JP5828823B2 - Cell culture module and cell culture method - Google Patents
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Description
本発明は、培養細胞を細胞培養担体から回収するのに好適な細胞培養モジュール及びこれを用いた細胞培養方法に関する。 The present invention relates to a cell culture module suitable for recovering cultured cells from a cell culture carrier and a cell culture method using the same.
近年、ヒト骨髄液からの幹細胞の分離技術、目的とする組織細胞への分化・誘導技術、三次元培養技術及び足場材料の開発等の進歩に伴い、細胞培養によって幹細胞から皮膚、骨、軟骨、血管、心臓弁及び靭帯等の生体組織を形成することが可能となり、臨床応用も行われている。また、胚性幹細胞（ＥＳ細胞）や人工多能性幹細胞（ＩＰＳ細胞）に関しても、再生医療への応用に対する注目が高まっている。 In recent years, with the development of stem cell separation technology from human bone marrow fluid, differentiation / induction technology to target tissue cells, three-dimensional culture technology, and development of scaffold materials, etc., cells, skin, bone, cartilage, Biological tissues such as blood vessels, heart valves and ligaments can be formed, and clinical applications are also being performed. Further, with regard to embryonic stem cells (ES cells) and induced pluripotent stem cells (IPS cells), attention is being focused on application to regenerative medicine.
上記のような幹細胞の培養は、通常、基板表面に複数のウェルが設けられた細胞培養担体を用いて行われる。このような細胞培養方法においては、培養した細胞は、ウェル内に接着しているため、細胞培養担体から剥離して回収しなければならない。
この剥離作業は、従来は、トリプシン処理等の酵素処理により行われていたが、酵素処理のような化学的な処理方法では、培養した細胞の一部は劣化、さらには死滅する場合もあり、また、処理操作が煩雑であるという課題も有していた。
Stem cell culture as described above is usually performed using a cell culture carrier in which a plurality of wells are provided on the substrate surface. In such a cell culture method, since the cultured cells are adhered in the wells, they must be detached from the cell culture carrier and recovered.
Conventionally, this peeling work has been performed by enzyme treatment such as trypsin treatment, but in a chemical treatment method such as enzyme treatment, some of the cultured cells may be deteriorated and even killed, In addition, there is a problem that the processing operation is complicated.
このような課題に鑑みて、本出願人は、酵素処理を行うことなく、物理的な方法で培養細胞を細胞培養担体から容易に剥離する手段として、水圧を利用した細胞培養モジュールを提案した（例えば、特許文献１，２参照）。
この細胞培養モジュールは、多孔体からなる細胞培養担体を密閉した空間に培養液を充填した状態で、細胞培養面の背面から水圧（液圧）をかけて、前記細胞培養担体から培養細胞を剥離し、この剥離された細胞を培養液とともに排出させて回収する構成を備えているものである。
In view of such a problem, the present applicant has proposed a cell culture module using water pressure as a means for easily detaching cultured cells from a cell culture carrier by a physical method without performing enzyme treatment ( For example, see Patent Documents 1 and 2).
This cell culture module peels cultured cells from the cell culture support by applying water pressure (hydraulic pressure) from the back of the cell culture surface in a state where a culture solution is filled in a sealed space of a cell culture support made of a porous material. In addition, the peeled cells are discharged together with the culture solution and collected.
上記のような細胞培養モジュールを用いれば、酵素処理による場合よりも効率的に培養した細胞を剥離・回収することができる。
しかしながら、培養した細胞を細胞培養担体から剥離する際、細胞を剥離するのに十分な水圧が得られなかったり、剥離した細胞が細胞培養担体の外周縁付近に再付着して残留したりする等、培養した細胞の回収率が高いとは言えなかった。また、水圧を高くすれば、回収率の向上は図られるが、そのためには、モジュール全体を高耐圧設計としなければならず、装置の大型化及び高コスト化を招くという課題も有していた。
If the cell culture module as described above is used, the cultured cells can be peeled and collected more efficiently than by the enzyme treatment.
However, when the cultured cells are detached from the cell culture carrier, sufficient water pressure is not obtained to peel off the cells, or the detached cells remain attached to the vicinity of the outer periphery of the cell culture carrier. The recovery rate of cultured cells was not high. Moreover, if the water pressure is increased, the recovery rate can be improved, but for that purpose, the entire module must be designed to have a high withstand voltage, and there is a problem that the apparatus is increased in size and cost. .
本発明は、上記課題を解決するためになされたものであり、細胞培養担体のウェル内での細胞培養後の処理において、簡便な処理操作により、前記ウェル内で培養された細胞を効率的かつ高い回収率で剥離して回収することができる細胞培養モジュール及びこれを用いた細胞培養方法を提供することを目的とするものである。 The present invention has been made to solve the above problems, and in the treatment after cell culture in the well of the cell culture carrier, the cells cultured in the well can be efficiently and easily treated by a simple treatment operation. It is an object of the present invention to provide a cell culture module that can be separated and recovered at a high recovery rate and a cell culture method using the same.
本発明に係る細胞培養モジュールは、表面に複数のウェルが設けられた円板状の細胞培養担体を載置する保持部材と、前記保持部材の細胞培養担体載置面に被せられる蓋部材とを備え、前記蓋部材の下面は、前記細胞培養担体の上面外周部及び前記保持部材に密着し、前記蓋部材下面と前記細胞培養担体表面との間に隙間が形成され、前記蓋部材には、前記細胞培養担体上に緩衝液を供給するために均等に配置され、下向きに貫通する複数の導入孔と、前記細胞培養担体上の緩衝液を排出するために中央に配置された中央孔とが設けられ、前記導入孔から供給された緩衝液が、前記隙間において渦流を生じると同時に、前記中央孔から排出されることを特徴とする。
このように、細胞培養担体上で緩衝液の渦流を生じさせることにより、その流れの力によって、培養された細胞を細胞培養担体から効率的かつ高い回収率で剥離して回収することができる。
The cell culture module according to the present invention comprises a holding member for mounting a disk-shaped cell culture carrier having a plurality of wells on the surface, and a lid member for covering the cell culture carrier mounting surface of the holding member. Provided, the lower surface of the lid member is in close contact with the upper surface outer peripheral portion of the cell culture carrier and the holding member, and a gap is formed between the lower surface of the lid member and the surface of the cell culture carrier, A plurality of introduction holes that are evenly arranged to supply a buffer solution on the cell culture carrier and penetrate downward, and a central hole that is arranged in the center to discharge the buffer solution on the cell culture carrier. The buffer solution that is provided and is supplied from the introduction hole is discharged from the central hole at the same time as generating a vortex in the gap.
In this way, by generating a vortex of the buffer solution on the cell culture carrier, the cultured cells can be efficiently detached and recovered from the cell culture carrier by the force of the flow.
前記導入孔は、傾き角度がその貫通孔の中心軸を通る鉛直方向断面において、前記細胞培養担体表面に対して２０°〜８０°であり、数が３〜４個であり、かつ、前記蓋部材の下面側における導入孔の出口の中心位置が、前記隙間の半径Ｒに対して中心からの距離が０．７Ｒ以上の外周部であることが好ましい。
前記導入孔をこのような態様で設けることにより、流路の配置設計や加工がしやすく、また、前記細胞培養担体上の隙間において、培養された細胞を細胞培養担体から確実に剥離するのに好適な渦流を生じやすい。
The introduction hole has an inclination angle of 20 ° to 80 ° with respect to the surface of the cell culture carrier in a vertical cross section passing through the central axis of the through hole, the number of the introduction holes is 3 to 4, and the lid The center position of the outlet of the introduction hole on the lower surface side of the member is preferably an outer peripheral portion whose distance from the center is 0.7 R or more with respect to the radius R of the gap.
By providing the introduction hole in such a manner, it is easy to design and process the flow path, and to surely detach the cultured cells from the cell culture carrier in the gap on the cell culture carrier. It is easy to generate a suitable vortex.
また、本発明に係る細胞培養方法は、上記の細胞培養モジュールを用いた細胞培養方法であって、表面に複数のウェルが設けられた円板状の多孔体からなる細胞培養担体を前記保持部材に載置し、前記細胞培養担体の上面外周部及び前記保持部材に蓋部材を密着固定した後、前記保持部材の細胞培養担体載置面に通じる供給流路から前記細胞培養担体の裏面側に第１の培養液を供給し、該細胞培養担体上面まで浸透させる第１の培養液の供給工程と、第１の培養液の供給を停止し、前記蓋部材の前記中央孔から前記細胞培養担体上面に未分化の細胞を含む第２の培養液を滴下し、細胞を播種する細胞播種工程と、前記供給流路から前記細胞培養担体の裏面側に第１の培養液を供給しながら、第１の培養液及び第２の培養液の培養液成分のみを前記中央孔から排出させ、前記ウェル内で細胞を凝集化させる細胞凝集化工程と、第１の培養液の供給を停止し、前記蓋部材の前記導入孔から緩衝液を供給し、凝集化した培養細胞を前記緩衝液とともに前記中央孔から排出させて回収する培養細胞回収工程とを備えていることを特徴とする。
このような培養方法によれば、間葉系幹細胞やＥＳ細胞の凝集塊を効率的に形成し、かつ、培養された細胞の細胞培養担体からの剥離及び回収まで連続的に簡便に行うことができる。
The cell culturing method according to the present invention is a cell culturing method using the above cell culturing module, wherein the holding member is a cell culturing carrier comprising a disc-shaped porous body having a plurality of wells on the surface. After the lid member is closely fixed to the outer periphery of the upper surface of the cell culture carrier and the holding member, from the supply flow channel leading to the cell culture carrier placement surface of the holding member to the back side of the cell culture carrier Supplying the first culture solution to infiltrate the cell culture support to the upper surface thereof, stopping the supply of the first culture solution, and from the central hole of the lid member to the cell culture support Dropping a second culture solution containing undifferentiated cells on the upper surface and seeding the cells, while supplying the first culture solution from the supply channel to the back side of the cell culture carrier, Only the culture fluid components of the first culture fluid and the second culture fluid The cell aggregating step for discharging the cells from the central hole and aggregating the cells in the well and the supply of the first culture solution were stopped, and the buffer solution was supplied from the introduction hole of the lid member to be aggregated. And a cultured cell recovery step of discharging the cultured cells together with the buffer solution from the central hole and recovering the cultured cells.
According to such a culture method, agglomerates of mesenchymal stem cells and ES cells can be efficiently formed, and the cultured cells can be easily and continuously removed and recovered from the cell culture carrier. it can.
本発明に係る細胞培養モジュールによれば、細胞培養担体のウェル内で細胞培養した後、培養した細胞を回収する際、酵素処理のように細胞の劣化を生じさせることなく、前記ウェル内で培養された細胞を効率的に剥離して、高い回収率で回収することができる。また、処理操作が簡便であるという利点も有している。
また、本発明に係る細胞培養方法によれば、上記のような細胞培養モジュールを用いて細胞培養を行うことにより、三次元細胞塊を効率的に培養することができ、さらに、培養された細胞の細胞培養担体からの剥離及び回収までを連続的に簡便に行うことができる。
According to the cell culture module of the present invention, after cell culture in the well of the cell culture carrier, when collecting the cultured cells, the cells are cultured in the wells without causing deterioration of the cells as in the enzyme treatment. The detached cells can be efficiently detached and recovered at a high recovery rate. Moreover, it has the advantage that processing operation is simple.
Further, according to the cell culture method according to the present invention, by performing cell culture using the cell culture module as described above, a three-dimensional cell mass can be efficiently cultured, and further, the cultured cells From the cell culture carrier to the separation and recovery can be continuously and easily performed.
以下、本発明について、図面を参照して詳細に説明する。
図１に、本発明に係る細胞培養モジュールの概略を示す。図１に示した細胞培養モジュールは、表面に複数のウェル１１が設けられた円板状の細胞培養担体１を載置する保持部材２と、保持部材２の細胞培養担体載置面（上面）に被せられる蓋部材３とからなる。
保持部材２の細胞培養担体載置面は、細胞培養担体１のサイズに対応した凹部が形成され、Ｏリング２１を介して、細胞培養担体１が載置される。
一方、蓋部材３の下面は、細胞培養担体１上面の外周部及び保持部材２にＯリング２２を介して密着し、蓋部材３下面と細胞培養担体１表面との間に隙間４が形成される。
Hereinafter, the present invention will be described in detail with reference to the drawings.
FIG. 1 shows an outline of a cell culture module according to the present invention. The cell culture module shown in FIG. 1 has a
A concave portion corresponding to the size of the cell culture carrier 1 is formed on the cell culture carrier placement surface of the
On the other hand, the lower surface of the
なお、蓋部材３の隙間４の径は、細胞培養担体１の径よりもわずかに小さいことが好ましい。ただし、蓋部材３が細胞培養担体１表面のウェル１１を覆うことがないようにする。
蓋部材３をこのように形成することにより、細胞培養担体１から剥離された培養細胞が細胞培養担体１の外縁部や背面側に周り込むことを防止することができる。
The diameter of the
By forming the
また、蓋部材３には、複数の導入孔３１と中央孔３２が設けられている。図２に、蓋部材３の一例の下面図を示す。
導入孔３１は、細胞培養担体１上に緩衝液５を供給する流路であり、蓋部材３に均等に配置され、斜め下向きに貫通するように設けられる。図２においては、３つの導入孔３１が、中心角１２０°をなすように均等な配置で設けられ、各導入孔３１は、上面から下面に向かって斜め内向きの貫通孔である。
また、中央孔３２は、細胞培養担体１上の緩衝液５を排出する流路であり、円板状の細胞培養担体１の中心上方、すなわち、蓋部材３の中央に設けられる。
The
The
The
上記のような構成を備えた細胞培養モジュールに、緩衝液５を導入孔３１から供給することにより、隙間４において渦流が生じ、これと同時に、緩衝液５は蓋部材３の中央の中央孔３２から排出される。
図３に、図２に示した蓋部材を用いた場合の細胞培養担体上での緩衝液の渦流方向のイメージを矢印で示す。
本発明に係る細胞培養モジュールを用いれば、細胞培養担体上で図３に示すような緩衝液の渦流を生じさせることができ、その流れの力によって、細胞培養担体のウェル内で培養された細胞を該細胞培養担体から容易に剥離し、細胞した培養を緩衝液とともに回収することができ、培養細胞の回収率の向上が図られる。
By supplying the
In FIG. 3, an image of the vortex direction of the buffer solution on the cell culture carrier when the lid member shown in FIG. 2 is used is indicated by an arrow.
When the cell culture module according to the present invention is used, a vortex of the buffer solution as shown in FIG. 3 can be generated on the cell culture carrier, and the cells cultured in the well of the cell culture carrier by the force of the flow. Can be easily detached from the cell culture carrier, and the cultured cell can be collected together with a buffer solution, thereby improving the collection rate of the cultured cells.
蓋部材３を斜め下向きに貫通する導入孔３１の傾きは、該貫通孔の中心軸を通る鉛直方向断面において、細胞培養担体１表面に対する角度θが２０°〜８０°であることが好ましい。
前記傾き角度θが８０°よりも大きい場合、渦流の勢いが弱くなり、培養された細胞が細胞培養担体１から剥離されない領域ができるおそれがある。一方、前記傾き角度θが２０°未満の場合、流路の配置が設計上困難となり、また、渦流の流速はそれほど大きくならないため、これ以上小さくする必要はない。
前記傾き角度θは、３０°〜６０°であることがより好ましい。
As for the inclination of the
When the inclination angle θ is larger than 80 °, the vortex flow becomes weak and there is a possibility that a region where cultured cells are not detached from the cell culture carrier 1 may be formed. On the other hand, when the inclination angle θ is less than 20 °, the arrangement of the flow path becomes difficult in design, and the flow velocity of the vortex does not increase so much, so it is not necessary to reduce it further.
The tilt angle θ is more preferably 30 ° to 60 °.
また、導入孔３１の数は、３〜４個であることが好ましい。
導入孔３１は、２個でも渦流が形成されるが、流速分布にむらが生じやすく、培養された細胞が細胞培養担体１から剥離されない領域ができるおそれがある。一方、導入孔３１が５個以上の場合、流速分布は均等になるが、導入孔３１の数の増加に伴って１個当たりの供給流量が減少し、渦流の流速が低下するため、培養された細胞を細胞培養担体１から剥離することができる流れが形成されないおそれがある。また、導入孔３１の径を小さくして、数を増加すると、緩衝液５が導入孔３１を流れる際の圧力損失が大きくなり、流速が低下し、また、加工コストも増大することとなる。
The number of
Even if two
蓋部材３の下面側における導入孔３１の出口の中心位置は、隙間４の半径Ｒに対して中心からの距離が０．７Ｒ以上の外周部であることが好ましい。
前記距離が０．７Ｒ未満とすると、導入孔３１の出口位置よりも外側における渦流の流速が小さすぎて、細胞培養担体１の外周部において培養された細胞を剥離するために十分な強さの流れが形成されないおそれがある。
前記距離は、０．８Ｒ〜０．９Ｒであることがより好ましい。
The center position of the outlet of the
If the distance is less than 0.7R, the flow velocity of the vortex outside the outlet position of the
The distance is more preferably 0.8R to 0.9R.
また、細胞培養担体１の直径が１０〜３０ｍｍ程度で、隙間４の半径Ｒが８〜２８ｍｍ程度の場合、導入孔３１の出口径は、０．３〜１．０ｍｍであることが好ましい。
出口径が０．３ｍｍ未満の場合、緩衝液６が導入孔３１を流れる際の圧力損失が大きく、供給流量が減少し、培養した細胞を細胞培養担体１から剥離することができるような強さの渦流が形成されにくい。一方、出口径が１．０ｍｍを超える場合、断面積の増加に伴って供給流速が低下し、この場合も、培養された細胞を細胞培養担体１から剥離するのに十分な強さの渦流が形成されないおそれがある。
なお、導入孔３１の孔径は、均一でもよいが、細胞培養担体１上の隙間４への出口径、すなわち、蓋部材３の下面側の孔径が小さくなるように先細状に形成すれば、緩衝液５が導入孔３１を流れる際の圧力損失を低減させることができる。
前記出口径は、０．４〜０．６ｍｍであることがより好ましい。
When the diameter of the cell culture carrier 1 is about 10 to 30 mm and the radius R of the
When the outlet diameter is less than 0.3 mm, the pressure loss when the buffer 6 flows through the
In addition, although the hole diameter of the
The outlet diameter is more preferably 0.4 to 0.6 mm.
なお、細胞培養担体１の下部には、細胞培養担体１に振動を与えたり、例えば、シーソーのような動きで傾きを変化させる機構が設けられていることが好ましい。
このように細胞培養担体１に動きを与えることにより、培養された細胞が細胞培養担体からより剥離しやすくなる。
In addition, it is preferable that a mechanism for applying vibration to the cell culture carrier 1 or changing the tilt by a motion such as a seesaw is provided below the cell culture carrier 1.
Thus, by giving a motion to the cell culture carrier 1, the cultured cells are more easily detached from the cell culture carrier.
緩衝液５は、培養する細胞の種類に応じて適宜調製されるが、通常、リン酸緩衝生理食塩水が用いられる。
The
細胞培養担体１の材質は、特に限定されるものではなく、プラスチックや、生体親和性及び生体適合性に優れ、細胞の足場として適しているとされるジルコニア、チタニア、アルミナ又はハイドロキシアパタイト等のセラミックス等を用いることができる。また、表面が多孔体からなり、三次元細胞塊を形成しやすく、また、細胞付着性が高い細胞培養担体であっても、上記の本発明に係る細胞培養モジュールによれば、培養された細胞を細胞培養担体から効率的かつ高い回収率で剥離することができる。 The material of the cell culture carrier 1 is not particularly limited, and plastics, ceramics such as zirconia, titania, alumina, or hydroxyapatite, which are excellent in biocompatibility and biocompatibility and are suitable as cell scaffolds. Etc. can be used. In addition, the cell culture module according to the present invention described above can be used even if the cell culture carrier according to the present invention is a cell culture carrier that has a porous surface and is easy to form a three-dimensional cell mass. Can be peeled from the cell culture carrier efficiently and with a high recovery rate.
特に、三次元細胞塊を効率的に凝集させるためには、細胞培養担体の裏面から培養液をウェル内に浸透可能である多孔体からなる細胞培養担体が好ましい。この場合の多孔体は、培養液等の浸透性や細胞培養担体自体の強度を保持の観点から、気孔率２０〜５０％、かつ、平均気孔径０．１５〜０．４５μｍであることが好ましい。なお、前記気孔率及び平均気孔径は、水銀ポロシメータを用いた水銀圧入法による測定値で表したものである。 In particular, in order to efficiently aggregate the three-dimensional cell mass, a cell culture carrier made of a porous body that can penetrate the culture solution into the well from the back surface of the cell culture carrier is preferable. In this case, the porous body preferably has a porosity of 20 to 50% and an average pore diameter of 0.15 to 0.45 μm from the viewpoint of maintaining the permeability of the culture solution and the strength of the cell culture carrier itself. . The porosity and average pore diameter are represented by values measured by a mercury intrusion method using a mercury porosimeter.
また、本発明に係る細胞培養方法は、上記の細胞培養モジュールを用いた細胞培養方法であって、表面に複数のウェルが設けられた円板状の多孔体からなる細胞培養担体を前記保持部材に載置し、前記細胞培養担体の上面外周部及び前記保持部材に蓋部材を密着固定した後、前記保持部材の細胞培養担体載置面に通じる供給流路から前記細胞培養担体の裏面側に第１の培養液を供給し、該細胞培養担体上面まで浸透させる第１の培養液の供給工程と、第１の培養液の供給を停止し、前記蓋部材の前記中央孔から前記細胞培養担体上面に未分化の細胞を含む第２の培養液を滴下し、細胞を播種する細胞播種工程と、前記供給流路から前記細胞培養担体の裏面側に第１の培養液を供給しながら、第１の培養液及び第２の培養液の培養液成分のみを前記中央孔から排出させ、前記ウェル内で細胞を凝集化させる細胞凝集化工程と、第１の培養液の供給を停止し、前記蓋部材の前記導入孔から緩衝液を供給し、凝集化した培養細胞を前記緩衝液とともに前記中央孔から排出させて回収する培養細胞回収工程とを経ることにより行われる。
このような工程を経ることにより、間葉系幹細胞やＥＳ細胞の凝集塊を効率的に形成し、かつ、培養された細胞の細胞培養担体からの剥離及び回収までを連続的に簡便に行うことができる。
The cell culturing method according to the present invention is a cell culturing method using the above cell culturing module, wherein the holding member is a cell culturing carrier comprising a disc-shaped porous body having a plurality of wells on the surface. After the lid member is closely fixed to the outer periphery of the upper surface of the cell culture carrier and the holding member, from the supply flow channel leading to the cell culture carrier placement surface of the holding member to the back side of the cell culture carrier Supplying the first culture solution to infiltrate the cell culture support to the upper surface thereof, stopping the supply of the first culture solution, and from the central hole of the lid member to the cell culture support Dropping a second culture solution containing undifferentiated cells on the upper surface and seeding the cells, while supplying the first culture solution from the supply channel to the back side of the cell culture carrier, Only the culture fluid components of the first culture fluid and the second culture fluid The cell aggregating step for discharging the cells from the central hole and aggregating the cells in the well and the supply of the first culture solution were stopped, and the buffer solution was supplied from the introduction hole of the lid member to be aggregated. This is carried out through a culture cell recovery step in which the cultured cells are discharged together with the buffer solution from the central hole and recovered.
Through these steps, agglomerates of mesenchymal stem cells and ES cells can be efficiently formed, and the cultured cells can be easily and continuously detached and collected from the cell culture carrier. Can do.
以下、上記細胞培養方法を各工程順に、図１を参照して詳細に説明する。
まず、第１の培養液の供給工程において、表面に複数のウェル１１が設けられた円板状の多孔体からなる細胞培養担体１を保持部材２に載置し、細胞培養担体１の上面外周部及び保持部材２に蓋部材３を密着固定する。そして、保持部材２の細胞培養担体載置面に通じる供給流路２３から細胞培養担体１の裏面側に第１の培養液を供給し、細胞培養担体上面まで第１の培養液を十分に浸透させる。
このように、細胞培養の前処理工程として、予め、細胞培養担体１全体に第１の培養液を十分に供給し、細胞培養担体上面まで第１の培養液に浸漬した状態としておく。
Hereinafter, the cell culture method will be described in detail with reference to FIG.
First, in the first culture solution supply step, the cell culture carrier 1 made of a disk-shaped porous body having a plurality of wells 11 provided on the surface is placed on the holding
As described above, as a pretreatment step for cell culture, the first culture solution is sufficiently supplied in advance to the entire cell culture carrier 1, and the upper surface of the cell culture carrier is immersed in the first culture solution.
細胞培養担体１は、培養の阻害要因となり得る気泡を含まない培養液を、複数のウェル１１全体に満遍なく供給する観点から、上述したような気孔率及び平均気孔径を備えた多孔体により構成することが好ましい。 The cell culture carrier 1 is composed of a porous body having a porosity and an average pore diameter as described above from the viewpoint of supplying a culture solution that does not contain bubbles, which can be an obstacle to culture, to the whole of the plurality of wells 11. It is preferable.
前記第１の培養液（培地）の種類は、特に限定されるものではないが、例えば、ＥＳ細胞を培養する場合は、ＤＭＥＭ、Ｆ１２、ＫＮＯＣＫＯＵＴ ＤＭＥＭ等が好ましい。さらに、これらの培地に、ＦＢＳ（ウシ血清）、非必須アミノ酸、ピルビン酸、グルタミン酸等の細胞を維持するのに必要な物質が添加される。
また、間葉系幹細胞を培養する場合には、例えば、ＭＥＭ、α−ＭＥＭ、ＤＭＥＭ、イーグル培地等が好ましい。さらに、これらの培地に、ＦＢＳ（ウシ血清）、グルタミン酸等の細胞を維持するのに必要な物質が添加される。
The type of the first culture medium (medium) is not particularly limited. For example, when culturing ES cells, DMEM, F12, KNOCKOUT DMEM, and the like are preferable. Furthermore, substances necessary for maintaining cells such as FBS (bovine serum), non-essential amino acids, pyruvate, and glutamate are added to these media.
Further, when culturing mesenchymal stem cells, for example, MEM, α-MEM, DMEM, Eagle medium, and the like are preferable. Furthermore, substances necessary for maintaining cells such as FBS (bovine serum) and glutamic acid are added to these media.
次に、細胞播種工程において、第１の培養液の供給を停止した後、蓋部材３の中央孔３２から細胞培養担体１上面に未分化の細胞を含む第２の培養液を滴下し、細胞を播種する。
このような滴下による播種においては、ウェル１１内外を問わず、細胞培養担体上面全体に細胞が播種されるが、細胞培養担体上面まで第１の培養液に浸漬した状態で、好ましくは４８時間以上静置することにより、ウェル外の細胞がウェル内に移動して付着し、ウェル内で凝集塊を形成させることができる。
すなわち、第１の培養液と、未分化の細胞を含む第２の培養液とを懸濁させた状態で未分化の細胞を細胞培養担体上面に播種することにより、負荷をかけることなく、播種した細胞をウェル内に導入することができ、スムーズな凝集化を促すことができる。
Next, in the cell seeding step, after the supply of the first culture solution is stopped, a second culture solution containing undifferentiated cells is dropped onto the upper surface of the cell culture carrier 1 from the
In such seeding by dripping, cells are seeded on the entire upper surface of the cell culture carrier regardless of inside or outside of the well 11, but preferably 48 hours or more in a state immersed in the first culture solution up to the upper surface of the cell culture carrier. By allowing the cells to stand, cells outside the well move into and adhere to the well, and aggregates can be formed in the well.
That is, by seeding undifferentiated cells on the upper surface of the cell culture carrier in a state where the first culture solution and the second culture solution containing undifferentiated cells are suspended, seeding is performed without applying a load. Cells can be introduced into the well, and smooth aggregation can be promoted.
前記第２の培養液（培地）の種類は、前記第１の培養液と同様のものでもよい。
また、培養する細胞の種類は、特に限定されるものではないが、本発明に係る培養方法は、特に、ＥＳ細胞や間葉系幹細胞に好適に適用することができる。ＥＳ細胞の場合は、１×１０4〜１×１０6個／ｃｍ2程度、また、間葉系幹細胞の場合は、１×１０4〜１×１０5個／ｃｍ2程度播種することが好ましい。
The type of the second culture solution (medium) may be the same as that of the first culture solution.
Further, the type of cells to be cultured is not particularly limited, but the culture method according to the present invention can be suitably applied particularly to ES cells and mesenchymal stem cells. In the case of ES cells, it is preferable to seed approximately 1 × 10 4 to 1 × 10 6 cells / cm 2 , and in the case of mesenchymal stem cells, it is preferable to seed approximately 1 × 10 4 to 1 × 10 5 cells / cm 2. .
次に、細胞凝集化工程において、供給流路２３から細胞培養担体１の裏面側に第１の培養液を供給しながら、第１の培養液及び第２の培養液の培養液成分のみを蓋部材３の中央孔３２から排出させ、ウェル内１１で細胞を凝集化させる。
このように培養液を流通させることにより、細胞培養担体１のウェル１１内の細胞は、常に第１の培養液の供給を受けることができるため、ウェル１１内での細胞の凝集化が効率的に行われる。
なお、第１の培養液の供給速度は、ウェル１１内で凝集化した細胞が剥離しないように、０．１ｍｌ／時間程度であることが好ましい。
Next, in the cell agglutination step, only the culture solution components of the first culture solution and the second culture solution are covered while supplying the first culture solution from the
By circulating the culture medium in this manner, the cells in the well 11 of the cell culture carrier 1 can always receive the supply of the first culture liquid, so that the aggregation of the cells in the well 11 is efficient. To be done.
Note that the supply rate of the first culture solution is preferably about 0.1 ml / hour so that cells aggregated in the well 11 are not detached.
そして、培養細胞回収工程において、第１の培養液の供給を停止した後、蓋部材３の導入孔３１から緩衝液を供給し、凝集化した培養細胞を緩衝液とともに中央孔３２から排出させて回収する。
このように緩衝液を供給することにより、上述したように、細胞培養担体１上の空隙４に渦流が生じ、この流れによってウェル１１内で凝集化した培養細胞を、細胞培養担体１から確実に剥離し、高回収率で回収することができる。また、細胞の培養から培養細胞の回収までを前記細胞培養モジュール内で、細胞培養担体１を取り外すことなく、連続的に行うことができるため、細胞培養の処理操作が簡便となる。
なお、緩衝液の供給速度は、空隙４内に渦流を効率よく発生する観点から、５〜１０ｍｌ／秒程度であることが好ましい。
In the cultured cell recovery step, after the supply of the first culture solution is stopped, the buffer solution is supplied from the
By supplying the buffer in this way, as described above, a vortex is generated in the
In addition, it is preferable that the supply speed | rate of a buffer solution is about 5-10 ml / second from a viewpoint of generating a vortex | eddy_current efficiently in the space |
以下、本発明を実施例に基づきさらに具体的に説明するが、本発明は下記の実施例により制限されるものではない。
［実施例１］
気孔率３５％のアルミナセラミックス多孔体からなり、ウェル径２５０μｍ、ウェル数１５００個の直径１６ｍｍの円板状の細胞培養担体のウェル内で、直径約２００μｍのマウスＥＳ細胞を三次元塊状に培養した。
この細胞培養担体を、図１に示すような構成からなる細胞培養モジュールにセットした。導入孔３１の数は３個、導入孔の孔径は出口０．６ｍｍ、入口２．０ｍｍ、傾き角度θ＝５０°、導入孔の出口の中心を、隙間４の半径Ｒ＝７ｍｍに対して中心からの距離が０．８Ｒ＝５．６ｍｍの位置とした。
EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example, this invention is not restrict | limited by the following Example.
[Example 1]
A mouse ES cell having a diameter of about 200 μm was cultured in a three-dimensional mass in a well of a disk-shaped cell culture carrier having a porosity of 35% and a well diameter of 250 μm and a number of wells of 1500 and 16 mm in diameter. .
This cell culture carrier was set in a cell culture module having a structure as shown in FIG. The number of introduction holes 31 is 3, the diameter of the introduction hole is 0.6 mm at the outlet, 2.0 mm at the inlet, the inclination angle θ = 50 °, and the center of the outlet of the introduction hole is centered with respect to the radius R of the
［実施例２〜５］
細胞培養モジュールを、下記表１に示すような導入孔の数、出口径、入口径、傾き角度θ、出口の中心位置とし、それ以外については、実施例１と同様にして、この細胞培養モジュールに三次元塊状の細胞を培養した細胞培養担体をセットした。
[Examples 2 to 5]
The cell culture module has the number of introduction holes, the outlet diameter, the inlet diameter, the inclination angle θ, and the center position of the outlet as shown in Table 1 below. A cell culture carrier on which three-dimensional massive cells were cultured was set.
［比較例１］
従来の細胞培養面の背面から水圧（液圧）をかけて、前記細胞培養担体から培養細胞を剥離する構成を備えた細胞培養モジュールに、実施例１と同様の三次元塊状の細胞を培養した細胞培養担体をセットした。
[Comparative Example 1]
The same three-dimensional massive cells as in Example 1 were cultured in a cell culture module having a configuration in which the cultured cells were peeled off from the cell culture carrier by applying water pressure (hydraulic pressure) from the back of the conventional cell culture surface. A cell culture carrier was set.
上記の各実施例及び比較例の細胞培養モジュールにおいて、緩衝液としてリン酸緩衝生理食塩水１０ｍｌを用い、圧力２ｋｇｆ／ｃｍ2で緩衝液を導入孔から供給し、培養細胞を細胞培養担体表面から剥離し、中央孔から排出した培養細胞を緩衝液とともに回収した。
この回収された培養細胞の数のウェル数に対する比率を回収率として求めた。
これらの結果を表１にまとめて示す。
In the cell culture module of each of the above Examples and Comparative Examples, 10 ml of phosphate buffered saline is used as a buffer solution, and the buffer solution is supplied from the introduction hole at a pressure of 2 kgf / cm 2 , and the cultured cells are removed from the surface of the cell culture carrier. The cultured cells that were detached and discharged from the central hole were collected together with the buffer.
The ratio of the number of cultured cells collected to the number of wells was determined as the recovery rate.
These results are summarized in Table 1.
表１に示したとおり、緩衝液を細胞培養担体表面側の所定の導入孔から渦流として供給する方式により（実施例１〜５）、ウェル内で培養した細胞を細胞培養担体表面から容易に剥離することができ、従来の培養細胞面の背面から水圧をかける方式（比較例１）に比べて回収率が向上することが認められた。特に、導入孔の数が３〜４個、出口径が０．４〜０．６ｍｍ、傾き角度θが３０°〜５０°、出口の中心位置が０．８Ｒ〜０．９Ｒの場合（実施例１，２）、回収率が高かった。 As shown in Table 1, by supplying a buffer solution as a vortex from a predetermined introduction hole on the cell culture carrier surface side (Examples 1 to 5), cells cultured in the well can be easily detached from the cell culture carrier surface. It was confirmed that the recovery rate was improved as compared with the conventional method (Comparative Example 1) in which water pressure was applied from the back of the cultured cell surface. In particular, when the number of introduction holes is 3 to 4, the exit diameter is 0.4 to 0.6 mm, the inclination angle θ is 30 ° to 50 °, and the center position of the exit is 0.8R to 0.9R (Example) 1, 2), the recovery rate was high.
１ 細胞培養担体
２ 保持部材
３ 蓋部材
４ 空隙
５ 緩衝液
１１ ウェル
２１，２２ Ｏリング
２３ 供給流路
３１ 導入孔
３２ 中央孔
DESCRIPTION OF SYMBOLS 1 Cell culture support |
Claims (3)
前記蓋部材の下面は、前記細胞培養担体の上面外周部及び前記保持部材に密着し、前記蓋部材下面と前記細胞培養担体表面との間に隙間が形成され、
前記蓋部材には、前記細胞培養担体上に緩衝液を供給するために均等に配置され、下向きに貫通する複数の導入孔と、前記細胞培養担体上の緩衝液を排出するために中央に配置された中央孔とが設けられ、
前記導入孔から供給された緩衝液が、前記隙間において渦流を生じると同時に、前記中央孔から排出されることを特徴とする細胞培養モジュール。 A holding member for mounting a disk-shaped cell culture carrier provided with a plurality of wells on the surface, and a lid member for covering the cell culture carrier mounting surface of the holding member;
The lower surface of the lid member is in close contact with the outer periphery of the upper surface of the cell culture carrier and the holding member, and a gap is formed between the lower surface of the lid member and the surface of the cell culture carrier,
The lid member is evenly arranged to supply a buffer solution on the cell culture carrier, a plurality of introduction holes penetrating downward, and a central member for discharging the buffer solution on the cell culture carrier A central hole is provided,
The cell culture module, wherein the buffer solution supplied from the introduction hole generates a vortex in the gap and is simultaneously discharged from the central hole.
表面に複数のウェルが設けられた円板状の多孔体からなる細胞培養担体を前記保持部材に載置し、前記細胞培養担体の上面外周部及び前記保持部材に蓋部材を密着固定した後、前記保持部材の細胞培養担体載置面に通じる供給流路から前記細胞培養担体の裏面側に第１の培養液を供給し、該細胞培養担体上面まで浸透させる第１の培養液の供給工程と、
第１の培養液の供給を停止し、前記蓋部材の前記中央孔から前記細胞培養担体上面に未分化の細胞を含む第２の培養液を滴下し、細胞を播種する細胞播種工程と、
前記供給流路から前記細胞培養担体の裏面側に第１の培養液を供給しながら、第１の培養液及び第２の培養液の培養液成分のみを前記中央孔から排出させ、前記ウェル内で細胞を凝集化させる細胞凝集化工程と、
第１の培養液の供給を停止し、前記蓋部材の前記導入孔から緩衝液を供給し、凝集化した培養細胞を前記緩衝液とともに前記中央孔から排出させて回収する培養細胞回収工程と
を備えていることを特徴とする細胞培養方法。 A cell culture method using the cell culture module according to claim 1 or 2,
After placing a cell culture carrier made of a disk-shaped porous body provided with a plurality of wells on the surface on the holding member, and fixing the lid member tightly to the outer periphery of the upper surface of the cell culture carrier and the holding member, A first culture solution supplying step of supplying a first culture solution from the supply channel leading to the cell culture carrier mounting surface of the holding member to the back surface side of the cell culture carrier, and allowing the first culture solution to permeate the upper surface of the cell culture carrier; ,
A cell seeding step of stopping the supply of the first culture liquid, dropping a second culture liquid containing undifferentiated cells on the upper surface of the cell culture carrier from the central hole of the lid member, and seeding the cells;
While supplying the first culture fluid from the supply channel to the back side of the cell culture carrier, only the culture fluid components of the first culture fluid and the second culture fluid are discharged from the central hole, A cell aggregation step of aggregating cells with,
A culture cell recovery step of stopping the supply of the first culture solution, supplying a buffer solution from the introduction hole of the lid member, and discharging the aggregated cultured cells together with the buffer solution from the central hole for recovery. A cell culture method comprising:
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