JP2019037159A - Cell content recovery method and cell content recovery apparatus - Google Patents

Abstract

To provide a cell content recovery method and a cell content recovery apparatus each of which enables a cell membrane to be perforated with a weak suction pressure and enables the immixing of impurities from outside of a cell to be suppressed.SOLUTION: The cell content recovery method of the present invention comprises at least: a first step of arranging, in solution 30, a cell 40 on one side A1 with a plate 20 comprising a through-hole 10 interposed between the one side A1 and the other side A2; a second step of bringing a cell membrane 41 of the cell into close contact with the through-hole by sucking the solution through the through-hole from the other side A2 with the plate interposed between the sides A1 and A2; a third step of dissolving a part of the cell membrane contacting a cell membrane solution through the through-hole by filling the other side with a cell membrane solution 31; a fourth step of perforating a hole 42 in the dissolved part of the cell membrane by the suction through the through-hole from the other side; and a fifth step of sucking a content 43 of the cell from the hole of the cell membrane.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method and an apparatus for recovering cell contents which can suppress contamination of impurities with a weak suction pressure.

A technique is known in which cells are closely attached to minute holes provided in a plate and aspiration is carried out through the holes to make a hole in a part of the cell membrane, and the contents of the cells are aspirated and recovered from the holes.
There is a patch clamp RT-PCR method etc. (nonpatent literature 1 and 2) as a method of analyzing the function of a cell, a life mechanism, etc. by analyzing the metabolite which is the contents of a cell, a gene, mRNA etc.
The present inventors are promoting a project to comprehensively analyze single cells in tissue by forming the holes in an array on the chip, and report the results (non-patent document). 3).

Lambollez, B., Audinat, E., Bochet, P., Crepel, F. and Rossier, J. (1992) Neuron 9 [2], 247-258. Saiful Islam, Unaμm Kjallquist, Annalena Molener, Pawel Zajac, Jian-Bing Fan, Peter Lonnerberg and Sten Linnarsson, (2017), Genome Research CSH Press, August 21. Takamura et al., “Quantitative analysis of single cell mRNA by planar patch clamp and verification of external contamination” [13a-B8-3] The Institute of Applied Physics September 2016

However, the above prior art has the following problems.
That is, when the contents of the cells are aspirated, there is a problem that the extracellular solution is also sucked together if the adhesion between the pores provided in the plate and the cells is not sufficient.
For example, when it is desired to analyze mRNA in cells, the solution present outside the cells contains mRNA derived from other cells or experimental environment as an impurity. Since the amount of mRNA to be analyzed is extremely small, contamination with impurities during aspiration 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 punctured to make a hole, if the aspiration pressure is too high, the amount of impurities mixed increases, and if the aspiration pressure is too low, the cell membrane does not form a hole, so adjustment of the aspiration pressure is There is a problem of difficulty.

  SUMMARY OF THE INVENTION In view of the above problems, the present invention provides a method and an apparatus for recovering cell contents which can puncture a cell membrane with a weak suction pressure and suppress contamination of impurities from outside the cell. To aim.

In the method for recovering cell contents of the present invention, a first step of arranging cells on one side across a plate provided with a through hole in a solution, and the other side across the plate via the through hole A second step of bringing the cell membrane of the cell into close contact with the through hole by aspirating the solution, and filling the other side with the cell membrane solution to contact the cell membrane solution via the through hole A third step of lysing a part of the cell membrane, a fourth step of making a hole in the cell membrane dissolution site by suctioning from the other side through the through hole, and the contents of the cell from the cell membrane hole The method is characterized by at least including a fifth step of suctioning an object.
Furthermore, the method is characterized by further comprising a sixth step of confining the contents of the aspirated cells in a closed space.
In addition, the diameter of the through hole is 5 μm or less.
Further, the present invention is characterized in that the contents of the cells are aspirated simultaneously or substantially simultaneously to a plurality of cells.
The apparatus for recovering cell contents of the present invention comprises a plate having a through hole and being disposed in a solution, and the through hole from the other side in a state where cells are disposed on one side across the plate. The suction means is provided for bringing the cell membrane of the cell into close contact with the through hole by suctioning the solution via the cell membrane, and the other side is filled with a cell membrane solution to dissolve the cell membrane through the through hole. A portion of the cell membrane in contact with the solution is dissolved, and then aspiration is made from the other side through the through hole to make a hole in the dissolution site of the cell membrane, and then the cell of the cell It is characterized by suctioning the contents.
Furthermore, it is characterized in that the contents of the aspirated cells are confined in a closed space.
In addition, the diameter of the through hole is 5 μm or less.
Further, the present invention is characterized in that the contents of the cells are aspirated simultaneously or substantially simultaneously to a plurality of cells.

In the present invention, the cell membrane is brought into close contact with the through holes provided in the plate, and then a part of the cell membrane is dissolved through the through holes using a cell membrane 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 suctioned through the through hole. In addition, since the cell membrane is aspirated with a weak pressure in close contact with the through hole, the solution outside the cell is difficult to move through the through hole and the contamination from outside the cell can be suppressed.
If the contents of the aspirated cells are confined in a closed space, the analysis of the contents can be performed accurately and sufficiently.
When the diameter of the through hole is about 5 μm or less, the dissolution treatment of a part of the cell membrane can be most effectively performed in a state where the cell membrane is in close contact with the through hole by suction.

Diagrams (a) to (e) schematically showing the steps of the apparatus and method for recovering cell contents The figure which shows the device configuration for carrying out the collection method of the cell contents It is a figure which shows the observation result of the fluorescence reduction at the time of extraction of the single cell and cell content which were captured on the through-hole by the attraction | suction from the lower layer, (a) before seeding, (b) is cell capture, (C) shows the shed of cells after extraction and (d) shows the shed of cells after extraction. Scale bar: 20 μm Figure (a) showing cells (HEK 293) aspirated and intimately attached to the through holes (a) Figure (b) showing the extracellular fluid replaced with a solution containing control mRNA and the solution on the aspiration side (C) shows the state of substitution with PBS solution of 10% NP-40 The graph which shows the success or failure of cell membrane disruption by the magnitude | size of negative pressure, and processing time. O indicates success, x indicates failure. Graph showing the amount of cell-derived EGFP mRNA and the amount of extracellular oligoDNA contamination due to differences in aspiration methods

The method and apparatus for recovering cell contents of the present invention will be described.
As shown in FIG. 1A, the operator first arranges the plate 20 provided with the through holes 10 in the solution 30, and arranges the cells 40 on one side A1 of the plate 20 (first step).
The diameter of the through holes 10 may be changed as appropriate according to the size of the cells 40, but a diameter of about 5 μm or less is preferable based on the size of general cells.
Further, a large number of through holes 10 may be arranged in an array on one plate 20.
The type of solution 30 used in the first step is not particularly limited. For example, Triton X-100, Triton X-114, Brij-35, Tween 20, Tween 80, Octyl glucoside, Octyl thioglycoside, SDS, CHAPS, CHAPSO Well-known solutions may be used.

Next, as shown in FIG. 1 (b), the operator holds the plate 20 and suctions the solution 30 from the other side A2 through the through hole 10 by a known suction means (the direction of suction is shown in the drawing) Shown by arrows). Along with this, the cell 40 moves to the plate 20 side together with the solution 30, and the cell membrane 41 adheres to the through hole 10 (second step).
Next, as shown in FIG. 1 (c), the operator fills the other side A 2 of the plate 20 with the cell membrane solution 31. As a result, a part of the cell membrane 41 in a portion in contact with the cell membrane solution 31 via the through hole 10 is dissolved (third step).
The cell membrane lysate 31 is not particularly limited as long as it has a function of lysing the cell membrane 41. For example, NP-40, Triton X-100, Triton X-114, Brij-35, Tween 20, Tween 80, Octyl glucoside There may be mentioned solutions containing known surfactants such as Octyl thioglycoside, SDS, CHAPS, CHAPSO, and enzymes such as Zymolyase, lysozyme, cellulase, thymolyase, and dorisulase.

Next, as shown in FIG. 1 (d), the worker aspirates the cell membrane lysate 31 from the other side A2 of the plate 20 through the through hole 10 to make a hole 42 in the dissolution site of the cell membrane 41 ( 4th step). Alternatively, the cell membrane lysate 31 may be replaced with a known buffer such as PBS (Phosphate Buffered Saline phosphate buffered saline).
Since the cell membrane 41 in the portion in contact with the cell membrane lysate 31 in the third step is dissolved, the hole 42 can be easily opened in the cell membrane with a weak suction pressure.
In the fourth step, when a hole 42 is made in the lysed portion of the cell membrane 41 by suction, the contents 43 of the cell are also simultaneously suctioned from the hole as shown in FIG. 1 (e) (fifth step).
Since the cell membrane 41 is in intimate contact with the periphery of the through hole 10, only the contents 43 of the cell move through the through hole 10 to the other side A2 of the plate 20 at the time of suction. In other words, since the solution 30 filling the outside of the cell 40 hardly passes through the through holes 10 and remains on one side A1 of the plate 20, the impurities contained in the solution 30 filling the outside of the cell 40 Can be prevented from being sucked.
It is preferable to confine the contents 43 of the aspirated cells in a closed space (sixth step).

Next, examples of the apparatus and method for recovering cell contents described in the above embodiment will be described.
In order to measure biomolecular profiles such as mRNA in single cells, internal biomolecules are extracted from single cells brought into close contact with fine through holes.
A substrate developed for the planar patch clamp and an extractor (FIG. 2) having the structure shown in FIG. 1 were used. The extraction apparatus is composed of upper and lower two layers of chambers, and both chambers are connected by fine through holes formed in the extraction substrate (plate).
Cells derived from human embryonic kidney cells (HEK 293 cells) were used as test cells. The fluorescent protein GFP was expressed in the cell contents and used as a marker for extraction experiments. The cells were first placed on the 2 μm diameter through holes (FIG. 3 (a)) provided in the plate of the planar patch clamp tip by primary aspiration (5 kPa) (FIG. 3 (b)). After that, the aspiration pressure was increased to 15 kPa as secondary aspiration for the purpose of cell membrane destruction, and while the position of the cell was maintained, the rapid decay of the intracellular GFP fluorescence intensity was confirmed (FIG. 3 (c)) ). This decrease in fluorescence intensity is the result of the cell membrane being destroyed by the aspiration pressure at the penetration point (FIG. 3 (d)) and the cell contents being extracted. Furthermore, this extraction solution was recovered, GFP mRNA was reverse transcribed, and quantitative analysis was performed using real-time PCR.

  As an advantage of the apparatus and method for recovering cell contents of the present invention, unlike the method of spatial transcriptome that disrupts cells in one space, the cell contents are aspirated from the through holes, so the information of other cells is obtained. It can be mentioned that it is difficult to mix. In order to evaluate this, the recovery amount of the cell contents of single cells and the amount of contamination (contamination) of mRNA contained in this extracellular solution were evaluated by qPCR. In order to reduce the amount of contamination, optimization of the suction method was performed.

Aspirate cells into minute through holes (Fig. 4 (a)), replace extracellular solution with a solution containing control mRNA having a sequence not present in human or mouse (Fig. 4 (b)), and touch the through holes. The cell membrane was disrupted, the cytoplasm was aspirated and extracted, and it was examined how much the extract was mixed. In breaking the cell membrane, a method of suctioning and breaking in the state of FIG. 4 (b) and a case where the solution on the extraction side (PBS) 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, when a surfactant was added to the solution on the extraction side, it was examined how the state of cell membrane disruption differs (FIG. 5). The negative column (suction pressure) is shown in the vertical column, and the water immersion time into the cell membrane lysate is shown in the horizontal column. It was found that the lower the negative pressure, the smaller the external contamination, and if it is 1 kPa, the cell membrane can be broken if it is contacted with a 1% NP-40 PBS solution for about 10 minutes.

Next, the amount of cell-derived EGFP mRNA and the amount of extraneous contaminating oligoRNA due to differences in aspiration methods are shown (FIG. 6). All cellular content aspirates can detect EGFP mRNA.
It was not detected from 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 the oligoRNA from the outside increased in proportion to the suction pressure. This implies that the sealability is poor due to the fine unevenness around the through hole created in the present embodiment, and it is suggested that the external RNA is mixed through the unevenness. In addition, a part of the cell membrane was dissolved and treated with a surfactant prior to aspiration, and the amount of contamination improved to a negligible level of about 70 pl compared to 20 μl for a sample that was aspirated with low aspiration pressure (1 kPa) did. It is expected that further improvement can be achieved by performing process improvement on the asperities around the through holes in addition to the combined use of the cell membrane treatment.

  The present invention relates to a method and an apparatus for recovering cell contents which can puncture a cell membrane with a weak suction pressure and can suppress contamination of impurities from outside the cell, which is a planar technique. The present invention relates to a method capable of recovering cell contents at many points simultaneously and is industrially applicable.

A1 One side
A2 the other side
10 through holes
20 plates
30 solution
31 cell membrane lysate
40 cells
41 cell membrane
42 holes
43 contents

Claims (8)

Placing the cells on one side of the plate with the through holes in the solution;
A second step of bringing the cell membrane of the cell into close contact with the through hole by suctioning the solution from the other side of the plate via the through hole;
A third step of lysing a part of the cell membrane in contact with the cell membrane lysate through the through hole by filling the other side with the cell membrane lysate;
A fourth step of forming a hole in a dissolution site of the cell membrane by suctioning from the other side through the through hole;
A method for recovering cell contents, comprising at least a fifth step of suctioning the contents of the cells from holes 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 aspirated cells in a closed space.
The diameter of the said through-hole is 5 micrometers or less, The collection | recovery method of the cell contents of Claim 1 or 2 characterized by the above-mentioned.
The method for recovering cell contents according to any one of claims 1 to 3, wherein the contents of the cells are aspirated simultaneously or substantially simultaneously to a plurality of cells.
A plate having through holes and disposed in the solution;
A suction means is provided for bringing the cell membrane of the cells into close contact with the through hole by sucking the solution from the other side through the through hole in a state where the cell is disposed on one side across the plate. Yes,
Filling the other side with a cell membrane solution to dissolve a part of the cell membrane in contact with the cell membrane solution via the through hole;
Then, a hole is formed in the dissolution site of the cell membrane by suctioning from the other side through the through hole,
Next, an apparatus for recovering cell contents, wherein the contents of the cells are aspirated from the holes of the cell membrane.
The apparatus for recovering cell contents according to claim 5, further comprising: confining the contents of the aspirated cells in a closed space.
The diameter of the said through-hole is 5 micrometers or less, The collection | recovery apparatus of the cell content of Claim 5 or 6 characterized by the above-mentioned.
The apparatus for recovering cell contents according to any one of claims 5 to 7, wherein the contents of the cells are aspirated simultaneously or substantially simultaneously to a plurality of cells.

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