼成後のガラスビーズ間の空隙における平均細孔径は、１７μｍであった。
<Production of glass filter>
(Example 1)
An alumina tube having an inner diameter of 24 mm and a height of 30 mm was prepared and stood on an alumina plate. As glass beads, soda-lime glass beads having an average particle diameter of about 150 μm and a dispersity of 90% diameter: 145.2 μm, 50% diameter: 148.6 μm, 10% diameter: 152.7 μm (made by Union Corporation) Precision uni-beads SPL-150) were prepared. The glass beads were filled into the alumina tube so that the height of the glass beads was 1 mm, and the glass beads were densely filled into the alumina tube by pressing with a stainless steel rod. Thereafter, the glass beads were baked at 660 to 710 ° C. for 1 hour. The average pore diameter in the space between the glass beads after firing was 17 μm.
（実施例２）
ガラスビーズの分散度が９０％径：１４３．１μｍ、５０％径：１５１．８μｍ、１０％径：１６０．７μｍであるソーダ石灰ガラスのビーズ（ユニオン社製ユニビーズＬＢ−１５０）を用い、実施例１と同様の手法により、ガラスフィルタを作製した。ガラスビーズ間の空隙における平均細孔径は、１８μｍであった。
(Example 2)
Example using soda-lime glass beads (Unibeads LB-150 manufactured by Union Co., Ltd.) having a dispersion degree of glass beads of 90% diameter: 143.1 μm, 50% diameter: 151.8 μm, 10% diameter: 160.7 μm A glass filter was produced in the same manner as in 1. The average pore diameter in the space between the glass beads was 18 μm.
＜ガラスフィルタの性能評価＞
（評価１）
がん細胞の大きさを模した１５μｍポリスチレン球を含む分散液と、赤血球の大きさを模した６μｍポリスチレン球を含む分散液を、それぞれ準備した。それぞれの分散液を実施例１及び２のガラスフィルタに通過させ、通過前後における分散液の吸光度を測定し、分散液の吸光度から粒子濃度（個／ｍｌ）を算出した。吸光度の測定には、紫外―可視分光光度計 ＵＶ−２４００（株式会社島津製作所）を用いた。測定結果を以下の表１に示す。なお、表１中の「ガラスフィルタ通過後の粒子数（％）」は、ガラスフィルタ通過前の分散液中のポリスチレン球の全粒子数を１００％とした場合の、ガラスフィルタ通過後のポリスチレン球の粒子数の％を表す。
<Performance evaluation of glass filter>
(Evaluation 1)
A dispersion containing 15 μm polystyrene spheres simulating the size of cancer cells and a dispersion containing 6 μm polystyrene spheres simulating the size of red blood cells were prepared. Each dispersion was passed through the glass filters of Examples 1 and 2, the absorbance of the dispersion before and after passage was measured, and the particle concentration (pieces / ml) was calculated from the absorbance of the dispersion. For the measurement of absorbance, an ultraviolet-visible spectrophotometer UV-2400 (Shimadzu Corporation) was used. The measurement results are shown in Table 1 below. The “number of particles after passing through the glass filter (%)” in Table 1 is the polystyrene sphere after passing through the glass filter when the total number of polystyrene spheres in the dispersion before passing through the glass filter is 100%. % Of the number of particles.
実施例１及び２のいずれのガラスフィルタにおいても、１５μｍポリスチレン球はほとんど通過しなかったのに対し、６μｍポリスチレン球は５０％以上通過することが確認された。特に、ガラスビーズの分散度が９０％径：１４５.２μｍ、５０％径：１４８.６μｍ、１０％径：１５２．７μｍである実施例１においては、６μｍポリスチレン球が９０％以上通過しており、ガラスビーズの分散度が９０％径：１４３．１μｍ、５０％径：１５１．８μｍ、１０％径：１６０．７μｍである実施例２より、６μｍポリスチレン球が多く通過したことが確認された。この結果より、ガラスビーズを融着させて形成したガラスフィルタに、がん細胞を含む血液を通過させることで、がん細胞を分離除去できること、及び、赤血球を含む血液にこのガラスフィルタを通過させることで、赤血球がガラスフィルタを通過することが示唆された。また、ガラスビーズの分散度が９０％径：１４５.２μｍ、５０％径：１４８.６μｍ、１０％径：１５２．７μｍであるガラスフィルタが、赤血球を通過させる性能が高いことが示唆された。 In any of the glass filters of Examples 1 and 2, it was confirmed that the 15 μm polystyrene sphere hardly passed, whereas the 6 μm polystyrene sphere passed 50% or more. In particular, in Example 1 in which the dispersity of the glass beads is 90% diameter: 145.2 μm, 50% diameter: 148.6 μm, 10% diameter: 152.7 μm, 6% polystyrene spheres pass through 90% or more. From Example 2 in which the degree of dispersion of the glass beads was 90% diameter: 143.1 μm, 50% diameter: 151.8 μm, and 10% diameter: 160.7 μm, it was confirmed that many 6 μm polystyrene spheres passed. From this result, cancer cells can be separated and removed by passing blood containing cancer cells through a glass filter formed by fusing glass beads, and this glass filter is passed through blood containing red blood cells. This suggested that red blood cells pass through the glass filter. In addition, it was suggested that a glass filter having a dispersion degree of glass beads of 90% diameter: 145.2 μm, 50% diameter: 148.6 μm, 10% diameter: 152.7 μm has high performance of allowing red blood cells to pass through.
（評価２）
評価１において、実施例１のガラスフィルタ通過後の１５μｍポリスチレン球分散液、６μｍポリスチレン球分散液のそれぞれについて、光学顕微鏡により写真を撮影した。その結果を図２に示す。図２において、（ａ）は、１５μｍポリスチレン球分散液を通過させた後のガラスビーズを示し、（ｂ）は６μｍポリスチレン球分散液を通過させた後のガラスビーズを示す。
(Evaluation 2)
In Evaluation 1, photographs were taken with an optical microscope for each of the 15 μm polystyrene sphere dispersion and the 6 μm polystyrene sphere dispersion after passing through the glass filter of Example 1. The result is shown in FIG. In FIG. 2, (a) shows the glass beads after passing the 15 μm polystyrene sphere dispersion liquid, and (b) shows the glass beads after passing the 6 μm polystyrene sphere dispersion liquid.
図２に示すように、実施例１のガラスフィルタ通過後、１５μｍポリスチレン球は、ガラスビーズ上に堆積しているのに対し、６μｍポリスチレン球は、ガラスビーズ上に堆積していないことが観察された。この結果より、ガラスビーズを融着させて形成したガラスフィルタにおいて、がん細胞を分離除去できること、及び、赤血球が通過可能であることが裏付けられた。 As shown in FIG. 2, after passing through the glass filter of Example 1, it was observed that 15 μm polystyrene spheres were deposited on the glass beads, whereas 6 μm polystyrene spheres were not deposited on the glass beads. It was. From these results, it was confirmed that cancer cells can be separated and removed and that red blood cells can pass through a glass filter formed by fusing glass beads.
（評価３）
６μｍポリスチレン球と１５μｍポリスチレン球の混合分散液を作製し、実施例１のフィルタを通過させ、通過前後の粒子濃度を測定した。測定は、プレパラートに分散液を塗布し、プレパラート全体に対して粒子の画像を複数撮影し、粒子径の分布を算出することにより行った。測定には、静止画像解析型粒子径分布測定装置モフォロギＧ３（株式会社Ｍａｌｖｅｒｎ）を用いた。その結果を、表２に示す。なお、表２中の「ガラスフィルタ通過前の粒子数（％）」及び「ガラスフィルタ通過後の粒子数（％）」は、それぞれ、ガラスフィルタ通過前の混合分散液中のポリスチレン球（１５μｍポリスチレン球と６μｍポリスチレン球の両方を含む）の全粒子数を１００％とした場合の、１５μｍポリスチレン球、６μｍポリスチレンのそれぞれの粒子数の％を表す。
(Evaluation 3)
A mixed dispersion of 6 μm polystyrene spheres and 15 μm polystyrene spheres was prepared, passed through the filter of Example 1, and the particle concentration before and after passage was measured. The measurement was performed by applying the dispersion liquid to the slide, taking a plurality of particle images for the entire slide, and calculating the particle size distribution. For the measurement, a still image analysis type particle size distribution measuring apparatus Morphogi G3 (Malvern, Inc.) was used. The results are shown in Table 2. In Table 2, “number of particles before passing through glass filter (%)” and “number of particles after passing through glass filter (%)” are respectively polystyrene spheres (15 μm polystyrene) in the mixed dispersion before passing through the glass filter. % Of the respective number of particles of 15 μm polystyrene sphere and 6 μm polystyrene when the total number of particles (including both spheres and 6 μm polystyrene spheres) is 100%.
１５μｍポリスチレン球及び６μｍポリスチレン球の混合分散液においても、１５μｍポリスチレンは実施例１のガラスフィルタをほとんど通過せず、６μｍポリスチレン球は実施例１のガラスフィルタを通過することが確認された。この結果より、ガラスビーズを融着させて形成したガラスフィルタにおいて、がん細胞と赤血球とを含む血液において、がん細胞を選択的に分離除去できることが示された。 Even in the mixed dispersion of 15 μm polystyrene spheres and 6 μm polystyrene spheres, it was confirmed that 15 μm polystyrene hardly passed through the glass filter of Example 1, and that 6 μm polystyrene spheres passed through the glass filter of Example 1. From this result, it was shown that cancer cells can be selectively separated and removed in blood containing cancer cells and red blood cells in a glass filter formed by fusing glass beads.
（評価４）
正常マウスの血液を１０倍希釈し、この１０倍希釈血液に、実施例２のガラスフィルタを通過させ、赤血球が通過するか否かについて評価した。評価は、ＢＩＯＲＥＶＯ（株式会社キーエンス）を用いて写真を撮影することにより行った。血液の希釈溶媒としてＰＢＳバッファーを用い、５００倍の倍率で撮影した写真を観察した。その結果を図３に示す。図３において、（ａ）はフィルタ通過前の血液を観察した図を示し、（ｂ）はガラスフィルタ通過後に血液を観察した図を示す。
(Evaluation 4)
The blood of normal mice was diluted 10-fold, and the 10-fold diluted blood was passed through the glass filter of Example 2 to evaluate whether erythrocytes passed. Evaluation was performed by taking a photograph using BIOREVO (Keyence Corporation). A photograph taken at a magnification of 500 times was observed using PBS buffer as a blood dilution solvent. The result is shown in FIG. In FIG. 3, (a) shows a view of the blood before passing through the filter, and (b) shows a view of the blood after passing through the glass filter.
図３に示すように、実施例２のガラスフィルタの通過後において、ガラスフィルタ通過前と比べて、赤血球の減少は確認されなかった。また、実施例２のガラスフィルタ通過後においても、赤血球の変形は確認されなかった。また、図には示さないが、ほとんど血液がガラスに付着しなかったことが確認された。この結果より、ガラスビーズを融着させて形成したガラスフィルタにより、実際の血液中の赤血球を通過させることが可能であること、及び、このガラスフィルタは、血液中に含まれる赤血球を破壊せず、血液中の細胞を分離するのに適していることが示された。 As shown in FIG. 3, after passing through the glass filter of Example 2, a decrease in red blood cells was not confirmed as compared to before passing through the glass filter. Further, even after passing through the glass filter of Example 2, deformation of red blood cells was not confirmed. Although not shown in the figure, it was confirmed that almost no blood adhered to the glass. From this result, it is possible to pass red blood cells in the actual blood by the glass filter formed by fusing the glass beads, and this glass filter does not destroy the red blood cells contained in the blood. It has been shown to be suitable for separating cells in the blood.
１０ ガラスビーズ
２０ 棒
３０ 管
４０ 板
10 glass beads 20 bars 30 tubes 40 plates
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| JP7039801B2 (en) | 2016-08-26 | 2022-03-23 | 学校法人東京理科大学 | Cell separation method and cell separation device |
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