JP2009291097A - Cell culture apparatus - Google Patents

Cell culture apparatus Download PDF

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JP2009291097A
JP2009291097A JP2008146320A JP2008146320A JP2009291097A JP 2009291097 A JP2009291097 A JP 2009291097A JP 2008146320 A JP2008146320 A JP 2008146320A JP 2008146320 A JP2008146320 A JP 2008146320A JP 2009291097 A JP2009291097 A JP 2009291097A
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cells
culture
cell
electrode
cell suspension
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Masaru Hakoda
優 箱田
Hiroki Hibino
浩樹 日比野
Yoshiaki Shiba
良昭 芝
Hiroshi Fukuda
宏 福田
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Gunma University NUC
Olympus Corp
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Gunma University NUC
Olympus Corp
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    • 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

Abstract

<P>PROBLEM TO BE SOLVED: To remove unnecessary cells from a cell suspension and to culture necessary cells in a high concentration. <P>SOLUTION: The cell culture apparatus 1 is equipped with a culture container 2 storing a cell suspension A, a circulation flow channel 3 that is connected to the culture container 2 and circulates the cell suspension A stored in the culture container 2 and an unnecessary cell adsorption part 5 that is arranged in the middle position of the circulation flow channel 3 and has an electrode 7 forming a high-frequency non-uniform electric field in the flow channel 3 and adsorbing unnecessary cells for culture by dielectrophoretic force. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

本発明は細胞培養装置に関するものである。   The present invention relates to a cell culture apparatus.

従来、骨髄液中の間葉系幹細胞等の接着細胞を濃縮・分離してから培養する方法としては、採取した骨髄液を遠心した後に、その上澄み液を除去し、残った沈殿部分のみを播種する方法が知られている（例えば、特許文献１参照）。
この方法においては、播種の際に間葉系幹細胞が均一になるように、前記沈殿部分を一旦容器に取ってから培養容器に播種する。こうして播種された間葉系幹細胞は、培養容器中で培養される。骨髄液中の間葉系幹細胞は、培養容器の底面に付着して増殖する性質を有している。 Conventionally, as a method of culturing after concentrating and separating adherent cells such as mesenchymal stem cells in bone marrow fluid, the collected bone marrow fluid is centrifuged, the supernatant is removed, and only the remaining precipitate is seeded Is known (see, for example, Patent Document 1).
In this method, in order to make the mesenchymal stem cells uniform at the time of seeding, the precipitate is once taken up in a container and then seeded in a culture container. The mesenchymal stem cells thus seeded are cultured in a culture vessel. Mesenchymal stem cells in the bone marrow fluid have the property of growing by attaching to the bottom of the culture vessel.

特開２００４−４９１４２号公報JP 2004-49142 A

しかし、上記の方法を採用した場合、沈殿部分には間葉系幹細胞が濃縮されるほかに、骨髄液中の赤血球も大量に集められる。このため、播種した際に培養容器中に赤血球が大量に散在することになる。赤血球は浮遊細胞であるが、静置された培養容器中で沈降し、培養容器の底一面を覆うため、間葉系幹細胞が培養容器の底に接着するのを阻害する。   However, when the above method is employed, mesenchymal stem cells are concentrated in the precipitated portion, and a large amount of red blood cells in the bone marrow fluid is also collected. For this reason, a large amount of red blood cells are scattered in the culture container when seeded. Although erythrocytes are floating cells, they settle in a stationary culture vessel and cover the entire bottom surface of the culture vessel, thus preventing mesenchymal stem cells from adhering to the bottom of the culture vessel.

従って、骨髄播種後、間葉系幹細胞を回収できるまでの初代培養期間は早くても１０日、遅い場合は約２週間かかっていた。このため、より短期間で効率よく間葉系幹細胞を培養・回収できるようにするために、赤血球等の浮遊細胞を除去して、より高濃度の間葉系幹細胞液を培養する培養装置が望まれていた。   Therefore, after bone marrow seeding, the primary culture period until the mesenchymal stem cells could be recovered took 10 days at the earliest and about 2 weeks at the latest. For this reason, a culture apparatus that removes floating cells such as erythrocytes and cultures a higher concentration of mesenchymal stem cell solution is desired in order to cultivate and collect mesenchymal stem cells efficiently in a shorter period of time. It was rare.

この発明は上述した事情に鑑みてなされてものであって、細胞懸濁液内から不要細胞を除外して必要な細胞を高濃度で培養することができる細胞培養装置を提供することを目的としている。   The present invention has been made in view of the above-described circumstances, and an object thereof is to provide a cell culture apparatus capable of culturing necessary cells at a high concentration by removing unnecessary cells from the cell suspension. Yes.

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、細胞懸濁液を貯留する培養容器と、該培養容器に接続され、該培養容器内に貯留されている細胞懸濁液を循環させる循環流路と、該循環流路の途中位置に設けられ、循環流路内に高周波不平等電界を形成して、誘電泳動力により培養に不要な細胞を吸着する電極を有する不要細胞吸着部とを備える細胞培養装置を提供する。 In order to achieve the above object, the present invention provides the following means.
The present invention relates to a culture container that stores a cell suspension, a circulation channel that is connected to the culture container and circulates the cell suspension stored in the culture container, and an intermediate position of the circulation channel. And a cell culturing apparatus provided with an unnecessary cell adsorbing part having an electrode that forms a high-frequency unequal electric field in the circulation channel and adsorbs cells unnecessary for culture by dielectrophoretic force.

本発明によれば、培養容器内に貯留されている細胞懸濁液が循環流路内を循環させられる際に、循環流路の途中位置に設けられた電極によって循環流路内に形成されている高周波不平等電界中を通過させられる。細胞懸濁液内の細胞は、その種類に基づいて異なる誘電泳動特性を有しているので、高周波不平等電界の作用により異なる誘電泳動力を受ける。   According to the present invention, when the cell suspension stored in the culture vessel is circulated in the circulation channel, the cell suspension is formed in the circulation channel by the electrode provided in the middle of the circulation channel. Is allowed to pass through a high-frequency unequal electric field. Since the cells in the cell suspension have different dielectrophoretic characteristics based on the type, they receive different dielectrophoretic forces due to the action of the high-frequency unequal electric field.

このことを利用して、細胞懸濁液中に存在する不要な細胞は、誘電泳動力により不要細胞吸着部の電極に吸着され、必要な細胞は電極に吸着されることなく循環流路を循環させられて培養容器に戻るようにすることができる。
発明者らは、研究の結果、電極に吸着された細胞が死滅することを発見した。したがって、循環流路を循環させられる間に電極に接着した細胞を死滅させることにより、培養容器内に存在する不要な細胞の濃度を低下させることができ、必要な細胞を高濃度で培養することが可能となる。 By utilizing this, unnecessary cells existing in the cell suspension are adsorbed to the electrode of the unnecessary cell adsorption part by the dielectrophoretic force, and the necessary cells circulate in the circulation channel without being adsorbed to the electrode. And returned to the culture vessel.
As a result of research, the inventors have found that cells adsorbed to the electrode die. Therefore, it is possible to reduce the concentration of unnecessary cells present in the culture vessel by killing the cells adhered to the electrode while circulating in the circulation channel, and to culture the necessary cells at a high concentration. Is possible.

上記発明においては、前記細胞懸濁液が、接着性の細胞と浮遊性の細胞とを含み、前記不要細胞吸着部が、浮遊性の細胞を電極に吸着することとしてもよい。
このようにすることで、培養容器内の浮遊性の細胞の濃度を低減し、接着性の細胞の濃度を増大させて、培養容器の底面等の接着面を浮遊性の細胞に占有されることを防止して、接着性の細胞を効率よく培養することができる。 In the above invention, the cell suspension may include an adhesive cell and a floating cell, and the unnecessary cell adsorption unit may adsorb the floating cell to the electrode.
By doing this, the concentration of floating cells in the culture vessel is reduced, the concentration of adhesive cells is increased, and the adhesive surface such as the bottom surface of the culture vessel is occupied by the floating cells. And adherent cells can be efficiently cultured.

本発明によれば、細胞懸濁液内から不要細胞を除外して必要な細胞を高濃度で培養す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 unnecessary cells can be excluded from the cell suspension and necessary cells can be cultured at a high concentration.

本発明の一実施形態に係る細胞培養装置１について、図１〜図６を参照して、以下に説明する。
本実施形態に係る細胞培養装置１は、図１に示されるように、例えば、骨髄液を培地に混合して得られた細胞懸濁液Ａを貯留する培養容器２と、該培養容器２に接続された循環流路３と、該循環流路３に設けられ、循環流路３内の細胞懸濁液Ａを流動させるポンプ４と、循環流路３の途中位置に配置され、細胞懸濁液Ａ内の浮遊性の細胞を吸着し、接着性の細胞を通過させる誘電フィルタ５とを備えている。 A cell culture device 1 according to an embodiment of the present invention will be described below with reference to FIGS.
As shown in FIG. 1, the cell culture device 1 according to the present embodiment includes, for example, a culture container 2 that stores a cell suspension A obtained by mixing bone marrow fluid with a culture medium, The connected circulation channel 3, the pump 4 provided in the circulation channel 3 for flowing the cell suspension A in the circulation channel 3, and disposed in the middle of the circulation channel 3, the cell suspension And a dielectric filter 5 that adsorbs the floating cells in the liquid A and allows the adhesive cells to pass therethrough.

循環流路は、培養容器２から流出させた細胞懸濁液Ａを培養容器２の外部で循環させて培養容器２内に戻すように環状に形成されている。
誘電フィルタ５は、フィルタ容器６と、該フィルタ容器６内を２つの領域６ａ，６ｂに区画するように配置される電極７と、該電極７に高周波電圧を加える電圧供給部８と、培養容器２側から流れてくる細胞懸濁液Ａを一方の領域６ａに流入させる流入口６ｃと、該一方の領域６ａを通過した細胞懸濁液Ａをフィルタ容器６外に流出させる第１の流出口６ｄと、前記一方の領域６ａから電極７を通過して他方の領域６ｂに流入した細胞懸濁液Ａをフィルタ容器６外に流出させる第２の流出口６ｅとを備えている。 The circulation channel is formed in an annular shape so that the cell suspension A that has flowed out of the culture vessel 2 is circulated outside the culture vessel 2 and returned to the culture vessel 2.
The dielectric filter 5 includes a filter container 6, an electrode 7 arranged so as to partition the filter container 6 into two regions 6a and 6b, a voltage supply unit 8 for applying a high frequency voltage to the electrode 7, and a culture container Inflow port 6c that allows cell suspension A flowing from the second side to flow into one region 6a, and first outlet port that causes cell suspension A that has passed through one region 6a to flow out of filter vessel 6 6d and a second outlet 6e through which the cell suspension A that has passed through the electrode 7 from the one region 6a and has flowed into the other region 6b flows out of the filter container 6.

また、第２の流出口６ｅには該第２の流出口６ｅから流出する細胞懸濁液Ａの流量を調節するバルブ９が設けられている。
第１，第２の流出口６ｄ，６ｅに接続する配管１０，１１は、前記ポンプ４に接続する前に合流されている。これにより、ポンプ４による吸引力によって、細胞懸濁液Ａは第１、第２の流出口６ｄ，６ｅの両方からそれぞれ吸引されて循環流路３内を流通させられるようになっている。 The second outlet 6e is provided with a valve 9 for adjusting the flow rate of the cell suspension A flowing out from the second outlet 6e.
The pipes 10 and 11 connected to the first and second outlets 6 d and 6 e are joined before being connected to the pump 4. Thus, the cell suspension A is sucked from both the first and second outlets 6d and 6e by the suction force of the pump 4, and can be circulated in the circulation channel 3.

電極７は、例えば、図２に示されるように、複数の直棒状電極部７ａ，７ｂを平行に間隔をあけて配列してなる２つの櫛歯状電極７Ａ，７Ｂを、各櫛歯状電極７Ａ，６７の直棒状電極部７ａ，７ｂが交互に配置されるように配置することにより構成されている。   For example, as shown in FIG. 2, the electrode 7 includes two comb-like electrodes 7A and 7B formed by arranging a plurality of straight rod-like electrode portions 7a and 7b at intervals in parallel. The straight rod-like electrode portions 7a and 7b of 7A and 67 are arranged so as to be alternately arranged.

このような櫛歯状電極７Ａ，７Ｂに高周波電圧を加えると、図３に示されるように、電界強度の勾配を有する高周波不平等電界Ｅが発生するようになっている。図３は電界強度を等高線により示しており、電界強度は、直棒状電極部７ａ，７ｂに近いほど強く、離れる程弱くなっている。   When a high frequency voltage is applied to such comb-like electrodes 7A and 7B, a high frequency unequal electric field E having a gradient of electric field strength is generated as shown in FIG. FIG. 3 shows the electric field strength with contour lines. The electric field strength is stronger as it is closer to the straight rod-like electrode portions 7a and 7b, and is weaker as it is farther away.

このように構成された本実施形態に係る細胞培養装置１の作用について以下に説明する。
本実施形態に係る細胞培養装置１によれば、細胞懸濁液Ａを培養容器２内に貯留した状態で、ポンプ４を作動させることにより、培養容器２内の細胞懸濁液Ａが循環流路３内に吸引されて循環させられる。そして、電圧供給部８の作動により、フィルタ容器６内の櫛歯状電極７Ａ，７Ｂに高周波電圧を加えることによって、直棒状電極部７ａ，７ｂの周囲に高周波不平等電界Ｅを発生させる。これにより、細胞懸濁液Ａ内に含まれている各細胞が高周波不平等電界Ｅ中を流動させられる際に、各細胞の誘電泳動特性に応じた誘電泳動力を受ける。 The operation of the cell culture device 1 according to this embodiment configured as described above will be described below.
According to the cell culture device 1 according to the present embodiment, the cell suspension A in the culture container 2 is circulated by operating the pump 4 while the cell suspension A is stored in the culture container 2. It is sucked into the passage 3 and circulated. Then, a high frequency unequal electric field E is generated around the straight bar electrode portions 7a and 7b by applying a high frequency voltage to the comb-like electrodes 7A and 7B in the filter container 6 by the operation of the voltage supply unit 8. Thereby, when each cell contained in the cell suspension A is caused to flow in the high frequency unequal electric field E, it receives a dielectrophoretic force according to the dielectrophoretic characteristics of each cell.

この場合に、細胞懸濁液Ａ中の不要な浮遊性の細胞、例えば、血球系の細胞が正の誘電泳動特性を有し、必要な接着性の細胞、例えば、間葉系幹細胞等の非血球系の細胞が負の誘電泳動特性を有するような周波数の高周波不平等電界Ｅに調節しておくことにより、循環経路３を流動してきた細胞懸濁液Ａがフィルタ容器６内に流入すると、血球系の細胞は、高周波不平等電界Ｅの電界強度が高くなる方向、すなわち、直棒状電極部７ａ，７ｂに向かう方向に引き寄せられて、あるものは直棒状電極部７ａ，７ｂに接触し、あるものは直棒状電極部７ａ，７ｂ間の隙間を通過して第２の流出口６ｅからフィルタ容器６外に流出する。一方、細胞懸濁液Ａ中に含まれる非血球系の細胞は、直棒状電極部７ａ，７ｂから離れる方向に移動して直棒状電極部７ａ，７ｂに接触することなく、第１の流出口６ｄからフィルタ容器６外に流出する。   In this case, unnecessary floating cells in the cell suspension A, such as blood cells, have positive dielectrophoretic properties, and necessary adherent cells such as mesenchymal stem cells, etc. When the cell suspension A that has flowed through the circulation path 3 flows into the filter container 6 by adjusting the high-frequency unequal electric field E having such a frequency that the cells of the blood cell system have negative dielectrophoretic characteristics, The cells of the blood cell line are attracted in the direction in which the electric field strength of the high frequency unequal electric field E increases, that is, in the direction toward the straight rod-shaped electrode portions 7a and 7b, and some contact with the straight rod-shaped electrode portions 7a and 7b, Some flow out of the filter container 6 through the second outlet 6e through the gap between the straight electrode portions 7a and 7b. On the other hand, the non-hemocytic cells contained in the cell suspension A move in the direction away from the straight rod-shaped electrode portions 7a and 7b and do not contact the straight rod-shaped electrode portions 7a and 7b, and thus the first outlet It flows out of the filter container 6 from 6d.

血球系の細胞が櫛歯状電極７Ａ，７Ｂに捉えられることなく透過するためには、櫛歯状電極７Ａ，７Ｂ近傍に引き留めようとする誘電泳動力より細胞懸濁液Ａの流れにより受ける力を大きくしておく必要があり、バルブ９の開度を適宜調節することで、この条件を設定することができる。   In order for blood cells to permeate without being caught by the comb-like electrodes 7A, 7B, the force received by the flow of the cell suspension A from the dielectrophoretic force that is to be retained in the vicinity of the comb-like electrodes 7A, 7B Must be increased, and this condition can be set by appropriately adjusting the opening of the valve 9.

図４および図５に、８０ｋＨｚ２０Ｖｐｐと４０Ｖｐｐの高周波電圧を加えた誘電フィルタ５に流入して、電極７を透過した透過液内の細胞、電極７を透過することなくフィルタ容器６から流出した保持液内の細胞および直棒状電極部７ａ，７ｂに接触した細胞（いずれも細胞は３−２Ｈ３）を５×１０５cells／ｍLの細胞密度で、８．５重量％濃度のスクロース＋０．３重量％濃度のグルコース溶液内で培養したときの培養１日目と２日目の比増殖速度を示す。 In FIG. 4 and FIG. 5, it flows into the dielectric filter 5 to which high frequency voltages of 80 kHz 20 V pp and 40 V pp are applied, flows out from the filter container 6 without passing through the electrode 7, and the cells in the permeate that have passed through the electrode 7. Cells in the retentate and cells in contact with the straight electrode portions 7a and 7b (both cells are 3-2H3) at a cell density of 5 × 10 5 cells / mL, 8.5% by weight sucrose + 0.3 The specific growth rate on the first day and the second day of culture when cultured in a glucose solution having a concentration of% by weight is shown.

また、誘電フィルタ５を通過させない細胞３−２Ｈ３の通常濃度（５×１０５cells／ｍL）での培養時の比増殖速度および低濃度（４×１０４cells／ｍL）での培養時の比増殖速度を図６に示す。通常の濃度での培養時には、図５の透過液および保持液内の細胞と同様に１日目の比増殖速度が２日目の比増殖速度より高いことがわかる。一方、低濃度での培養時には、図５のフィルタに接触した細胞と同様の比増殖速度の変化が示されている。 In addition, the specific growth rate at the time of culturing at a normal concentration (5 × 10 5 cells / mL) of cells 3-2H3 not allowed to pass through the dielectric filter 5 and the ratio at the time of culturing at a low concentration (4 × 10 4 cells / mL) The growth rate is shown in FIG. When culturing at a normal concentration, it can be seen that the specific growth rate on the first day is higher than the specific growth rate on the second day, as in the cells in the permeate and retentate of FIG. On the other hand, at the time of culturing at a low concentration, a change in specific growth rate similar to that of the cells in contact with the filter of FIG. 5 is shown.

この図５から、発明者らは、研究の結果、電極７に吸着された細胞が、電極７に接触することなく透過あるいは保持された細胞と比較して培養１日目の比増殖速度が著しく低くなることを発見した。特に、電極７に加える高周波電圧の電圧値が高い４０Ｖｐｐの方が２０Ｖｐｐよりも培養１日目の比増殖速度が著しく低くなることを発見した。   From FIG. 5, the inventors found that the specific growth rate on the first day of culture was remarkably higher as compared with the cells permeated or retained without contacting the electrode 7 as a result of the study. I found it to be lower. In particular, it has been found that the specific growth rate on the first day of culture is significantly lower at 40 Vpp where the voltage value of the high-frequency voltage applied to the electrode 7 is higher than at 20 Vpp.

また、電極７に接触させることなく低濃度で培養した場合においては、培養１日目で１．５程度の比増殖速度を示しているのに対し、電極７に接触した細胞は、培養１日目で０．５〜０．９の比増殖速度しか示していないことがわかる。この結果、電極７に接触した細胞は、その電圧が高い程、ダメージを受けて死滅あるいは活性が低下することがわかった。   In addition, when the cells are cultured at a low concentration without being brought into contact with the electrode 7, a specific growth rate of about 1.5 is shown on the first day of the culture, whereas the cells in contact with the electrode 7 are cultured on the first day of the culture. It can be seen that the eye shows only a specific growth rate of 0.5 to 0.9. As a result, it was found that the cells in contact with the electrode 7 were damaged and killed or decreased in activity as the voltage increased.

本実施形態に係る細胞培養装置１は、この現象を利用して、誘電フィルタ５内に流入した細胞の誘電泳動特性の差に基づいて不要な血球系の細胞を電極７に接触させ、必要な非血球系の細胞を電極７に接触させることなくフィルタ容器６内から流出させて、循環経路３を介して培養容器２内に戻す。この動作を繰り返すことにより、血球系の細胞については電極７に接触させてダメージを与え、死滅あるいは活性を低下させる一方、非血球系の細胞についてはダメージを与えることなく培養容器２に戻すことができる。そして、これにより、培養容器２内の不要な血球系の浮遊性の細胞の濃度を下げ、必要な非血球系の接着性の細胞の濃度を増大させて、純度の高い接着性の細胞群が得られるように培養することができるという利点がある。   The cell culture device 1 according to the present embodiment uses this phenomenon to bring unnecessary blood cells into contact with the electrode 7 based on the difference in the dielectrophoretic characteristics of the cells that have flowed into the dielectric filter 5. Non-hemocytic cells are allowed to flow out of the filter container 6 without being brought into contact with the electrode 7, and returned to the culture container 2 through the circulation path 3. By repeating this operation, blood cells are brought into contact with the electrode 7 to be damaged and killed or reduced in activity, while non-blood cells are returned to the culture vessel 2 without being damaged. it can. As a result, the concentration of unnecessary blood cell floating cells in the culture vessel 2 is decreased, and the concentration of necessary non-hemocytic adhesive cells is increased, so that a highly pure adhesive cell group can be obtained. There is an advantage that it can be cultured as obtained.

なお、接着性の細胞を十分に増殖させるためには、該細胞を培養容器２の底面等に接着させる必要があるため、循環流路３を循環させて不要な細胞を死滅あるいは活性を低下させる工程と、ポンプ４を停止して循環を停止し、細胞を沈降させて培養容器２の底面に接着させる工程とを繰り返し行うことが有効である。所定時間、例えば、１日静置することにより、接着性の細胞は培養容器２の底面に接着するが、一旦沈降した浮遊性の細胞はポンプ４の作動により再度舞い上がって循環流路３に流入する。
したがって、その後の循環動作においては、接着性の細胞の多くを循環させずに済み、浮遊性の細胞のみについて誘電フィルタ５による活性の低下処理を繰り返すことができる。 In order to sufficiently grow the adhesive cells, it is necessary to adhere the cells to the bottom surface of the culture vessel 2 and the like, so that unnecessary cells are killed or the activity is reduced by circulating the circulation channel 3. It is effective to repeat the process and the process of stopping the circulation by stopping the pump 4 and allowing the cells to settle and adhere to the bottom surface of the culture vessel 2. Adhesive cells adhere to the bottom surface of the culture vessel 2 by standing for a predetermined time, for example, for one day, but once suspended, the floating cells rise again by the operation of the pump 4 and flow into the circulation channel 3. To do.
Therefore, in the subsequent circulation operation, it is not necessary to circulate many of the adherent cells, and the activity reduction process by the dielectric filter 5 can be repeated only for the floating cells.

なお、本実施形態においては、骨髄液を培地に混合して得られた細胞懸濁液Ａについて、必要な接着性の細胞の高純度に増殖させる場合について説明したが、細胞ソースは骨髄液に限定されるものではなく、臍帯血、末梢血、脂肪組織等の生体組織を分解して得られた細胞懸濁液Ａ等の他の任意の細胞ソースを使用してもよい。   In the present embodiment, the cell suspension A obtained by mixing the bone marrow fluid with the culture medium has been described for the case where the necessary adherent cells are grown to high purity. However, the cell source is the bone marrow fluid. Without limitation, any other cell source such as cell suspension A obtained by decomposing biological tissue such as umbilical cord blood, peripheral blood, and adipose tissue may be used.

本発明の一実施形態に係る細胞培養装置を示す全体構成図である。It is a whole lineblock diagram showing the cell culture device concerning one embodiment of the present invention. 図１の細胞培養装置の誘電フィルタ内の電極の一例を示す図である。It is a figure which shows an example of the electrode in the dielectric filter of the cell culture apparatus of FIG. 図２の電極の周囲に形成される高周波不平等電界を等高線により示す図である。It is a figure which shows the high frequency unequal electric field formed around the electrode of FIG. 2 by a contour line. 図１の細胞培養装置において、２０Ｖｐｐの高周波電圧を加えた誘電フィルタ内の電極を透過した透過液および電極を透過しない保持液内の細胞、および電極に接触した細胞の培養１日目と培養２日目の比増殖速度を示すグラフである。In the cell culture apparatus of FIG. 1, the first day of culture and the culture 2 of the permeated liquid that has passed through the electrode in the dielectric filter to which a high frequency voltage of 20 Vpp is applied, the cells in the retentate that does not permeate the electrode, and the cells in contact with the electrode It is a graph which shows the specific growth rate of a day. 図１の細胞培養装置において、４０Ｖｐｐの高周波電圧を加えた誘電フィルタ内の電極を透過した透過液および電極を透過しない保持液内の細胞、および電極に接触した細胞の培養１日目と培養２日目の比増殖速度を示すグラフである。In the cell culture apparatus of FIG. 1, the first day of culture and the culture 2 of the permeated liquid that has passed through the electrode in the dielectric filter to which a high frequency voltage of 40 Vpp is applied, the cells in the retentate that does not permeate the electrode, and the cells in contact with the electrode It is a graph which shows the specific growth rate of a day. 誘電フィルタを使用することなく培養した通常濃度の細胞および低濃度の細胞の培養１日目と培養２日目の比増殖速度を示すグラフである。It is a graph which shows the specific growth rate of the culture | cultivation 1st day and the culture | cultivation 2nd day of the normal density | concentration cell cultured without using a dielectric filter, and the low density | concentration cell.

符号の説明Explanation of symbols

Ａ 細胞懸濁液
Ｅ 高周波不平等電界
１ 細胞培養装置
２ 培養容器
３ 循環流路
５ 誘電フィルタ（不要細胞吸着部）
７ 電極 A Cell suspension E High frequency unequal electric field 1 Cell culture device 2 Culture vessel 3 Circulating flow path 5 Dielectric filter (unnecessary cell adsorption part)
7 electrodes

Claims (2)

細胞懸濁液を貯留する培養容器と、
該培養容器に接続され、該培養容器内に貯留されている細胞懸濁液を循環させる循環流路と、
該循環流路の途中位置に設けられ、循環流路内に高周波不平等電界を形成して、誘電泳動力により培養に不要な細胞を吸着する電極を有する不要細胞吸着部とを備える細胞培養装置。 A culture vessel for storing the cell suspension;
A circulation channel connected to the culture vessel and circulating the cell suspension stored in the culture vessel;
A cell culturing apparatus provided with an unnecessary cell adsorbing portion provided at an intermediate position of the circulation channel, and having an electrode that forms a high-frequency unequal electric field in the circulation channel and adsorbs cells unnecessary for culture by dielectrophoretic force . 前記細胞懸濁液が、接着性の細胞と浮遊性の細胞とを含み、
前記不要細胞吸着部が、浮遊性の細胞を電極に吸着する請求項１に記載の細胞培養装置。 The cell suspension includes adhesive cells and floating cells,
The cell culture apparatus according to claim 1, wherein the unnecessary cell adsorption unit adsorbs floating cells to an electrode.
JP2008146320A 2008-06-03 2008-06-03 Cell culture apparatus Withdrawn JP2009291097A (en)

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Cited By (6)

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WO2012169493A1 (en) 2011-06-10 2012-12-13 株式会社日立製作所 Cell culture vessel, and culture device equipped with same
JP2012254057A (en) * 2011-06-10 2012-12-27 Hitachi Ltd Cell culture vessel, and culture device equipped with same
US8809054B2 (en) 2010-05-12 2014-08-19 Xpand Biotechnology B.V. Cell-culture-bag
US9121004B2 (en) 2010-02-26 2015-09-01 Korea Advanced Institute Of Science And Technology Cell culture unit and cell culture device including the same
JP2016111980A (en) * 2014-12-17 2016-06-23 大日本印刷株式会社 Cell selection method
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9121004B2 (en) 2010-02-26 2015-09-01 Korea Advanced Institute Of Science And Technology Cell culture unit and cell culture device including the same
US8809054B2 (en) 2010-05-12 2014-08-19 Xpand Biotechnology B.V. Cell-culture-bag
AU2011251055B2 (en) * 2010-05-12 2014-12-04 Scinus Cell Expansion B.V. Cell - culture - bag
WO2012169493A1 (en) 2011-06-10 2012-12-13 株式会社日立製作所 Cell culture vessel, and culture device equipped with same
JP2012254057A (en) * 2011-06-10 2012-12-27 Hitachi Ltd Cell culture vessel, and culture device equipped with same
US9487747B2 (en) 2011-06-10 2016-11-08 Hitachi, Ltd. Cell culture device
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