JP2010099052A - Method for isolating cell - Google Patents
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- JP2010099052A JP2010099052A JP2008275952A JP2008275952A JP2010099052A JP 2010099052 A JP2010099052 A JP 2010099052A JP 2008275952 A JP2008275952 A JP 2008275952A JP 2008275952 A JP2008275952 A JP 2008275952A JP 2010099052 A JP2010099052 A JP 2010099052A
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- 238000000034 method Methods 0.000 title abstract description 7
- 230000003204 osmotic effect Effects 0.000 claims abstract description 53
- 239000000243 solution Substances 0.000 claims abstract description 29
- 239000002504 physiological saline solution Substances 0.000 claims abstract description 21
- 239000012153 distilled water Substances 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 238000005406 washing Methods 0.000 claims description 13
- 238000003320 cell separation method Methods 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 239000007788 liquid Substances 0.000 abstract description 12
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 4
- 210000004027 cell Anatomy 0.000 description 48
- 230000004083 survival effect Effects 0.000 description 12
- 210000000130 stem cell Anatomy 0.000 description 9
- 210000003556 vascular endothelial cell Anatomy 0.000 description 9
- 238000004140 cleaning Methods 0.000 description 8
- 210000002950 fibroblast Anatomy 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 5
- 210000000170 cell membrane Anatomy 0.000 description 4
- 210000000577 adipose tissue Anatomy 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 2
- -1 but instead Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 102000038379 digestive enzymes Human genes 0.000 description 1
- 108091007734 digestive enzymes Proteins 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
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Abstract
Description
本発明は、細胞分離方法に関するものである。 The present invention relates to a cell separation method.
従来、ヒトの脂肪組織を採取して、消化酵素および生理食塩水とともに攪拌することにより脂肪組織内に含まれる脂肪由来細胞群を分離する技術が知られている（例えば、特許文献１参照。）。
この技術においては、分離された脂肪由来細胞群には、幹細胞、血管内皮細胞および線維芽細胞等が含まれるので、これらの細胞を種類毎に分離することが必要である。
Conventionally, a technique for separating adipose-derived cells contained in adipose tissue by collecting human adipose tissue and stirring it with digestive enzymes and physiological saline is known (see, for example, Patent Document 1). .
In this technique, since the separated adipose-derived cell group includes stem cells, vascular endothelial cells, fibroblasts, and the like, it is necessary to separate these cells for each type.
多数の細胞を含む細胞群から目的の細胞を分離する方法としては、抽出された細胞群を遠心分離機によって遠心処理することにより、密度勾配法によって細胞の種類毎に分離したり、添加する成長因子の種類を異ならせて、成長速度の相違を利用して細胞の種類毎に分離したり、あるいは、細胞の種類毎に異なる吸着特性を有する抗体を利用して吸着したりすることが行われる。
しかしながら、上記いずれの場合においても、特殊な試薬を使用したり、遠心分離機のような特別な装置を用いたり、あるいは時間をかけて培養したりする必要があるという不都合がある。
As a method of separating target cells from a cell group containing a large number of cells, the extracted cell group is centrifuged by a centrifuge to separate or add each cell type by density gradient method. Different types of factors can be used to separate each cell type using the difference in growth rate, or adsorption can be performed using antibodies having different adsorption characteristics for each cell type. .
However, in any of the above cases, there is an inconvenience that it is necessary to use a special reagent, to use a special apparatus such as a centrifuge, or to cultivate over time.
本発明は上述した事情に鑑みてなされたものであって、特殊な試薬や装置を用いることなく、迅速に所望の細胞を分離することができる細胞分離方法を提供することを目的としている。 This invention is made | formed in view of the situation mentioned above, Comprising: It aims at providing the cell separation method which can isolate | separate a desired cell rapidly, without using a special reagent and an apparatus.
上記目的を達成するために、本発明は以下の手段を提供する。
本発明は、複数種の細胞を含む細胞群に対して、生理食塩水とは浸透圧が異なる浸透圧液を供給する浸透圧液供給ステップと、該浸透圧液供給ステップにおいて浸透圧液を供給した後、所定の時間経過後に、前記細胞群を生理食塩水と同等の浸透圧を有する洗浄液によって洗浄する洗浄ステップとを含む細胞分離方法を提供する。
In order to achieve the above object, the present invention provides the following means.
The present invention provides an osmotic pressure supply step for supplying an osmotic pressure solution having a different osmotic pressure from physiological saline to a cell group including a plurality of types of cells, and an osmotic pressure solution is supplied in the osmotic pressure solution supply step. And a washing step of washing the cell group with a washing solution having an osmotic pressure equivalent to that of physiological saline after a lapse of a predetermined time.
発明者らは、細胞毎に浸透圧に対する耐性が異なることを発見した。
本発明によれば、浸透圧液供給ステップにおいて生理食塩水とは異なる浸透圧の浸透圧液を供給することにより、浸透圧に対する耐性が細胞毎に異なることを利用して、細胞を選択的に分離することができる。すなわち、生理食塩水は通常、０．８５〜０．９％濃度であるため、これとは浸透圧の異なる浸透圧液として、０．８５％濃度より低濃度の液体あるいは０．９％より高濃度の液体を利用することにより、細胞内外の浸透圧差によって、耐性の低い細胞の細胞膜を破壊して細胞を死滅させ、耐性の高い細胞のみを残すことができる。この場合に、特別な試薬や装置を使用することなく、また、長時間にわたる培養も行うことなく、所望の細胞を選択的に分離することができる。
The inventors have discovered that the resistance to osmotic pressure varies from cell to cell.
According to the present invention, by supplying an osmotic pressure liquid having an osmotic pressure different from that of physiological saline in the osmotic pressure liquid supply step, the cells are selectively used by utilizing the fact that the resistance to osmotic pressure differs for each cell. Can be separated. That is, since physiological saline is usually 0.85 to 0.9% concentration, as an osmotic pressure liquid having a different osmotic pressure, a liquid having a concentration lower than 0.85% concentration or higher than 0.9% is used. By using a liquid having a concentration, the cell membrane of a low-resistance cell can be destroyed and killed by an osmotic pressure difference between the inside and outside of the cell, leaving only a high-resistance cell. In this case, desired cells can be selectively separated without using a special reagent or apparatus and without culturing for a long time.
上記発明においては、前記洗浄ステップが、細胞群から分離する細胞の種類に応じて、前記浸透圧液を供給してから洗浄するまでの所要時間を調節することとしてもよい。
細胞によっては、浸透圧液に晒される時間によってその耐性を異ならせる場合もあり、このようにすることで、時間をかけて死滅する細胞を選択的に除去して分離することができる。
In the said invention, the said washing | cleaning step is good also as adjusting the required time after supplying the said osmotic pressure liquid after washing | cleaning according to the kind of cell isolate | separated from a cell group.
Depending on the cells, the resistance may vary depending on the time of exposure to the osmotic pressure solution. By doing so, cells that die over time can be selectively removed and separated.
また、上記発明においては、前記浸透圧液が、蒸留水であり、前記洗浄ステップが、浸透圧液供給の約５〜１０ｍｉｎ経過後に細胞群を洗浄することとしてもよい。
このようにすることで、浸透圧液として生理食塩水より浸透圧の低い蒸留水を使用することにより、細胞膜を介して浸透圧液を細胞内に浸透させ、浸透圧液の圧力によって耐性の低い細胞を破壊して、耐性の高い細胞を分離することができる。
Moreover, in the said invention, the said osmotic pressure liquid is distilled water, The said washing | cleaning step is good also as washing | cleaning a cell group after about 5-10min progress of osmotic pressure liquid supply.
In this way, by using distilled water having a lower osmotic pressure than physiological saline as the osmotic pressure solution, the osmotic pressure solution is permeated into the cell through the cell membrane, and the resistance is low due to the pressure of the osmotic pressure solution. Cells can be destroyed and highly resistant cells can be isolated.
また、上記発明においては、前記浸透圧液が、蒸留水よりも濃度の高く生理食塩水より濃度の低い食塩水であってもよい。
このように、浸透圧液として蒸留水より濃度の高く生理食塩水より濃度の低い食塩水を使用することにより、細胞膜を介して浸透圧液を細胞内に浸透させ、浸透圧液の圧力によって耐性の低い細胞を破壊して、耐性の高い細胞を分離することができる。
In the above invention, the osmotic pressure solution may be a saline solution having a higher concentration than distilled water and a lower concentration than physiological saline.
In this way, by using a saline solution having a concentration higher than that of distilled water and lower than that of physiological saline as the osmotic pressure solution, the osmotic pressure solution is permeated into the cell through the cell membrane and is resistant to the pressure of the osmotic pressure solution. Cells can be destroyed to isolate highly resistant cells.
本発明によれば、生体組織から分離される細胞群に含まれる死細胞数を低減し、不純物を含まない細胞群を得ることができるという効果を奏する。 According to the present invention, there is an effect that the number of dead cells contained in a cell group separated from a biological tissue can be reduced and a cell group containing no impurities can be obtained.
以下、本発明の一実施形態に係る細胞分離方法について、図面を参照して以下に説明する。
本実施形態に係る細胞分離方法は、複数種の細胞から特定種類の細胞を選択的に分離する方法であって、図１に示されるように、細胞群に蒸留水からなる浸透圧液を供給する浸透圧液供給ステップＳ１と、浸透圧液の供給後、所定の時間経過後に、細胞群を生理食塩水からなる洗浄液によって洗浄する洗浄ステップＳ２とを含んでいる。
Hereinafter, a cell separation method according to an embodiment of the present invention will be described with reference to the drawings.
The cell separation method according to the present embodiment is a method for selectively separating a specific type of cell from a plurality of types of cells, and as shown in FIG. 1, supplying an osmotic pressure solution made of distilled water to a cell group. An osmotic pressure supply step S1 and a cleaning step S2 for cleaning the cell group with a cleaning solution made of physiological saline after a predetermined time has elapsed after the osmotic pressure solution is supplied.
さらに具体的には、例えば、脂肪組織等の生体組織を切断して得られた組織片に浸透圧液を供給して組織片を浸漬させた状態で、所定時間にわたって維持した後、生理食塩水で洗浄する。蒸留水は、通常の生理食塩水、すなわち０．８５〜０．９％濃度の食塩水よりも十分に低い浸透圧を有しているので、このような浸透圧液が細胞群に対して供給されると、浸透圧液が各細胞の細胞膜を透過して細胞内に浸透する。 More specifically, for example, an osmotic solution is supplied to a tissue piece obtained by cutting a living tissue such as adipose tissue and the tissue piece is immersed in the tissue piece, and then maintained for a predetermined time. Wash with. Distilled water has an osmotic pressure sufficiently lower than that of normal physiological saline, that is, 0.85 to 0.9% concentration saline. Then, the osmotic pressure solution permeates the cells through the cell membrane of each cell.
発明者らは、細胞の種類に応じて浸透圧に対する耐性が異なることを発見した。
図２に示されるように、ＡＤＳＣ（脂肪由来幹細胞）、ＥＣ（血管内皮細胞）およびFibroblast（線維芽細胞）を蒸留水に浸漬した場合、その処理時間と生存率との関係が変化する。血管内皮細胞および線維芽細胞は浸透圧の低い浸透圧液に対する耐性が幹細胞より弱いので、５分〜１０分経過後には、その生存率は幹細胞の生存率に比べて十分に低くなる。
The inventors have found that resistance to osmotic pressure varies depending on the cell type.
As shown in FIG. 2, when ADSC (adipose-derived stem cells), EC (vascular endothelial cells) and Fibroblast (fibroblasts) are immersed in distilled water, the relationship between the treatment time and the survival rate changes. Since vascular endothelial cells and fibroblasts are less resistant to osmotic fluids with low osmotic pressure than stem cells, their survival rate is sufficiently lower than that of stem cells after 5 to 10 minutes.
特に、１０分経過後には、血管内皮細胞および線維芽細胞の生存率は２０％以下に低下するのに対し、幹細胞の生存率は６０％以上となっており、血管内皮細胞および線維芽細胞を死滅させて幹細胞を選択的に分離することができる。また、１５分経過後には、幹細胞の生存率は２５．５％であるのに対し、血管内皮細胞の生存率は１％、線維芽細胞の生存率は０％とすることができ、幹細胞をより確実に選択的に分離することができる。 In particular, after 10 minutes, the survival rate of vascular endothelial cells and fibroblasts is reduced to 20% or less, whereas the survival rate of stem cells is 60% or more. Stem cells can be selectively isolated by killing. Further, after 15 minutes, the survival rate of the stem cells is 25.5%, whereas the survival rate of the vascular endothelial cells can be 1% and the survival rate of the fibroblasts can be 0%. It is possible to selectively separate more reliably.
なお、本実施形態においては、浸透圧液として蒸留水を採用したが、これに代えて、蒸留水より濃度の高く生理食塩水より濃度の低い食塩水を採用してもよい。例えば、２０％濃度の生理食塩水に１５分間浸漬すると、図３に示されるように、血管内皮細胞のみを８０％以下の生存率に低下させ、４０分間浸漬すると、幹細胞および線維芽細胞の生存率を９０％以上に維持しながら、血管内皮細胞の生存率のみを７０％以下に低下させることができる。その結果、２０％濃度の生理食塩水に浸漬する時間を調節することにより、血管内皮細胞を除去した幹細胞および線維芽細胞を多く含む細胞群を選択的に分離することができる。 In this embodiment, distilled water is used as the osmotic pressure solution, but instead, saline having a higher concentration than distilled water and a lower concentration than physiological saline may be used. For example, when immersed in a 20% physiological saline for 15 minutes, as shown in FIG. 3, only the vascular endothelial cells are reduced to a survival rate of 80% or less, and when immersed for 40 minutes, the survival of stem cells and fibroblasts While maintaining the rate at 90% or higher, only the survival rate of vascular endothelial cells can be reduced to 70% or lower. As a result, it is possible to selectively separate a cell group containing a large amount of stem cells and fibroblasts from which vascular endothelial cells have been removed by adjusting the time of immersion in a 20% concentration physiological saline.
なお、本実施形態においては、蒸留水（０％濃度の食塩水）と２０％濃度の生理食塩水に浸漬した場合について説明したが、これに限定されるものではなく、蒸留水とリン酸緩衝生理食塩水との混合液を用いて任意の濃度の浸透圧液を構成することにしてもよい。また、食塩水に代えて、他の成分からなる浸透圧液を採用することにしてもよい。また、処理時間についても、上記実施形態に限定されるものではない。 In addition, in this embodiment, although the case where it immersed in distilled water (0% concentration saline) and 20% concentration physiological saline was demonstrated, it is not limited to this, Distilled water and phosphate buffer An osmotic solution having an arbitrary concentration may be formed using a mixed solution with physiological saline. Moreover, you may decide to employ | adopt the osmotic pressure liquid which consists of another component instead of salt solution. Further, the processing time is not limited to the above embodiment.
Ｓ１ 浸透圧液供給ステップ
Ｓ２ 洗浄ステップ
S1 Osmotic pressure liquid supply step S2 Cleaning step
Claims (4)
該浸透圧液供給ステップにおいて浸透圧液を供給した後、所定の時間経過後に、前記細胞群を生理食塩水と同等の浸透圧を有する洗浄液によって洗浄する洗浄ステップとを含む細胞分離方法。 An osmotic pressure supply step for supplying an osmotic pressure solution having a different osmotic pressure from physiological saline to a cell group including a plurality of types of cells;
A washing step of washing the cell group with a washing solution having an osmotic pressure equivalent to that of physiological saline after a predetermined time has elapsed after the osmotic pressure solution is supplied in the osmotic pressure solution supply step.
前記洗浄ステップが、浸透圧液供給の約５〜１０ｍｉｎ経過後に細胞群を洗浄する請求項１に記載の細胞分離方法。 The osmotic pressure solution is distilled water;
The cell separation method according to claim 1, wherein in the washing step, the cell group is washed after about 5 to 10 minutes have elapsed since the supply of the osmotic pressure solution.
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| WO2022239867A1 (en) * | 2021-05-14 | 2022-11-17 | 株式会社堀場アドバンスドテクノ | Microorganism detection apparatus and microorganism detection method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPWO2018084167A1 (en) * | 2016-11-01 | 2018-11-08 | テルモ株式会社 | Method for modifying an adherent cell culture |
| JP2019030322A (en) * | 2016-11-01 | 2019-02-28 | テルモ株式会社 | Modified method of cell culture in adhesive state |
| JP2022105168A (en) * | 2016-11-01 | 2022-07-12 | テルモ株式会社 | Method for modifying cell culture in adhered state |
| US11395863B2 (en) | 2016-11-01 | 2022-07-26 | Terumo Kabushiki Kaisha | Modification method for sheet-shaped cell culture |
| JP7282232B2 (en) | 2016-11-01 | 2023-05-26 | テルモ株式会社 | Methods for modifying adherent cell cultures |
| WO2022239867A1 (en) * | 2021-05-14 | 2022-11-17 | 株式会社堀場アドバンスドテクノ | Microorganism detection apparatus and microorganism detection method |
| EP4339273A4 (en) * | 2021-05-14 | 2025-06-18 | Horiba Advanced Techno, Co., Ltd. | MICROORGANISM DETECTION APPARATUS AND MICROORGANISM DETECTION METHOD |
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