JP6994711B2 - Cell content recovery method and recovery device - Google Patents
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Description
本発明は、弱い吸引圧で不純物の混入を抑えることができる細胞内容物の回収方法および回収装置に関する。 The present invention relates to a method and a recovery device for recovering cell contents, which can suppress the contamination of impurities with a weak suction pressure.
プレートに設けた微細な孔に細胞を密着させ、孔を介して吸引することで細胞膜の一部に穴をあけ、この穴から細胞の内容物を吸引・回収する技術が知られている。
細胞の内容物である代謝物、遺伝子、mRNA等を解析することにより、細胞の機能や生命機構の解析を行なう手法としてパッチクランプRT－PCR法等（非特許文献1及び2）がある。
本願発明者らは、上記孔をチップ上にアレイ状に形成することで組織中の単一細胞の解析を網羅的に行なうプロジェクトを推進しており、その成果を報告している（非特許文献3）。
A technique is known in which cells are brought into close contact with fine pores provided in a plate, a hole is made in a part of the cell membrane by sucking through the pores, and the contents of the cells are sucked and recovered from the holes.
There are patch clamp RT-PCR method and the like (
The inventors of the present application are promoting a project to comprehensively analyze a single cell in a tissue by forming the above holes in an array on a chip, and report the results (Non-Patent Documents). 3).
しかし、上記従来技術では次のような問題がある。
すなわち、細胞の内容物を吸引する際、プレートに設けた孔と細胞との密着が充分でない場合には細胞外の溶液も一緒に吸い込んでしまうという問題がある。
例えば細胞中のmRNAを解析したい場合に、この細胞の外部に存在する溶液には他の細胞や実験環境に由来したmRNAが不純物として含まれている。解析したいmRNAが極微量なため、吸引時に不純物が混入することで実験結果に大きな誤差が生じてしまい、場合によっては解析不能になる問題がある。
特に、細胞膜の一部を破って穴をあける際に、吸引圧が高すぎると不純物の混入量が多くなってしまい、吸引圧が低すぎると細胞膜に穴があかないため、吸引圧の調節が難しいという問題がある。
However, the above-mentioned conventional technique has the following problems.
That is, when sucking the contents of the cells, there is a problem that if the pores provided in the plate and the cells are not sufficiently adhered, the extracellular solution is also sucked together.
For example, when it is desired to analyze mRNA in a cell, the solution existing outside the cell contains mRNA derived from another cell or the experimental environment as an impurity. Since the amount of mRNA to be analyzed is extremely small, impurities are mixed in during suction, which causes a large error in the experimental results, and in some cases, there is a problem that analysis becomes impossible.
In particular, when a part of the cell membrane is broken to make a hole, if the suction pressure is too high, the amount of impurities mixed in will be large, and if the suction pressure is too low, the cell membrane will not have a hole, so the suction pressure can be adjusted. There is a problem that it is difficult.
本発明は、上記問題を考慮し、弱い吸引圧で細胞膜に穴をあけることができ、且つ細胞外からの不純物の混入を抑えることができる細胞内容物の回収方法および回収装置を提供することを目的とする。 In consideration of the above problems, the present invention provides a method and an apparatus for recovering cell contents, which can make a hole in a cell membrane with a weak suction pressure and can suppress contamination of impurities from the outside of the cell. The purpose.
本発明の細胞内容物の回収方法は、溶液内において、貫通孔を備えるプレートを挟んで一方の側に細胞を配置する第1ステップと、前記プレートを挟んで他方の側から前記貫通孔を介して前記溶液を吸引することで前記細胞の細胞膜を前記貫通孔に密着させる第2ステップと、前記他方の側を細胞膜溶解液で満たすことで前記貫通孔を介して当該細胞膜溶解液に接触する前記細胞膜の一部を溶解させる第3ステップと、前記他方の側から前記貫通孔を介して吸引することで前記細胞膜の溶解箇所に穴をあける第4ステップと、前記細胞膜の穴から前記細胞の内容物を吸引する第5ステップを少なくとも備えることを特徴とする。
また、更に、吸引した前記細胞の内容物を閉空間に閉じ込めておく第6ステップを備えることを特徴とする。
また、前記貫通孔の直径が5μm以下であることを特徴とする。
また、複数の細胞に対して同時若しくはほぼ同時に前記細胞の内容物を吸引することを特徴とする。
The method for recovering cell contents of the present invention comprises a first step of arranging cells on one side of a plate having a through hole in a solution, and the through hole from the other side of the plate. The second step of bringing the cell membrane of the cell into close contact with the through hole by sucking the solution, and the contact with the cell membrane lysate through the through hole by filling the other side with the cell membrane lysate. The third step of lysing a part of the cell membrane, the fourth step of making a hole in the lysis site of the cell membrane by sucking from the other side through the through hole, and the contents of the cell from the hole of the cell membrane. It is characterized by having at least a fifth step of sucking an object.
Further, it is characterized by comprising a sixth step of confining the contents of the sucked cells in a closed space.
Further, the diameter of the through hole is 5 μm or less.
It is also characterized in that the contents of the cells are aspirated to a plurality of cells at the same time or almost simultaneously.
本発明ではプレートに設けた貫通孔に細胞膜を密着させた上で、細胞膜溶解液を用いて貫通孔を介して細胞膜の一部を溶解させる。これにより細胞膜が薄くなるので、貫通孔を介して吸引した際に弱い吸引圧でも細胞膜に容易に穴をあけることができる。また、細胞膜を貫通孔に密着させた状態で弱い圧力で吸引するので、細胞外の溶液が貫通孔を通過して移動し辛くなり、細胞外からの不純物の混入を抑えることができる。
吸引した細胞の内容物を閉空間に閉じ込めておくことにすれば、内容物の解析処理を正確且つ充分に行なうことができる。
貫通孔の直径を5μm以下程度にすると、吸引によって細胞膜を貫通孔に密着させた状態で細胞膜の一部の溶解処理を最も効果的に行なうことができる。
In the present invention, the cell membrane is brought into close contact with the through hole provided in the plate, and then a part of the cell membrane is dissolved through the through hole using a cell membrane lysing solution. As a result, the cell membrane becomes thin, so that it is possible to easily make a hole in the cell membrane even with a weak suction pressure when sucking through the through hole. Further, since the cell membrane is sucked with a weak pressure in a state of being in close contact with the through hole, it becomes difficult for the extracellular solution to move through the through hole, and it is possible to suppress the contamination of impurities from the outside of the cell.
If the contents of the aspirated cells are confined in a closed space, the contents can be analyzed accurately and sufficiently.
When the diameter of the through hole is set to about 5 μm or less, it is possible to most effectively dissolve a part of the cell membrane in a state where the cell membrane is in close contact with the through hole by suction.
本発明の細胞内容物の回収方法及び回収装置について説明する。
図1(a)に示すように、作業者はまず貫通孔10を備えるプレート20を溶液30内に配置し、当該プレート20の一方の側A1に細胞40を配置する（第1ステップ）。
貫通孔10の直径は細胞40のサイズに応じて適宜変更すればよいが、一般的な細胞のサイズに基づくと直径5μm以下程度が好ましい。
また、一枚のプレート20に多数の貫通孔10をアレイ状に配列することにしてもよい。
第1ステップに用いる溶液30の種類は特に限定されるものではなく、例えばTriton X-100、Triton X-114, Brij-35, Tween 20, Tween 80, Octyl glucoside, Octyl thioglucoside, SDS, CHAPS, CHAPSO等、周知の溶液を用いればよい。
The method for recovering the cell contents and the recovery device of the present invention will be described.
As shown in FIG. 1 (a), the operator first places the
The diameter of the
Further, a large number of through
The type of
次に、図1(b)に示すように、作業者はプレート20を挟んで他方の側A2から貫通孔10を介して溶液30を周知の吸引手段で吸引する（吸引の向きを図中に矢印で示す）。これに伴い、細胞40は溶液30と共にプレート20側に移動していき、その細胞膜41が貫通孔10に密着する（第2ステップ）。
次に、図1(c)に示すように、作業者はプレート20の他方の側A2を細胞膜溶解液31で満たす。これにより貫通孔10を介して当該細胞膜溶解液31に接触する箇所の細胞膜41の一部が溶解する（第3ステップ）。
細胞膜溶解液31は、細胞膜41を溶解する機能を有する溶液であれば特に限定されないが、例えばNP-40、Triton X-100、Triton X-114, Brij-35, Tween 20, Tween 80, Octyl glucoside, Octyl thioglucoside, SDS, CHAPS, CHAPSO等の周知の界面活性剤やザイモリエイス(Zymolyase), リゾチーム，セルラーゼ，チモリアーゼ，ドリスラーゼ等の酵素を含有する溶液が挙げられる。
Next, as shown in FIG. 1 (b), the operator sandwiches the
Next, as shown in FIG. 1 (c), the operator fills the other side A2 of the
The
次に、図1(d)に示すように、作業者はプレート20の他方の側A2から貫通孔10を介して細胞膜溶解液31を吸引することで細胞膜41の溶解箇所に穴42をあける（第4ステップ）。なお、細胞膜溶解液31を例えばPBS(Phosphate Buffered Saline リン酸緩衝生理食塩水)等の周知のバッファーに置換しておいてもよい。
上記第3ステップで細胞膜溶解液31に接触する部分の細胞膜41は溶解しているため、弱い吸引圧で容易に細胞膜に穴42をあけることができる。
第4ステップで吸引により細胞膜41の溶解部分に穴42をあけると、図1(e)に示すように、当該穴から細胞の内容物43も同時に吸引される（第5ステップ）。
細胞膜41は貫通孔10の周囲に密着しているので、吸引時には細胞の内容物43だけが貫通孔10を通過してプレート20の他方の側A2に移動する。換言すると、細胞40の外部を満たしている溶液30は貫通孔10をほとんど通過せず、プレート20の一方の側A1に留まるので、細胞40の外部を満たしている溶液30に含まれている不純物が吸引される事態を防止できる。
なお、吸引した細胞の内容物43は閉空間に閉じ込めておくのが好ましい（第6ステップ）。
Next, as shown in FIG. 1 (d), the operator makes a
Since the
When a
Since the
The
次に、上記実施の形態で示した細胞内容物の回収装置および回収方法の実施例について説明する。
単一細胞内のmRNAなどの生体分子プロファイル測定を行うため、微細な貫通孔の上に密着させた単一細胞から内部の生体分子を抽出する。
プレーナーパッチクランプ用に開発した基板及び図1に示した構造を持つ抽出装置(図2)を用いた。抽出装置は上下二層のチャンバーから構成されており、両チャンバーは抽出基板（プレート）に形成した微細な貫通孔で接続されている。
テスト細胞としてヒト胎児腎細胞由来の細胞(HEK293細胞)を用いた。細胞内容物に蛍光タンパク質GFPを遺伝子発現させ抽出実験のマーカーとして利用した。細胞はまず一次吸引（5 kPa）により、プレーナーパッチクランプチップのプレートに設けた直径2μmの貫通孔（図3(a)）上へ配置した(図3(b))。その後、細胞膜破壊を目的に二次吸引として15 kPaまで吸引圧力を増大させることにより、細胞の位置を保持した状態で、細胞内のGFP蛍光強度の急速な減衰を確認した(図3(c))。この蛍光強度の減衰は細胞膜が貫通孔地点での吸引圧力により破壊され(図3(d))、細胞内容物が抽出された結果である。さらにこの抽出溶液を回収し、GFPのmRNAを逆転写し、リアルタイムPCRを用いた定量解析を行った。
Next, examples of the recovery device and recovery method for the cell contents shown in the above embodiment will be described.
In order to measure the profile of biomolecules such as mRNA in a single cell, the internal biomolecule is extracted from the single cell that is in close contact with the fine through hole.
The substrate developed for the planar patch clamp and the extraction device with the structure shown in Fig. 1 (Fig. 2) were used. The extraction device is composed of two upper and lower chambers, and both chambers are connected by fine through holes formed in the extraction substrate (plate).
Human fetal kidney cell-derived cells (HEK293 cells) were used as test cells. The fluorescent protein GFP was gene-expressed in the cell contents and used as a marker for extraction experiments. The cells were first placed by primary suction (5 kPa) over a 2 μm diameter through hole (Fig. 3 (a)) provided in the plate of the planar patch clamp tip (Fig. 3 (b)). After that, by increasing the suction pressure to 15 kPa as secondary suction for the purpose of cell membrane destruction, rapid attenuation of intracellular GFP fluorescence intensity was confirmed while maintaining the cell position (Fig. 3 (c)). ). This attenuation of fluorescence intensity is the result of the cell membrane being destroyed by the suction pressure at the through-hole point (Fig. 3 (d)) and the cell contents being extracted. Furthermore, this extract was recovered, GFP mRNA was reverse transcribed, and quantitative analysis was performed using real-time PCR.
本発明の細胞内容物の回収装置および回収方法の利点として、一つの空間で細胞を破砕するspatial transcriptomeの方法とは異なり、貫通孔から細胞内容物を吸引しているので他の細胞の情報が混入しにくいことがあげられる。これを評価するために、単一細胞の細胞内容物の回収量とこの細胞外の溶液に含まれるmRNAの混入（コンタミ）量をqPCRにより評価した。またコンタミ量を減らすために、吸引法の最適化を行った。 The advantage of the cell content recovery device and recovery method of the present invention is that unlike the spatial transcriptome method of disrupting cells in one space, the cell contents are sucked through the through holes, so that information on other cells can be obtained. It is difficult to mix. In order to evaluate this, the amount of recovered cell contents of a single cell and the amount of mRNA contamination contained in this extracellular solution were evaluated by qPCR. In addition, the suction method was optimized to reduce the amount of contamination.
微細な貫通孔に細胞を吸引し（図4(a)）、ヒトやマウスに存在しない配列を持つコントロールmRNAを含む溶液に細胞外溶液を置換し(図4(b))、貫通孔に接する細胞膜を破砕し細胞質を吸引抽出し、抽出液にどれだけ混入するかを調べた。細胞膜を破砕するにあたり、図4(b)の状態のまま吸引して破砕する方法と、抽出側の溶液（PBS）を界面活性剤溶液（1%NP-40）に置換して抽出した場合(図4(c))とで外部コンタミ（コントロールmRNAの混入）の程度を比較した。
まず、抽出側の溶液に界面活性剤を添加した場合、細胞膜破砕の様子がどのように異なるかを調べた（図5）。縦の列に陰圧（吸引圧）、横の行に細胞膜溶解液への浸水時間を示している。陰圧が低ければ低いほど外部コンタミは少ないはずで、1kPaであれば、10分ほど1%NP-40のPBS溶液に接触させておけば細胞膜を破砕できることが分かった。
The cells are aspirated into the fine through-holes (Fig. 4 (a)), the extracellular solution is replaced with a solution containing a control mRNA with a sequence not present in humans or mice (Fig. 4 (b)), and the cells come into contact with the through-holes. The cell membrane was crushed and the cytoplasm was aspirated and extracted, and the amount of contamination in the extract was investigated. When crushing the cell membrane, the method of suctioning and crushing the cell membrane in the state shown in Fig. 4 (b) and the case where the solution (PBS) on the extraction side is replaced with a surfactant solution (1% NP-40) for extraction ( The degree of external contamination (contamination of control mRNA) was compared with FIG. 4 (c)).
First, we investigated how the appearance of cell membrane crushing differs when a surfactant is added to the solution on the extraction side (Fig. 5). Negative pressure (suction pressure) is shown in the vertical column, and the immersion time in the cell membrane lysate is shown in the horizontal row. It was found that the lower the negative pressure, the less external contamination should be, and that 1 kPa can crush the cell membrane by contacting with 1% NP-40 PBS solution for about 10 minutes.
次に吸引法の違いによる細胞由来EGFP ｍRNAの量と細胞外からの混入オリゴRNAの量を示す(図6)。細胞内容物吸引サンプルはいずれもEGFP mRNAを検出できている。
吸引前後に培地を吸引したサンプルからは検出されず、環境からのコンタミ、およびキャリーオーバは無視できることが確認できた。外部からのオリゴRNAのコンタミは、吸引圧に比例して増えていることが分かった。これは、本実施例で作成した貫通孔の周囲には微細な凹凸があるためシール性が悪く、この凹凸を通して外部のRNAが混入していることを暗示している。また、吸引前に界面活性剤によって細胞膜の一部を溶解処理し、低い吸引圧（1 kPa）で吸引したサンプルではコンタミの量は吸引20 μlに比して70 pl程度と無視できるレベルに改善した。このような細胞膜処理の併用に加えて貫通孔の周囲の凹凸についてプロセス改良を施すことでさらに改善できるものと期待される。
Next, the amount of cell-derived EGFP mRNA and the amount of extracellularly contaminated oligo RNA by different suction methods are shown (Fig. 6). EGFP mRNA can be detected in all cell content aspiration samples.
It was not detected in the sample in which the medium was aspirated before and after aspiration, and it was confirmed that contamination from the environment and carryover were negligible. It was found that the contamination of oligo RNA from the outside increased in proportion to the suction pressure. This implies that the sealing property is poor due to the fine unevenness around the through hole created in this example, and that external RNA is mixed through the unevenness. In addition, in the sample in which a part of the cell membrane was dissolved with a surfactant before suction and sucked with a low suction pressure (1 kPa), the amount of contamination improved to about 70 pl, which is negligible compared to 20 μl of suction. did. In addition to the combined use of such cell membrane treatment, it is expected that the unevenness around the through hole can be further improved by improving the process.
本発明は、弱い吸引圧で細胞膜に穴をあけることができ、且つ細胞外からの不純物の混入を抑えることができる細胞内容物の回収方法および回収装置に関するものであり、プレーナー技術であることから同時に多数の点において細胞内容物の回収ができる方法に関するもので、産業上利用可能である。 The present invention relates to a method and a recovery device for recovering cell contents, which can make a hole in a cell membrane with a weak suction pressure and can suppress contamination of impurities from the outside of the cell, and is a planar technique. It relates to a method for recovering cell contents in many respects at the same time, and is industrially available.
A1 一方の側
A2 他方の側
10 貫通孔
20 プレート
30 溶液
31 細胞膜溶解液
40 細胞
41 細胞膜
42 穴
43 内容物
A1 One side
A2 The other side
10 through hole
20 plates
30 solution
31 Cell membrane lysate
40 cells
41 Cell membrane
42 holes
43 Contents
Claims (4)
前記プレートを挟んで他方の側から前記貫通孔を介して前記溶液を吸引することで前記細胞の細胞膜を前記貫通孔に密着させる第2ステップと、
前記他方の側を細胞膜溶解液で満たすことで前記貫通孔を介して当該細胞膜溶解液に接触する前記細胞膜の一部を溶解させる第3ステップと、
前記他方の側から前記貫通孔を介して吸引することで前記細胞膜の溶解箇所に穴をあける第4ステップと、
前記細胞膜の穴から前記細胞の内容物を吸引する第5ステップを少なくとも備えることを特徴とする細胞内容物の回収方法。
In the solution, the first step of placing cells on one side across a plate with through holes,
The second step of bringing the cell membrane of the cell into close contact with the through hole by sucking the solution from the other side of the plate through the through hole.
The third step of lysing a part of the cell membrane that comes into contact with the cell membrane lysate through the through hole by filling the other side with the cell membrane lysate,
The fourth step of making a hole in the lysis site of the cell membrane by sucking from the other side through the through hole,
A method for recovering a cell content, which comprises at least a fifth step of sucking the cell content from a hole in the cell membrane.
The method for recovering cell contents according to claim 1, further comprising a sixth step of confining the contents of the sucked cells in a closed space.
The method for recovering cell contents according to claim 1 or 2, wherein the diameter of the through hole is 5 μm or less.
The method for recovering cell contents according to any one of claims 1 to 3, wherein the contents of the cells are aspirated to a plurality of cells at the same time or almost simultaneously.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006129798A (en) | 2004-11-08 | 2006-05-25 | Onchip Cellomics Consortium | Cell chip and method for modifying cell and method for controlling cell |
| JP2008539711A (en) | 2005-05-03 | 2008-11-20 | オックスフォード・ジーン・テクノロジー・アイピー・リミテッド | Apparatus and method for analyzing individual cells |
| WO2015030201A1 (en) | 2013-08-30 | 2015-03-05 | 独立行政法人科学技術振興機構 | Planar patch clamp device and planar patch clamp system |
| WO2017131216A1 (en) | 2016-01-28 | 2017-08-03 | 国立大学法人京都大学 | Biopolymer fractionation chip, and biopolymer fractionation method and biopolymer analysis method using same |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2006129798A (en) | 2004-11-08 | 2006-05-25 | Onchip Cellomics Consortium | Cell chip and method for modifying cell and method for controlling cell |
| JP2008539711A (en) | 2005-05-03 | 2008-11-20 | オックスフォード・ジーン・テクノロジー・アイピー・リミテッド | Apparatus and method for analyzing individual cells |
| WO2015030201A1 (en) | 2013-08-30 | 2015-03-05 | 独立行政法人科学技術振興機構 | Planar patch clamp device and planar patch clamp system |
| WO2017131216A1 (en) | 2016-01-28 | 2017-08-03 | 国立大学法人京都大学 | Biopolymer fractionation chip, and biopolymer fractionation method and biopolymer analysis method using same |
Non-Patent Citations (1)
| Title |
|---|
| BMC Molecular Biology，UK，2010年，Vol.11，Article82 |
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