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JP7078949B2 - The Resolution and Collection Method of Cell Contents - Google Patents

The Resolution and Collection Method of Cell Contents Download PDF

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JP7078949B2
JP7078949B2 JP2017160963A JP2017160963A JP7078949B2 JP 7078949 B2 JP7078949 B2 JP 7078949B2 JP 2017160963 A JP2017160963 A JP 2017160963A JP 2017160963 A JP2017160963 A JP 2017160963A JP 7078949 B2 JP7078949 B2 JP 7078949B2
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cell
diaphragm
flow path
space
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JP2019037160A (en
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禅 高村
芳昭 浮田
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Japan Advanced Institute of Science and Technology
University of Yamanashi NUC
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Japan Advanced Institute of Science and Technology
University of Yamanashi NUC
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Description

本発明は、細胞内容物を極微量だけ吸引し、且つ吸引後の拡散を防ぐことができる細胞内容物の回収機構および回収方法に関する。 The present invention relates to a recovery mechanism and a recovery method for cell contents, which can suck a very small amount of cell contents and prevent diffusion after suction.

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（非特許文献1）等がある。
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2）。 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 is a patch clamp RT-PCR method (Non-Patent Document 1) and the like as a method for analyzing the function and life mechanism of cells by analyzing metabolites, genes, mRNA and the like which are the contents of cells.
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). 2).

Lambolez, B., Audinat, E., Bochet, P., Crepel, F. and Rossier, J.（1992） Neuron9[2], 247-258.Lambolez, B., Audinat, E., Bochet, P., Crepel, F. and Rossier, J. (1992) Neuron9 [2], 247-258. 高村ら， “プレーナーパッチクランプによる単一細胞mRNAの定量解析と外部汚染の検証” [13a-B8-3] 応用物理学会 2016年9月Takamura et al., “Quantitative analysis of single-cell mRNA by planar patch clamp and verification of external contamination” [13a-B8-3] Japan Society of Applied Physics, September 2016

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、細胞の大きさは1pL程度であり、細胞の内容物も同程度と極微量である。したがって、細胞内容物の体積以上を一度に吸引すると、吸引した細胞内容物が孔を通り抜けたあと広範囲に拡散して薄まってしまうという問題や、陰圧源の方まで流れていって失われてしまうという問題がある。
理想的には細胞内容物とほぼ同体積だけ吸引するのがよいが、極微量だけ吸引するように陰圧源の駆動を制御することが難しいという問題もある。 However, the above-mentioned conventional technique has the following problems.
That is, the size of the cell is about 1 pL, and the content of the cell is as small as the same. Therefore, if more than the volume of the cell contents is sucked at one time, the sucked cell contents will spread and dilute over a wide area after passing through the pores, and will flow to the negative pressure source and be lost. There is a problem that it will end up.
Ideally, it is better to suck in almost the same volume as the cell contents, but there is also a problem that it is difficult to control the drive of the negative pressure source so that only a very small amount is sucked.

本発明は、上記問題を考慮し、細胞内容物を極微量だけ吸引し、且つ吸引後の拡散を防ぐことができる細胞内容物の回収機構および回収方法を提供することを目的とする。 In consideration of the above problems, it is an object of the present invention to provide a recovery mechanism and a recovery method for cell contents, which can suck a very small amount of cell contents and prevent diffusion after suction.

本発明の細胞内容物の回収機構は、貫通孔を備えるプレートと、前記貫通孔の一方の端部と繋がる回収空間と、前記回収空間に繋がる流路と、前記2本の流路を開閉する弁と、前記流路内の液体を吸引して細胞を前記貫通穴の他方の端部に密着させるための吸引源と、前記回収空間内を負圧状態にするように変位可能なダイヤフラムを前記吸引源とは別に備えており、前記貫通孔の他方の端部に細胞を密着させ、且つ前記弁で前記流路を閉じた状態で前記ダイヤフラムを変位させて前記回収空間内を負圧状態にすることで、前記細胞の一部に穴をあけて前記貫通孔を介して前記細胞の内容物を前記回収空間内に吸引することを特徴とする。
また、前記ダイヤフラムの変位量を調節するための変位量調節構造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。
本発明の細胞内容物の回収方法は、貫通孔を備えるプレートと、前記貫通孔の一方の端部と繋がる回収空間と、前記回収空間に繋がる流路と、前記流路を開閉する弁と、前記流路内の液体を吸引して細胞を前記貫通穴の他方の端部に密着させるための吸引源と、前記回収空間内を負圧状態にするように変位可能なダイヤフラムが前記吸引源とは別に存在しており、前記貫通孔の他方の端部に細胞を密着させるステップと、前記弁で前記流路を閉じた状態で前記ダイヤフラムを変位させて前記回収空間内を負圧状態にすることで、前記細胞の一部に穴をあけて前記貫通孔を介して前記細胞の内容物を前記回収空間内に吸引するステップを備えることを特徴とする。
The cell content recovery mechanism of the present invention opens and closes a plate provided with a through hole, a recovery space connected to one end of the through hole, a flow path connected to the recovery space, and the two flow paths. The valve, a suction source for sucking the liquid in the flow path and bringing the cells into close contact with the other end of the through hole, and a diaphragm displaceable so as to bring the inside of the recovery space into a negative pressure state. It is provided separately from the suction source, and the cells are brought into close contact with the other end of the through hole, and the diaphragm is displaced with the flow path closed by the valve to bring the inside of the recovery space into a negative pressure state. This is characterized in that a hole is made in a part of the cell and the contents of the cell are sucked into the recovery space through the through hole.
Further, it is characterized by having a displacement amount adjusting structure for adjusting the displacement amount of the diaphragm.
Further, the diameter of the through hole is 5 μm or less.
The method for recovering the cell contents of the present invention includes a plate provided with a through hole, a recovery space connected to one end of the through hole, a flow path connected to the recovery space, and a valve for opening and closing the flow path. A suction source for sucking the liquid in the flow path to bring the cells into close contact with the other end of the through hole, and a diaphragm displaceable so as to bring the inside of the recovery space into a negative pressure state are the suction sources. Exists separately , the step of bringing the cell into close contact with the other end of the through hole and the displacement of the diaphragm with the flow path closed by the valve to bring the inside of the recovery space into a negative pressure state. This is characterized by comprising a step of making a hole in a part of the cell and sucking the contents of the cell into the recovery space through the through hole.

本発明ではダイヤフラムの変位量に応じて細胞内容物の回収量を調節できるので、細胞内容物を極微量だけ吸引することができる。
また、吸引した細胞内容物は回収空間内に留まるので、吸引後の拡散を防ぐことができる。
特に、ダイヤフラムの変位量を調節するための変位量調整構造を備えることにすれば、ダイヤフラムを最大限変位させることで細胞内容物を所望の量だけ吸引（回収）できるので、ダイヤフラムの変位を調節するための機構の駆動制御が容易になる。
貫通孔の直径を5μm以下程度にすると、吸引によって細胞を貫通孔に確実に密着させた状態で細胞内容物を吸引できるので、細胞外の溶液に含まれる不純物を吸引してしまう事態を防止できる。 In the present invention, since the recovery amount of the cell contents can be adjusted according to the displacement amount of the diaphragm, only a very small amount of the cell contents can be sucked.
In addition, since the aspirated cell contents remain in the recovery space, diffusion after aspiration can be prevented.
In particular, if a displacement amount adjusting structure for adjusting the displacement amount of the diaphragm is provided, the cell contents can be sucked (recovered) by the desired amount by maximizing the displacement of the diaphragm, so that the displacement of the diaphragm can be adjusted. The drive control of the mechanism for this is facilitated.
When the diameter of the through-hole is set to about 5 μm or less, the cell contents can be sucked in a state where the cells are firmly adhered to the through-hole by suction, so that it is possible to prevent the situation where impurities contained in the extracellular solution are sucked. ..

第1の実施の形態の細胞内容物の回収機構の構造を模式的に示した図(a)及びA-A’線矢視図(b)The figure (a) and the A-A'line arrow view (b) schematically showing the structure of the recovery mechanism of the cell contents of the first embodiment. 細胞内容物の回収機構の使用方法を示す図(a)～(d)Figures (a)-(d) showing how to use the cell content recovery mechanism 第2の実施の形態の細胞内容物の回収機構の構造を示す図(a)及びダイヤフラムの変位後の状態を示す図(b)及び(c)Figures (a) showing the structure of the cell content recovery mechanism of the second embodiment and figures (b) and (c) showing the state after displacement of the diaphragm.

［第1の実施の形態］
本発明の細胞内容物の回収機構の第1の実施の形態について説明する。
図1(a)及び(b)に示すように、細胞内容物の回収機構1はプレート10、回収空間20、流路30、弁40、ダイヤフラム50で概略構成されている。細胞60は単一の状態で細胞格納空間70に格納されている。
プレート10は貫通孔11を備えており、貫通孔11の一方の端部は回収空間20に繋がっており、他方の端部には細胞60が密着する。なお、一枚のプレート10に多数の貫通孔11をアレイ状に配列することにしてもよい。
貫通孔11の直径は細胞60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。 [First Embodiment]
The first embodiment of the cell content recovery mechanism of the present invention will be described.
As shown in FIGS. 1 (a) and 1 (b), the cell content recovery mechanism 1 is roughly composed of a plate 10, a recovery space 20, a flow path 30, a valve 40, and a diaphragm 50. The cell 60 is stored in the cell storage space 70 in a single state.
The plate 10 is provided with a through hole 11, one end of the through hole 11 is connected to the recovery space 20, and the other end is in close contact with the cell 60. A large number of through holes 11 may be arranged in an array on one plate 10.
The diameter of the through hole 11 may be appropriately changed according to the size of the cell 60, but is preferably about 5 μm or less in diameter based on the general cell size.

回収空間20は細胞内容物61を回収するための空間であり、貫通孔11の一方の端部と繋がっている。本実施の形態では、回収空間20内が仕切り板21によって区画化されており、mRNAを捕捉するために必要な処理が施されたビーズ80を各区画22内に格納している。ビーズ80の詳細は周知であるため説明を省略する。
流路30は回収空間20に繋がっており、流路30内を溶液や気体が通る仕組みになっている。本実施の形態では入口用と出口用の2本の流路30が回収空間20に繋がっている。
なお、回収空間20、流路30及び細胞格納空間70の内部は液体で満たされている。
弁40は流路30を開閉するための部材であり、本実施の形態では2本の流路30それぞれに弁40を取り付けている。弁40は周知の構造のものを利用できる。
ダイヤフラム50は回収空間20に繋がっており、変位することで回収空間20内を負圧状態にすることができる。 The recovery space 20 is a space for recovering the cell contents 61, and is connected to one end of the through hole 11. In the present embodiment, the recovery space 20 is partitioned by the partition plate 21, and the beads 80 that have been subjected to the treatment necessary for capturing mRNA are stored in each partition 22. Since the details of the beads 80 are well known, the description thereof will be omitted.
The flow path 30 is connected to the recovery space 20, and the mechanism is such that a solution or gas passes through the flow path 30. In this embodiment, two flow paths 30 for the inlet and the outlet are connected to the recovery space 20.
The inside of the recovery space 20, the flow path 30, and the cell storage space 70 is filled with a liquid.
The valve 40 is a member for opening and closing the flow path 30, and in the present embodiment, the valve 40 is attached to each of the two flow paths 30. The valve 40 can be of a well-known structure.
The diaphragm 50 is connected to the recovery space 20, and by displacing the diaphragm 50, the inside of the recovery space 20 can be in a negative pressure state.

次に、細胞内容物の回収方法について説明する。
図2(a)に示すように、作業者は入口側の弁40を「閉」、出口側の弁40を「開」にした状態で吸引する。なお、吸引源（陰圧源）を出口側の流路30の端部に取り付けている。吸引圧は例えば5 kPa程度の低圧でよい。
出口側の流路30を吸引することで回収空間20内の液体が吸引され、これにより貫通孔11を介して細胞格納空間70内の液体も吸引される。細胞格納空間70内の細胞60は液体と共に貫通孔11の他方の端部まで移動して密着する。
次に、図2(b)に示すように、作業者は入口用の弁40を開け、吸引することで回収空間20の内部を溶液（例えば1％のNP-40等）で満たす。
次に、図2(c)に示すように、作業者は入口用及び出口用の弁40を閉じることで回収空間20内を閉空間にする。
そして、図2(d)に示すように、作業者はダイヤフラム50を変位させ、回収空間20内を負圧状態にする。これにより細胞60の一部に穴があき、細胞内容物61は貫通孔11を通って回収空間20内に吸引される。
回収空間20内は閉空間になっているので、細胞内容物61をビーズ80と充分に反応させることができる。作業者は一定時間経過後、両方の弁40を開けてマニピュレータ等でビーズ80を回収する。 Next, a method for recovering the cell contents will be described.
As shown in FIG. 2A, the operator sucks the valve 40 on the inlet side in the “closed” state and the valve 40 on the outlet side in the “open” state. A suction source (negative pressure source) is attached to the end of the flow path 30 on the outlet side. The suction pressure may be a low pressure of, for example, about 5 kPa.
By sucking the flow path 30 on the outlet side, the liquid in the recovery space 20 is sucked, and thereby the liquid in the cell storage space 70 is also sucked through the through hole 11. The cells 60 in the cell storage space 70 move together with the liquid to the other end of the through hole 11 and adhere to each other.
Next, as shown in FIG. 2 (b), the operator opens the inlet valve 40 and sucks to fill the inside of the recovery space 20 with a solution (for example, 1% NP-40).
Next, as shown in FIG. 2 (c), the operator closes the inlet and outlet valves 40 to make the recovery space 20 a closed space.
Then, as shown in FIG. 2D, the operator displaces the diaphragm 50 to put the inside of the recovery space 20 in a negative pressure state. As a result, a part of the cell 60 is punctured, and the cell content 61 is sucked into the recovery space 20 through the through hole 11.
Since the recovery space 20 is a closed space, the cell contents 61 can be sufficiently reacted with the beads 80. After a certain period of time, the operator opens both valves 40 and collects the beads 80 with a manipulator or the like.

［第2の実施の形態］
次に、本発明の細胞内容物の回収機構の第2の実施の形態について説明するが、上記第1の実施の形態と同一の構成となる箇所については同一の符号を付してその説明を省略する。
図3(a)に示すように、本実施の形態の細胞内容物の回収機構2はダイヤフラム90が弁40の機能も兼ねる点と、ダイヤフラム90の変位量を調節するための変位量調節構造100を備える点に特徴を有する。
すなわち、図3(b)に示すようにダイヤフラム90のうち右側の部分が変位することで回収空間20内を負圧状態にし、図3(c)に示すようにダイヤフラム90の左側の部分が変位することで流路30を開閉する弁40として機能する。ダイヤフラム90の変位の調節は、ダイヤフラム90の下面側に存在する液体91の量を調節することで行なうことができる。
また、上部が開口した箱形状の変位量調整構造100をダイヤフラム90の右側及び左側の部分の下方に備えている。図3(b)に示すように、ダイヤフラム90の右側の部分が最大に変位した状態では変位量調整構造100の内面に密着する。つまり、ダイヤフラム90の右側部分は変位量調整構造100の体積より大きく変位することがない。したがって、例えば変位量調整構造100の体積を細胞内容物61の体積と同程度（例えば1pL程度）にしておけば、ダイヤフラム90の右側部分を最大限変位させるだけで細胞内容物61のほぼ全量を容易且つ正確に回収することができる。ダイヤフラム90の右側部分を最大限変位させるようにダイヤフラム90の下面側の液体91の量を調節することは容易である。同様に、図3(c)に示すようにダイヤフラム90の左側の部分を変位させて弁40として機能させる場合に、弁40の開閉度を調節することも容易になる。 [Second embodiment]
Next, the second embodiment of the cell content recovery mechanism of the present invention will be described, but the parts having the same configuration as the first embodiment will be described with the same reference numerals. Omit.
As shown in FIG. 3A, the cell content recovery mechanism 2 of the present embodiment has the point that the diaphragm 90 also functions as the valve 40 and the displacement amount adjusting structure 100 for adjusting the displacement amount of the diaphragm 90. It is characterized in that it is provided with.
That is, as shown in FIG. 3 (b), the right part of the diaphragm 90 is displaced to put the inside of the recovery space 20 into a negative pressure state, and as shown in FIG. 3 (c), the left part of the diaphragm 90 is displaced. By doing so, it functions as a valve 40 that opens and closes the flow path 30. The displacement of the diaphragm 90 can be adjusted by adjusting the amount of the liquid 91 present on the lower surface side of the diaphragm 90.
Further, a box-shaped displacement amount adjusting structure 100 having an open upper portion is provided below the right and left portions of the diaphragm 90. As shown in FIG. 3 (b), when the right portion of the diaphragm 90 is maximally displaced, it is in close contact with the inner surface of the displacement amount adjusting structure 100. That is, the right side portion of the diaphragm 90 is not displaced more than the volume of the displacement amount adjusting structure 100. Therefore, for example, if the volume of the displacement amount adjusting structure 100 is set to be about the same as the volume of the cell content 61 (for example, about 1 pL), almost the entire amount of the cell content 61 can be reduced by maximally displacing the right side portion of the diaphragm 90. It can be easily and accurately collected. It is easy to adjust the amount of liquid 91 on the underside of the diaphragm 90 so that the right portion of the diaphragm 90 is displaced as much as possible. Similarly, when the left side portion of the diaphragm 90 is displaced to function as the valve 40 as shown in FIG. 3 (c), it becomes easy to adjust the opening / closing degree of the valve 40.

次に、上記第1の実施の形態で示した細胞内容物の回収機構を用いた実施例について説明する。
1. 回収空間内にmRNA吸着ビーズをマイクロマニピュレーターにより配置し、貫通孔（直径2μm）を備えるプレートでカバーした。
2. 流路内と回収空間に溶媒を満たした。
3. 細胞チャンバー（細胞格納空間）に細胞を加えた。モデル細胞としてGFP安定高発現HEK293T細胞、およびノーマルのHEK293細胞を用いた。
4. 入口用流路の弁を閉じ、出口用流路の弁を開いた状態で回収空間内の溶媒を吸引し、細胞を貫通孔の他方の端部に密着させ、捕捉した。
5. 入口用及び出口用の弁を閉じた状態でダイヤフラムを動作させ、細胞内容物を回収空間内に抽出し、一定時間この状態を保持した。
6. マイクロマニピュレーターによりmRNA吸着ビーズをリアクションチューブに回収した。
7. 回収したｍRNAは速やかにSmartSeqV4を用いてｃDNAに変換し、次世代シーケンサで解析したところ、数千の遺伝子の発現を確認できた。 Next, an example using the cell content recovery mechanism shown in the first embodiment will be described.
1. The mRNA adsorption beads were placed in the recovery space by a micromanipulator and covered with a plate having a through hole (diameter 2 μm).
2. The flow path and the recovery space were filled with solvent.
3. Cells were added to the cell chamber (cell storage space). As model cells, HEK293T cells with stable and high expression of GFP and normal HEK293 cells were used.
4. With the inlet valve closed and the outlet valve open, the solvent in the recovery space was aspirated and the cells were brought into close contact with the other end of the through hole and captured.
5. The diaphragm was operated with the inlet and outlet valves closed, and the cell contents were extracted into the recovery space and maintained in this state for a certain period of time.
6. The mRNA adsorption beads were collected in a reaction tube by a micromanipulator.
7. The recovered mRNA was rapidly converted to cDNA using SmartSeqV4 and analyzed with a next-generation sequencer, and the expression of thousands of genes was confirmed.

本発明は、細胞内容物を極微量だけ吸引し、且つ吸引後の拡散を防ぐことができる細胞内容物の回収機構および回収方法に関するものであり、産業上利用可能である。 The present invention relates to a recovery mechanism and a recovery method for cell contents, which can suck a very small amount of cell contents and prevent diffusion after suction, and can be industrially used.

1 細胞内容物の回収機構
2 細胞内容物の回収機構
10 プレート
11 貫通孔
20 回収空間
21 仕切り板
22 区画
30 流路
40 弁
50 ダイヤフラム
60 細胞
61 細胞内容物
70 細胞格納空間
80 ビーズ
90 ダイヤフラム
91 液体
100 変位量調節構造 1 Cell content recovery mechanism
2 Cell content recovery mechanism
10 plates
11 Through hole
20 Collection space
21 divider
22 parcels
30 Channel
40 valve
50 diaphragm
60 cells
61 Cell contents
70 Cell storage space
80 beads
90 diaphragm
91 Liquid
100 Displacement amount adjustment structure

Claims (4)

貫通孔を備えるプレートと、
前記貫通孔の一方の端部と繋がる回収空間と、
前記回収空間に繋がる流路と、
前記流路を開閉する弁と、
前記流路内の液体を吸引して細胞を前記貫通穴の他方の端部に密着させるための吸引源と、
前記回収空間内を負圧状態にするように変位可能なダイヤフラムを前記吸引源とは別に備えており、
前記貫通孔の他方の端部に細胞を密着させ、且つ前記弁で前記流路を閉じた状態で前記ダイヤフラムを変位させて前記回収空間内を負圧状態にすることで、前記細胞の一部に穴をあけて前記貫通孔を介して前記細胞の内容物を前記回収空間内に吸引することを特徴とする細胞内容物の回収機構。
Plates with through holes and
A collection space connected to one end of the through hole,
The flow path connected to the collection space and
A valve that opens and closes the flow path,
A suction source for sucking the liquid in the flow path and bringing the cells into close contact with the other end of the through hole.
A diaphragm that can be displaced so as to bring the inside of the recovery space into a negative pressure state is provided separately from the suction source .
A part of the cells is brought into close contact with the other end of the through hole, and the diaphragm is displaced with the valve closed to bring the inside of the recovery space into a negative pressure state. A mechanism for recovering cell contents, which comprises making a hole in the cell and sucking the contents of the cells into the recovery space through the through hole.
 前記ダイヤフラムの変位量を調節するための変位量調節構造を備えることを特徴とする請求項1に記載の細胞内容物の回収機構。
The mechanism for recovering cell contents according to claim 1, further comprising a displacement amount adjusting structure for adjusting the displacement amount of the diaphragm.
 前記貫通孔の直径が5μm以下であることを特徴とする請求項1又は2に記載の細胞内容物の回収機構。
The mechanism for recovering cell contents according to claim 1 or 2, wherein the diameter of the through hole is 5 μm or less.
 貫通孔を備えるプレートと、
前記貫通孔の一方の端部と繋がる回収空間と、
前記回収空間に繋がる流路と、
前記流路を開閉する弁と、
前記流路内の液体を吸引して細胞を前記貫通穴の他方の端部に密着させるための吸引源と、
前記回収空間内を負圧状態にするように変位可能なダイヤフラムが前記吸引源とは別に存在しており、
前記貫通孔の他方の端部に細胞を密着させるステップと、
前記弁で前記流路を閉じた状態で前記ダイヤフラムを変位させて前記回収空間内を負圧状態にすることで、前記細胞の一部に穴をあけて前記貫通孔を介して前記細胞の内容物を前記回収空間内に吸引するステップを備えることを特徴とする細胞内容物の回収方法。
Plates with through holes and
A collection space connected to one end of the through hole,
The flow path connected to the collection space and
A valve that opens and closes the flow path,
A suction source for sucking the liquid in the flow path and bringing the cells into close contact with the other end of the through hole.
A diaphragm that can be displaced so as to bring the inside of the recovery space into a negative pressure state exists separately from the suction source .
The step of bringing the cell into close contact with the other end of the through hole,
By displacing the diaphragm with the valve closing the flow path to bring the inside of the recovery space into a negative pressure state, a hole is made in a part of the cell and the content of the cell is passed through the through hole. A method for recovering cell contents, which comprises a step of sucking a substance into the recovery space.
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