JP2685119B2 - Cell fractionation method and cell fractionation device - Google Patents
Cell fractionation method and cell fractionation deviceInfo
- Publication number
- JP2685119B2 JP2685119B2 JP6163869A JP16386994A JP2685119B2 JP 2685119 B2 JP2685119 B2 JP 2685119B2 JP 6163869 A JP6163869 A JP 6163869A JP 16386994 A JP16386994 A JP 16386994A JP 2685119 B2 JP2685119 B2 JP 2685119B2
- Authority
- JP
- Japan
- Prior art keywords
- cells
- cell fractionation
- pressure
- cell
- processing chamber
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
- C12M33/14—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus with filters, sieves or membranes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/40—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pressure
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Sustainable Development (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
【０００１】[0001]
【産業上の利用分野】本発明は、微細な個々の細胞を自
動的に分画処理する細胞分画方法及び細胞分画装置に関
する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a cell fractionation method and a cell fractionation apparatus for automatically fractionating minute individual cells.
【０００２】[0002]
【従来の技術】従来、バイオテクノロジー分野におい
て、プロトプラスト培養やプロトプラスト融合等を行う
ために個々のプロトプラストを単離したり、医療分野や
医学分野等において、血球成分を赤血球と白血球及び血
小板の夫々に分離するような場合に、図＊＊に示すよう
に、研究者等が顕微鏡下で確認しながら、マイクロマニ
ュピレーターやマイクロピペット等を操作して個々の細
胞を分離・抽出していた。しかし、かかる手作業による
分画処理は、熟練と多大な労力とを必要とする割には非
効率的であり、多量の細胞を短時間で処理することがで
きない問題があった。又、分画処理中に細胞を破壊して
しまい、有効な細胞を入手することが困難となる問題が
あった。2. Description of the Related Art Conventionally, in the field of biotechnology, individual protoplasts have been isolated for carrying out protoplast culture and fusion of protoplasts, and in the medical field and medical field, blood cell components have been separated into red blood cells, white blood cells and platelets. In such a case, as shown in Fig. **, researchers and the like operated and operated a micromanipulator, a micropipette, etc. while confirming under a microscope to separate and extract individual cells. However, such a manual fractionation process is inefficient in that it requires skill and a great deal of labor, and there is a problem that a large amount of cells cannot be treated in a short time. Further, there is a problem that cells are destroyed during the fractionation process, making it difficult to obtain effective cells.
【０００３】そこで、浮遊液中の細胞を個々に分光測定
して、細胞集団の性質を分析するフローサイトメトリー
法を適用したフローサイトメータ（例えば、日本分光工
業株式会社製「型番；Cyto ACE-150」) や、細胞培地の
表面に付着した細胞を蛍光分析や形態分析して自動分画
するレーザーサイトメータ（例えば、株式会社日科機製
「型番；ACAS 570」）等が開発されるようになった。Therefore, a flow cytometer to which a flow cytometry method for analyzing the properties of a cell population by individually spectroscopically measuring cells in a suspension is applied (for example, "Model number: Cyto ACE- manufactured by JASCO Corporation"). 150 ") and laser cytometers that automatically fractionate cells adhering to the surface of the cell culture medium by fluorescence analysis or morphological analysis (for example," Model number: ACAS 570 "manufactured by Nikkaki Co., Ltd.) became.
【０００４】[0004]
【発明が解決しようとする課題】しかしながら、上記の
フローサイトメータとレーザーサイトメータのようにフ
ローサイトメトリー法を適用した従来装置にあっては、
細胞の蛍光的・形態的な観点での分画処理を行うことが
できるが、細胞膜の硬さや細胞の変形能等の機能的な側
面での分画処理を行うことができなかった。更に、フロ
ーサイトメータは、光学的に細胞を分画するので、その
細胞の位置や向きが変化することによって、その細胞が
焦点面からずれ易く、高精度の分画処理を実現すること
が困難であった。一方、レーザーサイトメータは、大量
の試料細胞を分画するのに適さないという問題があっ
た。However, in the conventional apparatus to which the flow cytometry method is applied, such as the above-mentioned flow cytometer and laser cytometer,
Although it is possible to perform the fractionation treatment from the viewpoint of fluorescence and morphology of the cells, the fractionation treatment cannot be performed from the functional aspects such as the hardness of the cell membrane and the deformability of the cells. Further, since the flow cytometer optically fractionates cells, the cells are easily displaced from the focal plane due to changes in the position and orientation of the cells, making it difficult to realize highly accurate fractionation processing. Met. On the other hand, the laser cytometer has a problem that it is not suitable for fractionating a large amount of sample cells.
【０００５】本発明はこのような課題に鑑みて成された
ものであり、多量の細胞を自動的且つ高精度で分画処理
することのできる細胞分画方法及び細胞分画装置を提供
することを目的とする。The present invention has been made in view of the above problems, and provides a cell fractionation method and a cell fractionation apparatus capable of fractionating a large number of cells automatically and with high accuracy. With the goal.
【０００６】[0006]
【課題を解決するための手段】このような目的を達成す
るために本発明は、分画処理すべき細胞が流入側から流
出側へ通過し得る形状の多数の微細通路を半導体微細加
工技術により半導体基板に形成し、このように形成され
た半導体基板から成るフィルタ部材の前記流入側に、前
記細胞を含む浮遊細胞液を供給し、更に、前記流入側に
対して前記流出側を負圧にして、前記微細通路を介して
流入側から流出側へ通過した細胞を回収するようにし
た。In order to achieve such an object, the present invention uses a semiconductor fine processing technique to form a large number of fine passages having a shape through which cells to be fractionated can pass from an inflow side to an outflow side. A floating cell liquid containing the cells is supplied to the inflow side of the filter member formed on the semiconductor substrate and formed of the semiconductor substrate, and the outflow side is negatively pressured with respect to the inflow side. The cells that passed from the inflow side to the outflow side via the fine passage were collected.
【０００７】又、前記流出側と前記流出側との差圧を正
負交互に変化させる圧力を、前記流出側の処理室へ供給
することにより、フィルタ部材の微細通路の流れ方向を
交互に変化させるようにした。Further, by supplying to the processing chamber on the outflow side a pressure that alternately changes the differential pressure between the outflow side and the outflow side, the flow direction of the fine passages of the filter member is alternately changed. I did it.
【０００８】[0008]
【作用】このような細胞分画方法及び装置によれば、浮
遊細胞液の中の所望の細胞だけがフィルタ部材の前記多
数の微細通路を通過して流出側に出力し、残余の細胞等
の物質が微細通路を通過しないで流入側に残留すること
となり、流出側に出力した細胞を回収することによっ
て、多量の細胞を迅速且つ容易に分画処理することがで
きる。According to such a cell fractionation method and device, only the desired cells in the suspension cell fluid pass through the numerous fine passages of the filter member and are output to the outflow side, and residual cells and the like are discharged. The substance remains on the inflow side without passing through the fine passages, and by collecting the cells output to the outflow side, a large amount of cells can be fractionated rapidly and easily.
【０００９】又、前記流出側と前記流出側との差圧を正
負交互に変化させる圧力を、前記流出側の処理室へ供給
すると、微細通路に付着する細胞を離脱させたり、予め
細胞の付着を防止することができ、円滑な細胞分画を達
成することができる。Further, when a pressure that alternately changes the positive and negative pressure differences between the outflow side and the outflow side is supplied to the processing chamber on the outflow side, cells adhering to the fine passages are detached or cells adhere in advance. Can be prevented and smooth cell fractionation can be achieved.
【００１０】そして、微細通路の形状を、前記分画処理
すべき細胞の硬さ、形状、変形能、活性の少なくとも１
つの特性に基づいて形成することによって、様々な態様
が可能となる。例えば、プロトプラストや血球等の分画
処理に多大な効果を発揮する。The shape of the fine passage is determined by at least one of hardness, shape, deformability and activity of the cells to be fractionated.
By forming based on one characteristic, various modes are possible. For example, it exerts a great effect on the fractionation treatment of protoplasts and blood cells.
【００１１】[0011]
【実施例】以下、本発明による細胞分画装置の一実施例
を図面と共に説明する。まず、図１に基づいて実施例の
装置構成を説明する。本発明の要部エレメントである細
胞分画ユニット１と、細胞浮遊溶液（即ち、分画処理さ
れるべき細胞を生理食塩水などの溶液中に混合させたも
の）を収容するためのマイクロシリンジ２と、細胞を流
動搬送するための生理食塩水等の液を収容するためのタ
ンク３と、分画処理中に不要となった溶液を回収するた
めの洗浄用タンク４と、細胞浮遊液及び流動搬送液の流
速と流れ方向を制御するための負圧タンク５と、夫々の
構成エレメント間を連結する連結管Ｌ1 〜Ｌ3 の一側端
に設けられた第１ないし第４の開閉弁６，７，８，９が
備えられ、負圧タンク５と第２の開閉弁７とを連結する
連結管の途中に、分画処理された細胞を回収するための
回収容器１０を介在させるようになっている。DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS An embodiment of the cell fractionation device according to the present invention will be described below with reference to the drawings. First, the device configuration of the embodiment will be described with reference to FIG. A cell fractionation unit 1, which is an essential element of the present invention, and a microsyringe 2 for accommodating a cell suspension solution (that is, cells to be fractionated are mixed in a solution such as physiological saline). A tank 3 for containing a liquid such as physiological saline for carrying cells in a fluidized manner, a washing tank 4 for collecting a solution that is no longer needed during the fractionation process, a cell suspension and a flow. a negative pressure tank 5 for controlling the flow rate and flow direction of the carrier liquid, the first to fourth on-off valve provided on one end of the connecting pipe L 1 ~L 3 for connecting the respective configuration elements 6 , 7, 8 and 9 are provided, and a collection container 10 for collecting the fractionated cells is interposed in the middle of a connecting pipe connecting the negative pressure tank 5 and the second opening / closing valve 7. Has become.
【００１２】即ち、タンク３と細胞分画ユニット１の第
１の連結口１ａとが連結管Ｌ1 によって連結され、連結
管Ｌ1 の途中に設けられている第１の開閉弁６により流
路を開閉制御するようになっている。又、細胞分画ユニ
ット１の第２の連結口１ｂに連結される連結管Ｌ2 の一
方の分岐管が、第２の開閉弁７を介して回収容器１０に
連結され更に回収容器１０と負圧タンク５を連結し、第
２の開閉弁７によりその流路を開閉制御する。更に、連
結管Ｌ2 の他方の分岐管が、第３の開閉弁８を介して洗
浄用タンク４に連結され、第３の開閉弁８によりその流
路を開閉制御する。更に又、マイクロシリンジ２の上側
連結口と洗浄用タンク４とが、連結管Ｌ3 によって連結
され、連結管Ｌ3 の途中に設けられている第４の開閉弁
９によってその流路を開閉制御するようになっている。
そして、これらの開閉弁６〜９は、電磁弁などが適用さ
れることにより、後述する中央制御ユニット２３中のマ
イクロコンピュータ等によって電気的に開閉制御され
る。Namely, the flow path by the first on-off valve 6 and the first connection hole 1a of the tank 3 and cell fractionation unit 1 is connected by a connecting pipe L 1, is provided in the middle of the connecting pipe L 1 It is designed to control opening and closing. Further, one branch pipe of the connecting pipe L 2 connected to the second connecting port 1b of the cell fractionation unit 1 is connected to the collecting container 10 via the second opening / closing valve 7 and further connected to the collecting container 10. The pressure tank 5 is connected, and the second opening / closing valve 7 controls the opening / closing of the flow path. Further, the other branch pipe of the connecting pipe L 2 is connected to the cleaning tank 4 via the third opening / closing valve 8, and the third opening / closing valve 8 controls opening / closing of the flow path. Furthermore, the upper connecting opening of microsyringe 2 and the cleaning tank 4 are connected by a connecting pipe L 3, opening and closing controls the flow path by the fourth on-off valve 9 provided in the middle of the connecting pipe L 3 It is supposed to do.
The on-off valves 6 to 9 are electrically controlled to be opened and closed by a microcomputer or the like in a central control unit 23 described later by applying an electromagnetic valve or the like.
【００１３】更に、図１において、洗浄用タンク４に
は、当該タンク４内を吸引することによって負圧にする
第１のポンプ４ａと、内部圧力を計測する圧力センサ４
ｂが設けられ、負圧タンク５には、当該タンク５内を吸
引することによって負圧にする第２のポンプ５ａと、内
部圧力を計測する圧力センサ５ｂが設けられている。そ
して、第２，第３，第４の開閉弁７，８，９を個々に開
閉制御することによって、各連結管Ｌ2 ，Ｌ3 内の圧力
を制御するようになっている。Further, in FIG. 1, the cleaning tank 4 has a first pump 4a for sucking the inside of the tank 4 to make it a negative pressure, and a pressure sensor 4 for measuring the internal pressure.
b is provided, and the negative pressure tank 5 is provided with a second pump 5a that sucks the inside of the tank 5 to make it a negative pressure and a pressure sensor 5b that measures an internal pressure. The pressure in each of the connecting pipes L 2 and L 3 is controlled by individually controlling the opening / closing of the second, third and fourth on-off valves 7, 8 and 9.
【００１４】更に、各構成エレメントの構造を詳述す
る。細胞分画ユニット１は、図２の要部縦断面図に示す
ように、下側平板部材１１と、この下側平板部材１１に
対向配置される上側平板部材１２と、下側平板部材１１
と上側平板部材１２の内側面に挟着されるフィルタ部材
１３と、これらの下側平板部材１１と上側平板部材１２
の側端周囲を完全に密着・封止する側端部材１４で構成
されている。尚、この実施例では、各部材１１〜１３
は、周知の嵌着部品などを用いて図示の如く一体的に組
合わされた状態で細胞の分画処理に使用されるが、分画
処理の前後において上記の嵌着部品を取り外すことによ
り、個々に分解することができるようになっている。こ
のように、かかる細胞分画ユニット１は分割可能となっ
ている。但し、使用の態様に応じて、この細胞分画ユニ
ット１は、予め接着材等によってこれらの部材１１〜１
３を一体的に固着して成る分解不可の構造にしてもよ
い。Further, the structure of each constituent element will be described in detail. The cell fractionation unit 1 includes a lower flat plate member 11, an upper flat plate member 12 arranged to face the lower flat plate member 11, and a lower flat plate member 11 as shown in a longitudinal sectional view of a main part of FIG.
And a filter member 13 sandwiched between the inner side surfaces of the upper flat plate member 12, the lower flat plate member 11 and the upper flat plate member 12
The side end member 14 completely adheres and seals the periphery of the side end. In this embodiment, each member 11-13
Is used in the cell fractionation process in a state in which it is integrally combined as shown using well-known fitting parts, but by removing the fitting parts before and after the fractionation process, It can be disassembled into. In this way, the cell fractionation unit 1 can be divided. However, depending on the mode of use, the cell fractionation unit 1 may have these members 11 to 1 preliminarily attached with an adhesive or the like.
A non-decomposable structure formed by integrally fixing 3 may be used.
【００１５】下側平板部材１１は、透明ガラスや透明プ
ラスチックなどの透光性を有する硬質材料によって成型
され、フィルタ部材１３に接触する内側面ＰL が極めて
滑らかに平面研磨されている。The lower flat plate member 11 is molded from a hard material having a light-transmitting property such as transparent glass or transparent plastic, and the inner side surface P L which contacts the filter member 13 is extremely smoothly flat-polished.
【００１６】上側平板部材１２は、下側平板部材１１と
の対向形状が同一（例えば、円形や矩形）成型されると
共に、ガラスやプラスチックなどの硬質材料によって成
型され、フィルタ部材１３に接触する内側面ＰU が極め
て滑らかに平面研磨されている。そして、これら上側平
板部材１２と下側平板部材１１との側壁が筒状（例え
ば、これらの部材１１，１２に合致する円筒状や矩形筒
状）の側端部材１４内に嵌め合わされて密封される。更
に、上側平板部材１２の上部側端には、連結管Ｌ1 の先
端を密着して連結するための上記連結口１ａと、連結管
Ｌ2 の先端を密着して連結するための上記連結口１ｂ
と、マイクロシリンジ２の下端口２ａを密着して連結す
るための連結口１ｃが一体に成型されている。ここで、
これらの連結口１ａ，１ｂ，１ｃと連結管Ｌ1 ，Ｌ2 及
びマイクロシリンジ２の下端口２ａは着脱可能となって
おり、操作者が、分画処理に際してこれらを手作業にて
連結するようになっている。したがって、分画処理対象
である細胞の種類に応じて、種類の異なる細胞分画ユニ
ット１を選択的に使用することができるようになってい
る。The upper flat plate member 12 is molded in the same shape as the lower flat plate member 11 (for example, a circular shape or a rectangular shape), and is molded of a hard material such as glass or plastic so as to come into contact with the filter member 13. The side surface P U is extremely smoothly flat-polished. Then, the side walls of the upper flat plate member 12 and the lower flat plate member 11 are fitted and sealed in a tubular (for example, a cylindrical or rectangular tubular shape matching the members 11 and 12) side end member 14. It Further, on the upper end of the upper flat plate member 12, the connecting port for connecting in close contact with the connection hole 1a for connecting in close contact with the tip of the connecting pipe L 1, the front end of the connecting pipe L 2 1b
And a connection port 1c for closely connecting and connecting the lower end port 2a of the microsyringe 2 is integrally molded. here,
The connection ports 1a, 1b, 1c and the connection pipes L 1 , L 2 and the lower end port 2a of the microsyringe 2 are attachable / detachable, so that the operator can manually connect them during the fractionation process. It has become. Therefore, the cell fractionation units 1 of different types can be selectively used according to the type of cells to be fractionated.
【００１７】フィルタ部材１３は、半導体デバスを製造
するために用いられる所謂シリコンウエハなどのシリコ
ン平板で形成され且つ、下側平板部材１１と上側平板部
材１２及び側端部材１４によって画成されて成る処理室
１５内の略中央に収容可能な大きさに形成されている。
更にフィルタ部材１３の構造を図３〜図５に基づいて詳
述する。図３は下側平板部材１１の内側面に密着される
下側端の形状を、図４は上側平板部材１２の内側面に密
着される上側端の形状を示す。フィルタ部材１３の略中
央部分の上側端から下側端に貫通して、試料細胞の直径
に較べて極めて大きな内径（数ｍｍ〜数ｃｍ）の貫通孔
１６が穿設され、更に図３に示すように、下側端には、
この貫通孔１６の周囲を囲む一定高さ且つ厚さの塀状の
突条部１７が形成され、この突条部１７によって仕切ら
れた内側（浮遊細胞液を流入させる側となる）に、貫通
孔１６と連通する空間を設けるための凹段部（浮遊細胞
液を流入させるための処理室の一部となる）１８が形成
されている。更に図４に示すように、上側端には、貫通
孔１６を中心として、比較的浅い凹段部１９が形成され
ている。更に図５に示すように、突条部１７の突端部１
７ａには、数μｍないし数百μｍの範囲のいずれかの所
定幅Ｗ及び深さの凹欠溝１７ｂが形成されている。即
ち、夫々の凹欠溝１７ｂが突条部１７の厚さ方向に向け
て（凹段部１８から外側に向けて）切欠き形成され、且
つ突端部１７ａの長手方向に一定ピッチ間隔ＰＨ（但
し、Ｗ＜ＰＨ）で設けられている。尚、突端部１７ａの
突端面は極めて平坦に研磨されている。The filter member 13 is formed of a silicon flat plate such as a so-called silicon wafer used for manufacturing a semiconductor device, and is defined by a lower flat plate member 11, an upper flat plate member 12 and a side end member 14. The processing chamber 15 is formed to have a size that can be accommodated substantially in the center thereof.
Further, the structure of the filter member 13 will be described in detail with reference to FIGS. 3 shows the shape of the lower end that is in close contact with the inner surface of the lower flat plate member 11, and FIG. 4 shows the shape of the upper end that is in close contact with the inner surface of the upper flat plate member 12. A through hole 16 having an inner diameter (several mm to several cm) extremely larger than the diameter of the sample cell is formed so as to penetrate from the upper end to the lower end of the substantially central portion of the filter member 13, and further shown in FIG. So that the lower end is
A fence-shaped ridge 17 having a constant height and a thickness is formed surrounding the through hole 16 and penetrates inside (partially inflowing the floating cell liquid) partitioned by the ridge 17. A concave step portion (which becomes a part of the processing chamber for inflowing the floating cell liquid) 18 for forming a space communicating with the hole 16 is formed. Further, as shown in FIG. 4, a relatively shallow concave step portion 19 is formed at the upper end with the through hole 16 as the center. Further, as shown in FIG.
A concave groove 17b having a predetermined width W and a depth in the range of several μm to several hundreds of μm is formed in 7a. That is, each recessed groove 17b is formed in a notch toward the thickness direction of the protruding portion 17 (outward from the recessed step portion 18), and a fixed pitch PH (provided that the protruding end portion 17a is in the longitudinal direction). , W <PH). The tip surface of the tip 17a is ground to be extremely flat.
【００１８】よって、貫通孔１６と上側平板部材１２の
連結口１ｃとを相互に対向するようにして、フィルタ部
材１３が上側平板部材１２と下側平板部材１１との間に
挟着されることにより、多数の凹欠溝１７ｂと下側平板
部材１１の内側面とで画成されて成る多数の微細流路が
構成され、後述するように、供給手段となるマイクロシ
リンジ２内の細胞浮遊液を貫通孔１６を通して流入させ
ると、大きさや硬さや変形能等の所定条件を満足する細
胞だけがこれらの微細流路を通って、流出側の処理室１
５へ流出するようになっている。Therefore, the filter member 13 is sandwiched between the upper flat plate member 12 and the lower flat plate member 11 so that the through hole 16 and the connecting port 1c of the upper flat plate member 12 face each other. As a result, a large number of fine flow paths defined by the large number of recessed grooves 17b and the inner surface of the lower flat plate member 11 are formed, and as described later, the cell suspension in the microsyringe 2 serving as a supply means. Flow through the through holes 16, only cells satisfying predetermined conditions such as size, hardness, deformability, etc. pass through these fine flow paths, and the processing chamber 1 on the outflow side 1
It is supposed to flow out to 5.
【００１９】尚、凹欠溝１７ｂの形状（即ち、幅Ｗと溝
の深さと形）及びピッチ間隔ＰＨは、分画処理すべき試
料細胞の直径やその変形能や硬さ等の形態性、及び上記
の微細流路を通過するに際して変形しても損傷や死滅し
ない許容範囲（活性）等を考慮して様々に設計され、所
謂半導体製造技術におけるエッチング技術を適用するこ
とによって、このような多数の微細な凹欠溝１７ｂを高
精度で一定形状に形成している。又、フィルタ部材１３
の下側端と下側平板部材１１の内側面ＰL との接触面、
及びフィルタ部材１３の上側端と上側平板部材１２の内
側面ＰU との接触面の夫々にシリコングリスなどを介在
させることにより、分画処理されるべき細胞が貫通孔１
６と凹欠溝１７ｂのみを通過するように気密性を向上さ
せることが望ましい。The shape of the recessed groove 17b (that is, the width W and the depth and shape of the groove) and the pitch interval PH are the morphological characteristics such as the diameter of the sample cell to be fractionated, its deformability and hardness. In addition, various designs are made in consideration of an allowable range (activity) that does not cause damage or death even when deformed when passing through the above-described fine flow path, and by applying etching technology in so-called semiconductor manufacturing technology, The fine recessed groove 17b is formed in a constant shape with high accuracy. In addition, the filter member 13
A contact surface between the lower end of the lower flat plate member 11 and the inner side surface P L of the lower flat plate member 11,
By interposing silicon grease or the like on each of the contact surfaces between the upper end of the filter member 13 and the inner side surface P U of the upper flat plate member 12, the cells to be fractionated are processed through the through hole 1
It is desirable to improve the airtightness so that it passes only 6 and the recessed groove 17b.
【００２０】又、夫々の凹欠溝１７ｂの側壁は、凹段部
１８側（細胞を流入させる側）が広く、処理室１５側
（細胞を流入させる側）が狭くなるようなテーパー状の
平面に形成してもよいし、更に又、全体としてテーパー
状であるがその側壁を若干突状若しくは凹状に湾曲した
テーパー面にしてもよい。このように側壁をテーパー状
にすると、試料細胞の損傷等を低減することができる。
更に、凹段部１９を形成すること無く、貫通孔１６と連
結口１ｃとを略直接的に連通させるようにしてもよい。
又、突条部１７は、凹段部１８側と処理部１５側を上述
のように略隔絶するものであるので、この実施例に示す
ように、矩形状の形に限定されるものではなく、例えば
環状その他の形状にしてもよい。更に又、この実施例で
は、突条部１７を１個だけ形成したフィルタ部材１２を
示すがこれに限定されず、貫通孔１６を中心にして、径
の異なる複数の突条部を、分画すべき細胞が流出する方
向に同心円状に形成したフィルタ部材であってもよい。
このような複数の突条部を設けると、夫々の突条部の凹
欠溝を様々に設計することによって、分画処理の多様性
と分画精度の向上などを図ることができる。The side wall of each recessed groove 17b is a tapered flat surface such that the recessed step portion 18 side (the cell inflow side) is wide and the processing chamber 15 side (the cell inflow side) is narrow. Alternatively, the side wall may be a tapered surface that is curved so as to be slightly projecting or concave, although it has a tapered shape as a whole. When the side wall is tapered in this way, damage to the sample cells and the like can be reduced.
Further, the through hole 16 and the connecting port 1c may be made to communicate with each other substantially directly without forming the concave step portion 19.
Further, since the ridge portion 17 substantially separates the concave step portion 18 side and the processing portion 15 side as described above, it is not limited to the rectangular shape as shown in this embodiment. For example, it may have a ring shape or another shape. Furthermore, in this embodiment, the filter member 12 in which only one ridge portion 17 is formed is shown, but the present invention is not limited to this, and a plurality of ridge portions having different diameters are divided around the through hole 16. It may be a filter member formed concentrically in the direction in which the power cells flow out.
By providing such a plurality of ridges, it is possible to improve the variety of fractionation processing and the accuracy of fractionation by designing the recessed grooves of the respective ridges in various ways.
【００２１】再び図１及び図２に基づいて装置構成を説
明すると、細胞分画ユニット１は固定ステージ（図示せ
ず）に固定され、下側平板部材１１の外側から処理室１
５内を拡大視する顕微鏡２０と、その顕微鏡２０の拡大
映像を撮影するビデオカメラ２１と、ビデオカメラ２１
からの出力映像信号を画像処理することによってモニタ
２２に映像を再生させる中央制御ユニット２３と、連結
管Ｌ2 の一側に連結され中央制御ユニット２３の制御に
よって細胞分画ユニット１内の圧力を調節する差圧調整
ユニット２４が設けられている。Referring again to FIG. 1 and FIG. 2, the apparatus configuration will be described. The cell fractionation unit 1 is fixed to a fixed stage (not shown), and the treatment chamber 1 is placed from the outside of the lower flat plate member 11.
5, a microscope 20 for magnifying the inside of the camera 5, a video camera 21 for photographing an enlarged image of the microscope 20, and a video camera 21.
A central control unit 23 for reproducing an image on the monitor 22 by image-processing an output video signal from the control unit 23 and a pressure in the cell fractionation unit 1 under the control of the central control unit 23 connected to one side of the connecting pipe L 2. A differential pressure adjusting unit 24 for adjusting is provided.
【００２２】ここで、顕微鏡２０とビデオカメラ２１
は、下側平板部材１１に対する対向間隔と対向位置を自
在に調整するＸＹＺ可動ステージ（図示せず）に取り付
けられている。差圧調整ユニット２４は、図６に示すよ
うに、連結管Ｌ2 の管内圧力を検出する圧力センサ２４
ａと、連結管Ｌ2 の一側に設けられた流路切換え弁２４
ｂを介して連結管Ｌ2 に連設されたシリンダ２４ｃと、
シリンダ２４ｃのピストン２４ｄの進退移動量をパルス
モータ２４ｅの正逆転角によって制御するアクチュエー
タ２４ｆと、パルスモータ２４ｅへの供給電力の極性を
図７の波形図に示す如く変化させることによってその正
転と逆転を制御するファンクションジェネレータ２４ｇ
と、圧力センサ２４ａの計測値の変化（即ち、連結管Ｌ
2 内の圧力変化）を調節するためにファンクションジェ
ネレータ２４ｇからパルスモータ２４ｅへの供給電力の
極性を自動調整する圧力制御回路２４ｈを備えている。
尚、アクチュエータ２４ｆは、パルスモータ２４ｅの駆
動軸に連設されたスクリューと、一端がピストン２４ｄ
に固着され且つ上記スクリューに螺合するネジ部とを有
し、上記スクリューの正逆回転に応じてピストン２４ｄ
を進退移動させるのに伴ってシリンダ２４ｃ内の圧力を
制御することにより、連結管Ｌ2 内の圧力を加減制御す
る。そして、この差圧調整ユニット２４は、分画処理の
際に、細胞分画ユニット１内のフィルタ部材１３の凹欠
溝１７ｂに細胞が目詰まりするのを防止するため等に使
用される。Here, the microscope 20 and the video camera 21
Is attached to an XYZ movable stage (not shown) that freely adjusts the facing interval and the facing position with respect to the lower flat plate member 11. As shown in FIG. 6, the differential pressure adjusting unit 24 includes a pressure sensor 24 that detects the internal pressure of the connecting pipe L 2.
a and a flow path switching valve 24 provided on one side of the connecting pipe L 2.
a cylinder 24c connected to the connecting pipe L 2 via b,
An actuator 24f that controls the forward / backward movement amount of the piston 24d of the cylinder 24c by the forward / reverse rotation angle of the pulse motor 24e and the normal rotation by changing the polarity of the electric power supplied to the pulse motor 24e as shown in the waveform diagram of FIG. Function generator 24g for controlling reverse rotation
And the change in the measured value of the pressure sensor 24a (that is, the connecting pipe L
A pressure control circuit 24h is provided for automatically adjusting the polarity of the electric power supplied from the function generator 24g to the pulse motor 24e in order to adjust the pressure change (inside 2 ).
The actuator 24f has a screw connected to the drive shaft of the pulse motor 24e and a piston 24d at one end.
And a screw portion that is fixed to the screw and that is screwed into the screw.
By controlling the pressure in the cylinder 24c and along with the advancing and retracting, to moderate control the pressure in the connecting pipe L 2. The differential pressure adjusting unit 24 is used for preventing cells from being clogged in the recessed groove 17b of the filter member 13 in the cell fractionation unit 1 during the fractionation process.
【００２３】そして、かかる構成エレメントと複数の連
結管を有する細胞分画装置は、図８に示すような筐体２
５内に収容されて装置化されている。尚、筐体２５に
は、中央制御ユニット２３内のマイクロプロセッサ等に
対して種々の動作を指令するための操作用キーボード等
が設けられている。A cell fractionation device having such a constituent element and a plurality of connecting pipes has a casing 2 as shown in FIG.
It is housed in 5 and is made into a device. The housing 25 is provided with an operation keyboard for instructing various operations to the microprocessor and the like in the central control unit 23.
【００２４】次に、かかる構成の細胞分画装置の動作を
説明する。まず、装置の電源をオンにすると、中央制御
ユニット２３とビデオカメラ２１が起動して、顕微鏡２
０で拡大された細胞分画ユニット１内の映像をモニタ２
２に再生表示させる。そして、操作者が筐体２５に設け
られている操作キーボードを操作して前記ＸＹＺステー
ジの位置を適宜に移動させることによって、撮影対象と
その拡大率及び焦点位置を最適状態に調節することがで
き、鮮明な映像をモニタ２２に表示させることができ
る。更に、操作者は、予めタンク３に生理食塩水を注入
しておくと共に、回収容器１０を第２の開閉弁７と負圧
タンク５の間に連結しておき、更に、細胞分画ユニット
１の連結口１ａ，１ｂ，１ｃに、第１，第２の連結管Ｌ
1 ，Ｌ2 とマイクロシリンジ２を連結しておく。又、第
３の連結管Ｌ3 も予めマイクロシリンジ２の試料注入口
に連結しておく。Next, the operation of the cell fractionation device having such a configuration will be described. First, when the power of the device is turned on, the central control unit 23 and the video camera 21 are activated, and the microscope 2
Monitor the image in the cell fractionation unit 1 enlarged by 0
2 Playback display. Then, the operator can operate the operation keyboard provided on the housing 25 to appropriately move the position of the XYZ stage to adjust the object to be photographed, its magnification, and the focus position to the optimum state. Therefore, a clear image can be displayed on the monitor 22. Furthermore, the operator has previously injected physiological saline into the tank 3 and connected the collection container 10 between the second opening / closing valve 7 and the negative pressure tank 5, and further, the cell fractionation unit 1 To the connection ports 1a, 1b, 1c of the first and second connection pipes L
1 , L 2 and the microsyringe 2 are connected in advance. The third connecting pipe L 3 is also connected to the sample injection port of the microsyringe 2 in advance.
【００２５】次に、操作者が筐体２５の操作キーボード
により分画処理の開始を指令すると、中央制御ユニット
２３の制御により以下の動作が行われる。まず、第１〜
第４の開閉弁６，７，８，９の全てが閉鎖状態となる。
更に、流路切換え弁２４ｂは回収容器１０と連結管Ｌ2
の間を連通状態にして、差圧調整ユニット２４との接続
を遮断状態にする。Next, when the operator gives an instruction to start the fractionation process using the operation keyboard of the casing 25, the following operation is performed under the control of the central control unit 23. First of all,
All the fourth open / close valves 6, 7, 8, 9 are closed.
Further, the flow path switching valve 24b is connected to the recovery container 10 and the connecting pipe L 2
The two are connected to each other to disconnect the connection with the differential pressure adjusting unit 24.
【００２６】次に、第１，第２のポンプ４ａ，５ａを駆
動して洗浄用タンク４と負圧タンク５内を所定の負圧状
態に設定すると共に、分画処理中は、制御ユニット２３
が、これらのタンク４，５内の圧力を常に所定の負圧値
に保持するように、第１，第２のポンプ４ａ，５ａをオ
ンオフ制御する。Next, the first and second pumps 4a, 5a are driven to set the cleaning tank 4 and the negative pressure tank 5 to a predetermined negative pressure state, and the control unit 23 is operated during the fractionation process.
However, the first and second pumps 4a and 5a are on / off controlled so that the pressures in the tanks 4 and 5 are always maintained at a predetermined negative pressure value.
【００２７】次に、第１の開閉弁６と第３の開閉弁８を
開放状態にし、洗浄用タンク４の負圧吸引力によって、
タンク３内の生理食塩水を、細胞分画ユニット１の処理
室１５及びこれに連設している連結管Ｌ1 ，Ｌ2 内に流
入させ且つ充満させる。そして、処理室１５に生理食塩
水が充満した状態で第３の開閉弁８は閉じられる。この
際に余分に吸引した生理食塩水は洗浄用タンク４内に蓄
積される。Next, the first opening / closing valve 6 and the third opening / closing valve 8 are opened, and by the negative pressure suction force of the cleaning tank 4,
The physiological saline in the tank 3 is caused to flow into and fill the processing chamber 15 of the cell fractionation unit 1 and the connecting pipes L 1 and L 2 connected to the processing chamber 15. Then, the third opening / closing valve 8 is closed while the processing chamber 15 is filled with the physiological saline. At this time, the extra saline aspirated is accumulated in the cleaning tank 4.
【００２８】次に、第４の開閉弁９を一定時間だけ開放
にして、洗浄用タンク４の負圧吸引力によって、タンク
３の生理食塩水を細胞分画ユニット１の処理室１５を介
してマイクロシリンジ２内に一定量注入させる。この際
にも、余分に吸引した生理食塩水は洗浄用タンク４内に
蓄積される。Next, the fourth on-off valve 9 is opened for a certain period of time, and the physiological saline in the tank 3 is passed through the processing chamber 15 of the cell fractionation unit 1 by the negative suction force of the cleaning tank 4. A certain amount is injected into the microsyringe 2. Also at this time, the extra saline solution is accumulated in the cleaning tank 4.
【００２９】このように、生理食塩水をマイクロシリン
ジ２と細胞分画ユニット１の処理室１５内及び連結管Ｌ
1 ，Ｌ2 内に充満させた後、第１の開閉弁６が閉じられ
ることによって、全ての開閉弁６，７，８，９が一旦閉
じられる。As described above, the physiological saline is supplied to the microsyringe 2, the processing chamber 15 of the cell fractionation unit 1 and the connecting pipe L.
After filling 1 and L 2 , the first on-off valve 6 is closed, so that all the on-off valves 6, 7, 8, 9 are once closed.
【００３０】次に、操作者が、マイクロシリンジ２の上
部に設けられている試料注入口から細胞浮遊液を注入す
ることによって生理食塩水と混合させる。尚、マイクロ
シリンジ２中に気泡が入らないように操作することが望
ましい。Next, the operator mixes the cell suspension with physiological saline by injecting the cell suspension through the sample injection port provided on the upper portion of the microsyringe 2. In addition, it is desirable to operate so that air bubbles do not enter the microsyringe 2.
【００３１】そして、操作者が試料注入の完了を操作キ
ーボードで指令すると、再び、第２の開閉弁７が開放状
態となる。したがって、差圧発生手段となる負圧タンク
５の負圧吸引力によって、マイクロシリンジ２から分画
処理ユニット１ないし第２の連結管Ｌ2 及び回収容器１
０による気密流路内に差圧が掛かり（負圧タンク５側ほ
ど負圧となる）、マイクロシリンジ２内の細胞浮遊溶液
が生理食塩水と共にフィルタ部材１３の貫通孔１６に流
入し、更に、凹欠溝１７ｂを通過した細胞が第２の連結
管Ｌ2 を介して回収容器１０へ到達して回収・蓄積され
る。When the operator commands the completion of the sample injection using the operation keyboard, the second on-off valve 7 is opened again. Therefore, the negative pressure suction force of the negative pressure tank 5 serving as the differential pressure generating means causes the microsyringe 2 to flow from the microsyringe 2 to the fractionation processing unit 1 or the second connecting pipe L 2 and the collection container 1.
A differential pressure is applied to the airtight flow path due to 0 (the negative pressure becomes closer to the negative pressure tank 5 side), the cell suspension solution in the microsyringe 2 flows into the through hole 16 of the filter member 13 together with the physiological saline, and The cells that have passed through the recessed groove 17b reach the collection container 10 via the second connecting pipe L 2 and are collected and accumulated.
【００３２】ここで、フィルタ部材１３の凹欠溝１７ｂ
の機能を図９及び図１０と共に説明する。例えば、この
実施例の細胞分画装置をプロトプラストの単離に使用す
る場合を説明すると、そのプロトプラストの直径と、硬
さと、変形能などの機能性と、活性及び、分画処理時に
設定される負圧条件を考慮した形状の凹欠溝１７ｂが形
成された突条部２２を有するフィルタ部材１３が使用さ
れる。そして、上述した分画処理を行うと、図９に示す
ように、所定直径以上の異種細胞や細胞壁の除去されて
いない硬質細胞ＰＲ１は、凹欠溝１７ｂを通過すること
ができないので、突条部１７の内側に滞留しまい、正規
の直径及び変形能を有する細胞ＰＲ２のみが凹欠溝１７
ｂを通過して、回収容器１０に回収される。Here, the recessed groove 17b of the filter member 13 is formed.
The function of will be described with reference to FIGS. 9 and 10. For example, the case of using the cell fractionation device of this Example for isolation of protoplasts will be described. The diameter, hardness, functionality such as deformability, activity of protoplasts, and activity and fractionation processing are set. A filter member 13 having a ridge portion 22 in which a concave groove 17b having a shape considering the negative pressure condition is formed is used. Then, when the above-described fractionation process is performed, as shown in FIG. 9, the heterogeneous cells having a predetermined diameter or more and the hard cells PR1 from which the cell wall is not removed cannot pass through the recessed groove 17b, so that the protrusions are formed. Only the cells PR2, which have the regular diameter and deformability, are retained inside the portion 17 and have the concave groove 17
It is recovered in the recovery container 10 after passing through b.
【００３３】又、この実施例の細胞分画装置を、血球成
分から赤血球と白血球と血小板に分離するのに使用する
ような場合には、まず血球成分をマイクロシリンジ２に
注入してから上述の分画処理を行う。第１回目の分画処
理においては、赤血球と血小板は通過させるが白血球は
通過させない大きさの凹欠溝１７ｂを有するフィルタ部
材１３を使用する。この結果、回収容器１０には赤血球
ＰＲ３と血小板ＰＲ４を有する浮遊細胞液が抽出され、
細胞分画ユニット１のフィルタ部材１３の内側に白血球
ＰＲ５が残存することとなる。そして、この第１回目に
使用した細胞分画ユニット１中のフィルタ部材１３を別
種類のフィルタ部材１３’に取替えて次の第２回目の分
画処理を行う。この第２回目の処理では、血小板は通過
させるが赤血球は通過させない形状の凹欠溝１７ｂ’が
形成されている突条部１７’を有するフィルタ部材を使
用し、第１回目の処理で抽出した赤血球ＰＲ３と血小板
ＰＲ４を有する浮遊細胞液を再びマイクロシリンジ２に
注入して分画処理を行う。この結果、赤血球ＰＲ４は凹
欠溝１７ｂ’を通過せず、血小板ＰＲ３のみが回収容器
１０に回収される。When the cell fractionation apparatus of this embodiment is used to separate red blood cells, white blood cells, and platelets from blood cell components, the blood cell components are first injected into the microsyringe 2 and then the above-mentioned procedure is performed. Perform fractionation processing. In the first fractionation process, the filter member 13 having the recessed groove 17b of a size that allows red blood cells and platelets to pass but does not allow white blood cells to pass is used. As a result, the floating cell liquid containing red blood cells PR3 and platelets PR4 is extracted in the collection container 10,
The white blood cells PR5 remain inside the filter member 13 of the cell fractionation unit 1. Then, the filter member 13 in the cell fractionation unit 1 used for the first time is replaced with another type of filter member 13 ', and the second fractionation process is performed next. In this second treatment, a filter member having a ridge portion 17 'in which a concave groove 17b' having a shape that allows passage of platelets but not passage of red blood cells is formed is used and extracted in the first treatment. A suspension cell solution containing red blood cells PR3 and platelets PR4 is injected again into the microsyringe 2 to perform a fractionation process. As a result, the red blood cells PR4 do not pass through the recessed groove 17b ', and only the platelets PR3 are collected in the collection container 10.
【００３４】このように、所定形状の凹欠溝１７が形成
されたフィルタ部材１３に浮遊細胞液を流すことによっ
て、大きさ、硬さ、変形能等の機能、活性の差に基づい
て、所望の細胞を分画処理することができる。又、この
実施例の細胞分画装置では、負圧タンク５の負圧によっ
て、浮遊細胞液をフィルタ部材１３に自動的に流すの
で、大量の細胞を高速に分画処理することができる。As described above, by allowing the floating cell liquid to flow through the filter member 13 in which the recessed groove 17 having a predetermined shape is formed, it is possible to obtain a desired value based on the difference in functions such as size, hardness and deformability, and activity. Can be fractionated. Further, in the cell fractionation apparatus of this embodiment, the negative pressure of the negative pressure tank 5 automatically causes the floating cell liquid to flow through the filter member 13, so that a large amount of cells can be fractionated at high speed.
【００３５】更に、予め標準となる形状の凹欠溝１７が
形成されたフィルタ部材１３を細胞分画ユニット１に適
用し、マイクロシリンジ２に注入した浮遊細胞液につい
て上記同様の分画処理を行うと、負圧タンク５に設定さ
れている負圧は常時一定であるので、凹欠溝１７の内側
（貫通孔１６側）と外側（処理室１５側）との差圧が、
その浮遊細胞液内の細胞固有の大きさ、硬さ、変形能等
の機能、活性に応じた圧力となり、更に、マイクロシリ
ンジ２中の浮遊細胞液の減少速度がその差圧と相関を有
することとなる。よって、マイクロシリンジ２中の浮遊
細胞液の減少速度を計測することによって、分画処理に
より抽出した細胞の大きさ、硬さ、変形能等の機能、活
性の特性を容易に知ることができるという効果もある。
更に、凹欠溝１７を通過する細胞の移動状態をモニタ２
２に再生表示されている映像によって観察することによ
っても、細胞の大きさ、硬さ、変形能等の機能、活性の
特性を容易に知ることができる。Further, the filter member 13 in which the standard concave groove 17 is formed is applied to the cell fractionation unit 1, and the suspension cell liquid injected into the microsyringe 2 is subjected to the fractionation treatment similar to the above. Since the negative pressure set in the negative pressure tank 5 is always constant, the differential pressure between the inside (through hole 16 side) and the outside (processing chamber 15 side) of the recessed groove 17 is
The pressure depends on the size and hardness of the cells in the floating cell fluid, functions such as deformability, and activity, and the rate of decrease of the floating cell fluid in the microsyringe 2 has a correlation with the pressure difference. Becomes Therefore, by measuring the reduction rate of the floating cell fluid in the microsyringe 2, it is possible to easily know the function, such as the size, hardness, deformability, and activity of the cells extracted by the fractionation process. There is also an effect.
Furthermore, the movement state of cells passing through the recessed groove 17 is monitored 2
By observing with the image reproduced and displayed in 2, it is possible to easily know the characteristics of the function and activity such as the size, hardness and deformability of cells.
【００３６】次に、差圧調整ユニット２４の動作を説明
する。このユニット２４は、操作者が必要に応じて選択
的に起動させることができるようになっており、例え
ば、粘性の大きな細胞を分画処理する場合に、この細胞
がフィルタ部材１３の凹欠溝１７に目詰まりするのを防
止するために動作させたり、目詰まりした凹欠溝１７か
ら細胞を離脱させて開放状態にすることにより、所望の
細胞を通過させ易くするため等に動作させると効果的で
ある。Next, the operation of the differential pressure adjusting unit 24 will be described. The unit 24 can be selectively activated by the operator as needed, and for example, when the viscous cells are fractionated, the cells are notched in the groove of the filter member 13. It is effective to operate to prevent clogging of 17 or to release desired cells from the clogged groove 17 to make them open so that desired cells can easily pass through. Target.
【００３７】即ち、或る細胞を含む浮遊細胞溶液につい
て分画処理する際に、操作者が前記操作キーボードによ
り動作開始を指令すると、中央制御ユニット２３の指示
により、流路切換え弁２４ｂが切替わって連結管Ｌ2 に
シリンダ２４ｃを連通させ、更に、図７に示すような、
時間幅τF の正極性と時間幅τR の負極性が周期的に変
化する電力がファンクションジェネレータ２４ｇからパ
ルスモータ２４ｅへ供給され、その電力の極性反転に従
ってパルスモータ２４ｅが正逆回転するのに伴ってピス
トン２４ｄが進退移動することにより、シリンダ２４ｃ
内に充満している流動搬送液が連結管Ｌ2 内へ往復移動
する。したがって、シリンダ２４ｃ内の流動搬送液の移
動に伴って、フィルタ部材１３の突条部１７の内側と外
側で正負の差圧が交互に発生して、突条部１７の凹欠溝
１７ｂに付着している細胞を離脱させたり、付着を防止
することができる。又、上記の時間幅τF とτR の比率
を変化させたり、τF ＋τR の周期を変化させたり、更
に正極性と負極性の電力値を変化させて、ピストン２４
ｄのストローク及び移動速度を変化させることにより、
フィルタ部材１３の突条部１７の内側と外側で正負の差
圧を様々に変化させることができるので、凹欠溝１７ｂ
に付着している細胞を離脱させたり、その付着防止を確
実に達成することができる。更に又、操作者が操作用キ
ーボードにより中央制御ユニット２３に対してこのよう
な様々な差圧発生条件を指定すると、中央制御ユニット
２３が圧力制御回路２４ｅにかかる差圧発生条件を指令
し、圧力制御回路２４ｅが圧力センサ２４ａの計測値に
基づいてファンクションジェネレータ２４ｇに差圧発生
条件を満足する電力を出力させる帰還制御を行うので、
上記の細胞離脱や付着防止を自動的に行うことができ
る。That is, when the floating cell solution containing a certain cell is fractionated, the operator commands the start of operation by the operation keyboard, and the flow path switching valve 24b is switched by the instruction of the central control unit 23. To connect the cylinder 24c to the connecting pipe L 2 , and further, as shown in FIG.
Electric power in which the positive polarity of the time width τ F and the negative polarity of the time width τ R change periodically is supplied from the function generator 24g to the pulse motor 24e, and the pulse motor 24e rotates forward and backward in accordance with the polarity reversal of the electric power. As the piston 24d moves back and forth accordingly, the cylinder 24c
The fluid carrier liquid filling the inside reciprocates into the connecting pipe L 2 . Therefore, with the movement of the flowing carrier liquid in the cylinder 24c, positive and negative differential pressures are alternately generated inside and outside the protruding portion 17 of the filter member 13 and adhere to the recessed groove 17b of the protruding portion 17. The detached cells can be detached and adhesion can be prevented. In addition, the ratio of the time widths τ F and τ R is changed, the period of τ F + τ R is changed, and the power values of the positive polarity and the negative polarity are further changed to change the piston 24.
By changing the stroke and movement speed of d,
Since the positive and negative differential pressures can be variously changed between the inside and the outside of the protruding portion 17 of the filter member 13, the recessed groove 17b can be formed.
The cells adhering to the cells can be detached, or their attachment can be prevented without fail. Furthermore, when the operator designates such various differential pressure generation conditions to the central control unit 23 by the operation keyboard, the central control unit 23 commands the pressure control circuit 24e to generate the differential pressure generation condition, and Since the control circuit 24e performs feedback control for causing the function generator 24g to output electric power that satisfies the differential pressure generation condition based on the measurement value of the pressure sensor 24a,
The above-mentioned cell detachment and adhesion prevention can be automatically performed.
【００３８】尚、この実施例では、差圧調整ユニット２
４を、細胞分画ユニット１の下流側に位置する連結管Ｌ
2 に設けたが、他の変形例として、細胞分画ユニット１
の上流側に位置する連結管Ｌ1 の一端部（例えば、第１
の開閉弁６と連結口１ａの間）に設けてもよい。更に
又、図６に示すように細胞分画ユニット１の下流側に位
置する連結管Ｌ2 の一端部と細胞分画ユニット１の上流
側に位置する連結管Ｌ1の一端部とに２個の差圧調整ユ
ニット２４を設け、夫々の差圧調整ユニット２４のピス
トン２４ｄの進退移動を逆位相で行わせることによっ
て、フィルタ部材１３の突条部１７の内側と外側で正負
の差圧を交互に変化させるようにしてもよい。In this embodiment, the differential pressure adjusting unit 2
4 is a connecting pipe L located on the downstream side of the cell fractionation unit 1.
Although it is provided in FIG. 2 , as another modification, the cell fractionation unit 1
One end of the connecting pipe L 1 located upstream of (eg, the first
Between the on-off valve 6 and the connection port 1a). Further, as shown in FIG. 6, two pieces are provided at one end of the connecting pipe L 2 located downstream of the cell fractionation unit 1 and at one end of the connecting pipe L 1 located upstream of the cell fractionation unit 1. By providing the differential pressure adjusting units 24, and the pistons 24d of the respective differential pressure adjusting units 24 are moved forward and backward in opposite phases, positive and negative differential pressures are alternately applied inside and outside the ridges 17 of the filter member 13. It may be changed to.
【００３９】[0039]
【発明の効果】以上説明したように本発明によれば、分
画処理すべき細胞が流入側から流出側へ通過し得る形状
の多数の微細通路を半導体微細加工技術により半導体基
板に形成し、このように形成された半導体基板から成る
フィルタ部材の前記流入側に、前記細胞を含む浮遊細胞
液を供給し、更に、前記流入側に対して前記流出側を負
圧にして、前記微細通路を介して流入側から流出側へ通
過した細胞を回収するようにしたので、浮遊細胞液の中
の所望の細胞だけがフィルタ部材の前記多数の微細通路
を通過して流出側に出力し、残余の細胞等の物質が微細
通路を通過しないで流入側に残留することとなり、流出
側に出力した細胞を回収することによって、多量の細胞
を迅速且つ容易に分画処理することができる。As described above, according to the present invention, a large number of fine passages having a shape through which cells to be fractionated can pass from the inflow side to the outflow side are formed on a semiconductor substrate by a semiconductor fine processing technique. The floating cell liquid containing the cells is supplied to the inflow side of the filter member formed of the semiconductor substrate thus formed, and the outflow side is negatively pressured with respect to the inflow side, and Since the cells that have passed from the inflow side to the outflow side are collected through, only the desired cells in the suspension cell liquid pass through the numerous fine passages of the filter member and are output to the outflow side, and the remaining cells Substances such as cells remain on the inflow side without passing through the fine passages, and by collecting the cells output on the outflow side, a large amount of cells can be fractionated rapidly and easily.
【００４０】又、前記流出側と前記流出側との差圧を正
負交互に変化させる圧力を、前記流出側の処理室へ供給
することにより、フィルタ部材の微細通路の流れ方向を
交互に変化させるようにしたので、微細通路に付着する
細胞を離脱させたり、予め細胞の付着を防止することが
でき、円滑な細胞分画を達成することができる。Further, by supplying to the processing chamber on the outflow side a pressure that alternately changes the differential pressure between the outflow side and the outflow side, the flow direction of the fine passages of the filter member is alternately changed. Since this is done, cells adhering to the fine passages can be detached or cells can be prevented from adhering in advance, and a smooth cell fractionation can be achieved.
【００４１】そして、微細通路の形状を、前記分画処理
すべき細胞の硬さ、形状、変形能、活性の少なくとも１
つの特性に基づいて形成することによって、様々な態様
が可能となり、例えば、プロトプラストや血球等の分画
処理に多大な効果を発揮する。The shape of the fine passage is determined by at least one of hardness, shape, deformability and activity of the cells to be fractionated.
By forming it based on one characteristic, various modes are possible and, for example, a great effect is exerted on the fractionation treatment of protoplasts, blood cells and the like.
【００４２】[0042]
【図１】本発明による細胞分画装置の一実施例のシステ
ム構成図である。FIG. 1 is a system configuration diagram of an embodiment of a cell fractionation device according to the present invention.
【図２】細胞分画ユニットの構造を示す要部縦断面図で
ある。FIG. 2 is a longitudinal sectional view of an essential part showing the structure of a cell fractionation unit.
【図３】フィルタ部材の構造を示す斜視図である。FIG. 3 is a perspective view showing a structure of a filter member.
【図４】フィルタ部材の構造を更に示す斜視図である。FIG. 4 is a perspective view further showing the structure of the filter member.
【図５】フィルタ部材に設けられた突条部及び凹欠溝の
形状とそれらの機能を示す部分破断斜視図である。FIG. 5 is a partially cutaway perspective view showing the shapes of protruding portions and recessed grooves provided on the filter member and their functions.
【図６】差圧調整ユニットの構成を示す説明図でる。FIG. 6 is an explanatory diagram showing a configuration of a differential pressure adjusting unit.
【図７】差圧調整ユニットの動作を説明するための波形
図である。FIG. 7 is a waveform diagram for explaining the operation of the differential pressure adjusting unit.
【図８】一実施例の細胞分画装置の外観図である。FIG. 8 is an external view of a cell fractionation device according to an example.
【図９】一実施例の細胞分画装置の機能説明図である。FIG. 9 is a functional explanatory diagram of the cell fractionation device according to the embodiment.
【図１０】一実施例の細胞分画装置の機能を更に説明す
るための説明図である。FIG. 10 is an explanatory diagram for further explaining the function of the cell fractionation device according to the embodiment.
【図１１】従来技術を説明するための説明図である。FIG. 11 is an explanatory diagram for explaining a conventional technique.
１…細胞分画ユニット、２…マイクロシリンジ、３…タ
ンク、４…洗浄用タンク、５…負圧タンク、６，７，
８，９…開閉弁、１０…回収容器、１１…下側平板部
材、１２…上側平板部材、１３…フィルタ部材、１４…
側端部材、１５…処理室、１６…貫通孔、１７…突条
部、１７ｂ…凹欠溝、２０…顕微鏡、２１…ビデオカメ
ラ、２２…モニタ、２３…中央制御ユニット、２４…差
圧調整ユニット、Ｌ1 ，Ｌ2 ，Ｌ3 …連結管。1 ... Cell fractionation unit, 2 ... Micro syringe, 3 ... Tank, 4 ... Washing tank, 5 ... Negative pressure tank, 6, 7,
8, 9 ... Open / close valve, 10 ... Recovery container, 11 ... Lower flat plate member, 12 ... Upper flat plate member, 13 ... Filter member, 14 ...
Side end member, 15 ... Processing chamber, 16 ... Through hole, 17 ... Ridge portion, 17b ... Recessed groove, 20 ... Microscope, 21 ... Video camera, 22 ... Monitor, 23 ... Central control unit, 24 ... Differential pressure adjustment Unit, L 1 , L 2 , L 3 ... Connection pipe.
フロントページの続き (72)発明者 神谷 清 静岡県浜松市市野町1126番地の１ 浜松 ホトニクス株式会社内 (72)発明者 水口 義則 静岡県浜松市市野町1126番地の１ 浜松 ホトニクス株式会社内 (56)参考文献 特開 平１−265882（ＪＰ，Ａ) 特開 昭59−151886（ＪＰ，Ａ)Front Page Continuation (72) Inventor Kiyoshi Kamiya 1 Hamamatsu Photonics Co., Ltd., 1126 Ichinomachi, Hamamatsu, Shizuoka Prefecture (72) Inventor Yoshinori Mizuguchi 1 1126 Ichinocho, Hamamatsu, Shizuoka Prefecture Hamamatsu Photonics Co., Ltd. (56 ) Reference JP-A-1-265882 (JP, A) JP-A-59-151886 (JP, A)
Claims (14)
へ通過し得る形状の多数の微細通路を半導体微細加工技
術により半導体基板に形成して成るフィルタ部材の前記
流入側に、前記細胞を含む浮遊細胞液を供給すると共
に、前記流入側に対して前記流出側を負圧にし、前記微
細通路を介して流入側から流出側へ通過した細胞を回収
することを特徴とする細胞分画方法。1. A filter member formed by forming a plurality of microscopic passages in a semiconductor substrate by a semiconductor microfabrication technique into which a cell to be fractionated can pass from an inflow side to an outflow side. And a negative pressure on the outflow side with respect to the inflow side, and collecting the cells passing from the inflow side to the outflow side through the fine passages. Method.
べき細胞の硬さ、形状、変形能、活性の少なくとも１つ
の特性に基づいて形成することを特徴とする請求項１に
記載の細胞分画方法。2. The shape of the fine passage is formed on the basis of at least one characteristic of hardness, shape, deformability and activity of the cells to be fractionated. Cell fractionation method.
ストであることを特徴とする請求項１に記載の細胞分画
方法。3. The cell fractionation method according to claim 1, wherein the cells to be fractionated are protoplasts.
ことを特徴とする請求項１に記載の細胞分画方法。4. The cell fractionation method according to claim 1, wherein the cells to be fractionated are blood cells.
へ通過し得る形状の多数の微細通路が半導体微細加工技
術により半導体基板に形成されて成るフィルタ部材と、 前記細胞を含む浮遊細胞液を前記フィルタ部材の流入側
へ供給する供給手段と、 前記流入側に対して前記流出
側を負圧にすることにより、前記細胞を前記微細通路を
介して流入側から流出側へ通過させる差圧発生手段と、
を備えることを特徴とする細胞分画装置。5. A filter member comprising a large number of fine passages formed in a semiconductor substrate by a semiconductor fine processing technique so that cells to be fractionated can pass from an inflow side to an outflow side, and floating cells containing the cells. Supply means for supplying a liquid to the inflow side of the filter member, and a difference that causes the cells to pass from the inflow side to the outflow side through the fine passages by making the outflow side negative pressure with respect to the inflow side. Pressure generating means,
A cell fractionation device comprising:
へ通過し得る形状の多数の微細通路が半導体微細加工技
術により半導体基板に形成されて成るフィルタ部材を仕
切りとして密封収容することにより、前記流入側の処理
室と前記流出側の処理室とを有する細胞分画ユニット
と、 前記細胞を含む浮遊細胞液を前記細胞分画ユニットの前
記流入側の処理室へ供給する供給手段と、 前記流出側の処理室を負圧吸引にすることにより、前記
流入側の処理室中の細胞を前記微細通路を介して流出側
の処理室へ通過させる差圧発生手段と、を備えることを
特徴とする細胞分画装置。6. A filter member, which is formed by a semiconductor microfabrication technique on a semiconductor substrate and has a number of fine passages through which cells to be fractionated can pass from an inflow side to an outflow side, is hermetically accommodated as a partition. A cell fractionation unit having the treatment chamber on the inflow side and the treatment chamber on the outflow side, and a supply means for supplying a suspension cell solution containing the cells to the treatment chamber on the inflow side of the cell fractionation unit, Differential pressure generating means for allowing cells in the inflow-side processing chamber to pass to the outflow-side processing chamber through the fine passages by negatively suctioning the outflow-side processing chamber. And a cell fractionation device.
の処理室の内圧との差圧を正負交互に変化させる圧力
を、前記流出側の処理室へ供給する差圧調整手段を設け
たことを特徴とする請求項６に記載の細胞分画装置。7. A differential pressure adjusting means for supplying to the outflow-side processing chamber a pressure for alternately changing positive and negative the differential pressure between the internal pressure of the outflow-side processing chamber and the internal pressure of the outflow-side processing chamber. The cell fractionation device according to claim 6, wherein
の処理室の内圧との差圧を正負交互に変化させる圧力
を、前記流入側の処理室へ供給する差圧調整手段を設け
たことを特徴とする請求項７に記載の細胞分画装置。8. A differential pressure adjusting means for supplying to the processing chamber on the inflow side a pressure for alternately changing positive and negative the differential pressure between the internal pressure of the processing chamber on the outflow side and the internal pressure of the processing chamber on the outflow side. The cell fractionation device according to claim 7, wherein
の処理室の内圧との差圧を正負交互に変化させる圧力
を、前記流入側及び前記流出側の処理室へ供給する差圧
調整手段を設けたことを特徴とする請求項７に記載の細
胞分画装置。9. A differential pressure for supplying, to the inflow side and the outflow side processing chambers, a pressure for alternately changing positive and negative the differential pressure between the internal pressure of the outflow side processing chamber and the internal pressure of the outflow side processing chamber. The cell fractionation device according to claim 7, further comprising adjusting means.
る大きさに変化させる前記圧力を、前記処理室へ供給す
ることを特徴とする請求項７ないし請求項９のいずれか
１項に記載の細胞分画装置。10. The differential pressure adjusting means supplies the pressure for changing the differential pressure into positive and negative different magnitudes to the processing chamber, according to any one of claims 7 to 9. The described cell fractionation device.
る時間周期で変化させる前記圧力を、前記処理室へ供給
することを特徴とする請求項７ないし請求項９のいずれ
か１項に記載の細胞分画装置。11. The differential pressure adjusting means supplies the pressure for changing the differential pressure in positive and negative time periods different to each other to the processing chamber, according to any one of claims 7 to 9. The described cell fractionation device.
する圧力センサを有し、該圧力センサの計測値に基づい
て前記圧力を所定条件に設定する帰還制御手段を備える
ことを特徴とする請求項７ないし請求項１１のいずれか
１項に記載の細胞分画装置。12. The differential pressure adjusting means includes a pressure sensor for sequentially detecting the differential pressure, and feedback control means for setting the pressure to a predetermined condition based on a measurement value of the pressure sensor. The cell fractionation device according to any one of claims 7 to 11.
ルタ部材を透過観測可能な光透過部が設けられ、該光透
過部を介して細胞分画ユニット内部を撮影する撮影手段
が設けられていることを特徴とする請求項６に記載の細
胞分画装置。13. The cell fractionation unit is provided with a light transmission part capable of observing the filter member through transmission, and an image pickup means is provided for photographing the inside of the cell fractionation unit through the light transmission part. The cell fractionation device according to claim 6, wherein
状は、前記分画処理すべき細胞の硬さ、形状、変形能、
活性の少なくとも１つの特性に基づいて形成されること
を特徴とする請求項５又は請求項６のいずれか１項に記
載の細胞分画装置。14. The shape of the fine passages of the filter member is the hardness, shape, deformability of the cells to be fractionated,
The cell fractionation device according to claim 5, wherein the cell fractionation device is formed on the basis of at least one characteristic of activity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6163869A JP2685119B2 (en) | 1994-07-15 | 1994-07-15 | Cell fractionation method and cell fractionation device |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP6163869A JP2685119B2 (en) | 1994-07-15 | 1994-07-15 | Cell fractionation method and cell fractionation device |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0823967A JPH0823967A (en) | 1996-01-30 |
| JP2685119B2 true JP2685119B2 (en) | 1997-12-03 |
Family
ID=15782323
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP6163869A Expired - Fee Related JP2685119B2 (en) | 1994-07-15 | 1994-07-15 | Cell fractionation method and cell fractionation device |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2685119B2 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3738899B2 (en) | 2000-12-07 | 2006-01-25 | 株式会社 エフェクター細胞研究所 | Trace sample processing equipment |
| TWI241343B (en) * | 2000-12-07 | 2005-10-11 | Effector Cell Inst Inc | Well unit for detecting cell chemotaxis and separating chemotactic cells |
| TW200506364A (en) | 2003-04-09 | 2005-02-16 | Effector Cell Inst Inc | Apparatus for detecting cell chemo-taxis |
| JP6057317B2 (en) * | 2012-04-05 | 2017-01-11 | セイコーエプソン株式会社 | Separation device |
| CN107058075B (en) * | 2017-06-20 | 2023-10-20 | 商丘师范学院 | Plant cell protoplast purification instrument and purification method |
-
1994
- 1994-07-15 JP JP6163869A patent/JP2685119B2/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0823967A (en) | 1996-01-30 |
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