JPH04148676A - Perfusion culture - Google Patents
Perfusion cultureInfo
- Publication number
- JPH04148676A JPH04148676A JP27198790A JP27198790A JPH04148676A JP H04148676 A JPH04148676 A JP H04148676A JP 27198790 A JP27198790 A JP 27198790A JP 27198790 A JP27198790 A JP 27198790A JP H04148676 A JPH04148676 A JP H04148676A
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- Prior art keywords
- culture
- cell
- cells
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- 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.)
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Abstract
Description
【発明の詳細な説明】
産業上の利用分野
本発明はホロファイバーを利用して細胞を灌流培養する
新しい方法、より詳しくは細胞培養用媒質として高分子
栄養を供給でき、且つ培養細胞環境の経時的悪化をでき
るだけ防止して、長期間に亘る高密度細胞培養を可能と
し、更に目的生産物をより高収率で製造可能とする改良
された灌流培養方法に関する。DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention provides a new method for perfusion culture of cells using holofibers, more specifically, a method that can supply macromolecular nutrients as a cell culture medium and improve the aging of the cultured cell environment. The present invention relates to an improved perfusion culture method that enables high-density cell culture over a long period of time while preventing deterioration of the cell quality as much as possible, and also enables production of desired products at higher yields.
従来の技術
従来より、動物細胞、微生物細胞、植物細胞等の各種細
胞を高密度で大量に培養する方法の一つとしてホロファ
イバー(中空糸)を培養槽として利用した灌流培養方法
が知られている。Conventional technology The perfusion culture method, which uses a holofiber (hollow fiber) as a culture tank, has been known as one of the methods for culturing various types of cells such as animal cells, microbial cells, and plant cells at high density and in large quantities. There is.
この方法は、ホロファイバーの壁が半透膜として選択的
透過性を有することを利用して、適当なカラムに充填さ
れた多数本の束状ホロファイバー(キャピラリー)の外
表面上に細胞を設置し、該キャピラリーの内側空間(I
nner CapillarySpace・IC8)に
栄養素等の細胞培養用媒質を潅流させることによって、
上記栄養素等をキャピラリー壁を経て細胞に送り、逆に
細胞が存在するキャピラリー外側空間(Extra C
apillary 5pace。This method uses the selective permeability of the holofiber wall as a semipermeable membrane to place cells on the outer surface of a large number of bundled holofibers (capillaries) packed in an appropriate column. and the inner space of the capillary (I
By perfusing cell culture media such as nutrients into the inner Capillary Space IC8),
The above nutrients, etc. are sent to the cells through the capillary wall, and conversely, they are delivered to the outer space of the capillary where the cells exist (Extra C
apilary 5pace.
EC8)に産生される細胞生産物を固壁を経て上記IC
8を流れる潅流液中に流出させようとするものである。The cell products produced in EC8) are transferred to the above IC through a solid wall.
8 into the flowing perfusate.
上記方法は、細胞の生体外培養に適した微小環境を保持
し得、また細胞を三次元的に所謂有機体状構造に成長さ
せ得る点より、優れたものであるが、潅流液中に存在さ
せて細胞に供給すべき栄養素等は、上記キャピラリーの
半透膜を通過できる大きさ、即ち分画分子量以下に限定
され、従ってそれ以上の高分子物質を連続的に供給する
ことはできないという致命的欠点があった。The above method is superior in that it can maintain a microenvironment suitable for in vitro culture of cells and can grow cells three-dimensionally into a so-called organism-like structure. The nutrients, etc. that should be supplied to the cells are limited to a size that can pass through the semipermeable membrane of the capillary, that is, below the molecular weight cutoff, and therefore it is impossible to continuously supply macromolecular substances larger than that, which is a fatal problem. There were certain shortcomings.
また、上記方法では細胞が存在するEC8に、培養細胞
が産生じた老廃物や死細胞等が経時的に蓄積し、更に死
細胞の分解等により蛋白分解酵素等の細胞増殖阻止因子
や目的生産物の分解因子等も上記EC8に経時的に蓄積
され、之等が培養細胞環境を比較的速やかに悪化させる
と共に、培養細胞の高密度化を達成し得ない不利があり
、長期に亘る細胞培養を不可能としたり、目的生産物の
収率の向上をはかり得ない不利もあった。In addition, in the above method, waste products and dead cells produced by cultured cells accumulate over time in the EC8 where cells exist, and furthermore, due to the decomposition of dead cells, cell growth inhibitory factors such as proteases and the desired production Decomposition factors, etc. of substances accumulate over time in the EC8, which deteriorates the cultured cell environment relatively quickly, and has the disadvantage of not being able to achieve a high density of cultured cells, making it difficult to culture cells for a long period of time. There were also disadvantages in that it was impossible to improve the yield of the desired product.
発明が解決しようとする課題
本発明の目的は、上記従来のホロファイバーを用いた細
胞の灌流培養方法に見られる欠点を解消し、殊にホロフ
ァイバーの分画分子量よりも大きい分子量をもつ栄養素
やその他の細胞培養用試薬等を常時供給できると共に、
増殖細胞の産生ずる乳酸やアンモニウムイオン等の老廃
物は可及的速やかに細胞存在空間より排出させ、かくし
て新鮮な培養環境を常時維持して迅速且つ円滑な細胞の
増殖を行なわせ、最終到達細胞密度を向上させ、また細
胞増殖維持期間の長期化をはかり、ひいては目的生産物
の収量及び収率の向上を計ることにある。Problems to be Solved by the Invention The purpose of the present invention is to eliminate the drawbacks of the above-mentioned conventional cell perfusion culture method using holofibers, and in particular, to solve the problems of the above-mentioned methods for perfusion culture of cells using holofibers. In addition to being able to constantly supply other cell culture reagents,
Waste products such as lactic acid and ammonium ions produced by proliferating cells are discharged from the cell space as quickly as possible, thus constantly maintaining a fresh culture environment, allowing rapid and smooth cell proliferation, and ensuring that cells reach the final stage. The objective is to improve the density, prolong the cell growth maintenance period, and ultimately improve the yield and yield of the desired product.
課題を解決するための手段
本発明者らは上記目的より鋭意研究を重ねた結果、従来
のホロファイバー灌流培養方法において、細胞存在空間
(E CS)側より細胞非存在空間(IC8)側に培養
用媒質を流す方法を想到し、この方法によれば、上記目
的が悉く達成されることを見出し、ここに本発明を完成
するに至った。Means for Solving the Problems The present inventors have conducted intensive research for the above-mentioned purpose, and as a result, in the conventional holofiber perfusion culture method, culture is performed from the cell-existing space (ECS) side to the cell-free space (IC8) side. The present inventors have devised a method of flowing a medium for use, and have discovered that all of the above objects can be achieved by this method, thus completing the present invention.
即ち、本発明によればホロファイバーを利用して細胞を
灌流培養するに当り、該ホロファイバーの細胞存在空間
(E CS)側より細胞非存在空間(IC8)側へ細胞
培養用媒質を流すことを特徴とする灌流培養方法、並び
に灌流培養期間中の任意の時期に、細胞存在空間(E
CS)に蓄積される老廃物、死細胞、生細胞の増殖阻止
因子及び目的生産物の分解因子の少なくとも1種を、上
記EC3外に強制的に排出する上記灌流培養方法が提供
される。That is, according to the present invention, when perfusion culturing cells using a holofiber, a cell culture medium is allowed to flow from the cell presence space (ECS) side to the cell non-existence space (IC8) side of the holofiber. A perfusion culture method characterized by the following, and a cell existence space (E
The above-mentioned perfusion culture method is provided, in which at least one of waste products, dead cells, living cell growth-inhibiting factors, and target product decomposition factors accumulated in CS) is forcibly discharged from the EC3.
本発明方法において、用いられるホロファイバーとして
は、従来よりこの種細胞の灌流培養に用いられている各
種のものをいずれも使用できる。In the method of the present invention, any of the various types of holofibers conventionally used for perfusion culture of cells of this type can be used.
その例としては、例えばセルロース、セルロースエステ
ル類、ポリサルフォン、ポリアクリロニトリル、ポリメ
タクリル酸メチル、ポリアミド、ポリベンズイミダゾー
ル1、シリコーンポリカーボネート、ガラス等を素材と
して、溶融紡糸法、乾式紡糸法、湿式紡糸法等により製
造される中空糸を例示できる。之等は通常直径1肛以下
の毛管で、中空糸の壁が半透膜として機能する性質を共
通して有している。上記の内でも特にダウ・ケミカル社
製の酢酸セルロース膜(内径:180〜200μm1外
径=230〜250μm1分画分子量:約30000)
やアミコン社製のポリサルフォン膜(内径:約200
μm、外径:約350μm。Examples include melt spinning, dry spinning, wet spinning, etc. using cellulose, cellulose esters, polysulfone, polyacrylonitrile, polymethyl methacrylate, polyamide, polybenzimidazole 1, silicone polycarbonate, glass, etc. An example is a hollow fiber manufactured by. These are usually capillaries with a diameter of less than 1 annular, and they have in common the property that the hollow fiber wall functions as a semipermeable membrane. Among the above, cellulose acetate membrane made by Dow Chemical Company (inner diameter: 180-200 μm 1 outer diameter = 230-250 μm 1 molecular weight cut-off: approximately 30,000)
Polysulfone membrane manufactured by Amicon (inner diameter: approx. 200 mm)
μm, outer diameter: approximately 350 μm.
分画分子量:約10000.約30000、約5000
0)は好適である。また上記ホロファイバーは、これを
コラーゲン、フィブロネクチン(F N)等の細胞保持
に適した基質で被覆したものであってもよく、これは特
に接着依存性細胞の培養に適している。Molecular weight cutoff: approx. 10,000. Approximately 30,000, approximately 5,000
0) is preferred. Further, the above-mentioned holofiber may be coated with a substrate suitable for cell retention such as collagen or fibronectin (FN), and this is particularly suitable for culturing adhesion-dependent cells.
本発明方法は、基本的には以下の如くして実施される。The method of the present invention is basically carried out as follows.
即ち、上記ホロファイバーの複数本を束にして適当なカ
ラムに充填して細胞培養槽を形成させ、まず上記カラム
内のホロファイバー外表面上に培養用細胞を設置すると
共にこの細胞存在空間(E CS)に細胞培養用媒質を
導入させる。該媒質は上記細胞存在空間(E CS)に
て細胞に連続的に供給されると共に、ホロファイバーの
壁を通過して内部空間(細胞非存在空間、IC8)に至
り、該■C8を経てカラム外へ連続的に排出される。乳
酸やアンモニウムイオン等、更にプロテアーゼ等の細胞
生成物は、上記媒質の排出に伴われてホロファイバー壁
を通過し細胞非存在空間(IC8)を経て、カラム外に
排出される。尚、本発明方法は上記と逆の構成、即ちホ
ロファイバーの内部空間に細胞を存在させてここに細胞
培養用媒質を連続的に供給し、ホロファイバー壁を経て
外部空間に排出させる構成を採ることもできる。That is, a plurality of the above-mentioned holofibers are bundled and packed into a suitable column to form a cell culture tank, and first, cells for culture are placed on the outer surface of the holofibers in the above-mentioned column, and this cell existence space (E CS) is introduced with cell culture medium. The medium is continuously supplied to the cells in the cell-existing space (ECS), passes through the wall of the holofiber, reaches the internal space (cell-free space, IC8), and passes through C8 to the column. Continuously discharged outside. Cell products such as lactic acid, ammonium ions, and protease are discharged from the column through the hollow fiber wall and the cell-free space (IC8) as the medium is discharged. The method of the present invention adopts a configuration opposite to the above, that is, a configuration in which cells are allowed to exist in the internal space of the holofiber, a cell culture medium is continuously supplied thereto, and the medium is discharged to the external space through the holofiber wall. You can also do that.
上記本発明方法において用いられる、細胞培養用媒質と
しては、培養すべき細胞の種類に応じて適宜決定でき、
之等はいずれも細胞培養に要求される各種の栄養素を含
むものであればよく、その具体例としては、例えばME
M培地、DMEM培地、RPMI−1640培地、F−
12培地、NCTC−109培地、ウェイマウス
(Waymouth’s) M B培地、ウィリアムス
(William’s ) E培地等を例示テキル。The cell culture medium used in the above method of the present invention can be determined as appropriate depending on the type of cells to be cultured.
Any of these may contain the various nutrients required for cell culture, and specific examples include ME.
M medium, DMEM medium, RPMI-1640 medium, F-
12 medium, NCTC-109 medium, Waymouth's MB medium, Williams' E medium, and the like.
殊に本発明方法は、上記の構成、即ち細胞培養用媒質を
細胞存在空間(E CS)よりホロファイバー壁を経て
細胞非存在空間(IC8)に潅流させる構成を採用した
ことに基づいて、特に細胞培養用媒質として高分子物質
をも利用でき、之等を常に連続的に細胞に供給させ得る
利点がある。該高分子物質としては、使用するホロファ
イバーの分画分子量よりも分子量の大きい物質、例えば
トランスフェリン(分画分子量二80000)、血清ア
ルブミン(同:69000)、α−サイクロデキストリ
ン(α−CD、血清グロブリンのCohnの分画■(ト
ランスフェリン、セルロプラスミン、ハプトグロブリン
等の複合体)、肝細胞成長因子(HGF、同10300
0)、血小板由来成長因子(PDGF、同30000)
、神経成長因子(NGF、26000)、フィブロネク
チン(F N、同220000〜250000)、イン
ターロイキン2 (IL−2、同13000〜1400
0)、顆粒球コロニー刺激因子(G−C3F、同300
00)、マクロファージコロニー刺激因子(M−C8F
、同60000〜85000)、顆粒球・マクロファー
ジコロニ刺激因子(GM−C8F、同20000)等を
例示できる。In particular, the method of the present invention is based on the above configuration, that is, the configuration in which the cell culture medium is perfused from the cell-existing space (ECS) through the holofiber wall into the cell-free space (IC8). Polymeric substances can also be used as a cell culture medium, which has the advantage of being constantly supplied to the cells. The polymeric substances include substances with a molecular weight larger than the molecular weight cutoff of the hollow fiber used, such as transferrin (molecular weight cutoff: 280,000), serum albumin (molecular weight cutoff: 69,000), α-cyclodextrin (α-CD, serum Cohn's fraction of globulin (complex of transferrin, ceruloplasmin, haptoglobulin, etc.), hepatocyte growth factor (HGF, 10300
0), platelet-derived growth factor (PDGF, 30,000)
, nerve growth factor (NGF, 26,000), fibronectin (FN, 220,000 to 250,000), interleukin 2 (IL-2, 13,000 to 1,400)
0), granulocyte colony stimulating factor (G-C3F, 300
00), macrophage colony stimulating factor (M-C8F
, 60,000 to 85,000), and granulocyte/macrophage colony stimulating factor (GM-C8F, 20,000).
之等の高分子物質の本発明方法への利用は、例えば次の
如き利点を有する。即ち、トランスフェリンは殆どの動
物培養細胞の生存、増殖、分化に不可欠であり、栄養物
質としての鉄を1分子当り2原子結合(K8.=10
〜IOM)L、更にリセプタ−(T f R)との結合
を介して細胞に鉄を効率よく与える働きがある。血清ア
ルブミンは培養細胞に対して、必須脂肪酸である長鎖不
飽和脂肪酸(オレイン酸、リノール酸等)の担体として
脂肪酸の細胞への供給を制御して、細胞の増殖に促進的
に働く。r L−2は、該IL−2に対するレセプター
を細胞膜表面に有する各種の細胞、例えばNK細胞、T
細胞性白血病細胞、T細胞等の増殖を誘導する。HG
Fはインシュリン及びEGF (表皮細胞増殖因子)の
共存下で成熟肝細胞の増殖を促進する。The use of such polymeric substances in the method of the present invention has the following advantages, for example. That is, transferrin is essential for the survival, proliferation, and differentiation of most cultured animal cells, and it binds iron as a nutritional substance to two atoms per molecule (K8.=10
-IOM)L, and also has the function of efficiently providing iron to cells through binding with receptors (T f R). Serum albumin serves as a carrier for long-chain unsaturated fatty acids (oleic acid, linoleic acid, etc.) that are essential fatty acids for cultured cells, and controls the supply of fatty acids to the cells, thereby promoting cell proliferation. rL-2 can be used in various cells that have receptors for IL-2 on the cell membrane surface, such as NK cells and T cells.
Induces proliferation of cellular leukemia cells, T cells, etc. H.G.
F promotes proliferation of mature hepatocytes in the presence of insulin and EGF (epidermal growth factor).
上記本発明方法により培養される細胞としては、特に限
定されるものではないが、例えば代表的にはヒト繊維芽
細胞、平滑筋細胞、グリア細胞、3T3細胞、NK細胞
、T細胞等を例示できる。The cells to be cultured by the method of the present invention are not particularly limited, but typical examples include human fibroblasts, smooth muscle cells, glial cells, 3T3 cells, NK cells, and T cells. .
以下、本発明方法を添付図面を用いて説明する。The method of the present invention will be explained below with reference to the accompanying drawings.
第1図乃至第3図は本発明方法の実施に適した装置の概
略図であり、第1図は循環培養系(C1osed Cu
1ture)を、第2図はIC8循環培養+新鮮培地供
給系(IC5Overflow Cu1ture)を、
第3図はIC8及びEC8循環培養+新鮮培地供給+培
養産物回収系(ICS & EC5OverflowC
ulture )をそれぞれ示す。1 to 3 are schematic diagrams of an apparatus suitable for carrying out the method of the present invention, and FIG. 1 shows a circulating culture system (C1osed Cu
Figure 2 shows the IC8 circulation culture + fresh medium supply system (IC5Overflow Culture).
Figure 3 shows IC8 and EC8 circulating culture + fresh medium supply + culture product recovery system (ICS & EC5 OverflowC
culture) are shown respectively.
各図において(1)は循環ポンプを、(2)はガス交換
器を、(3)はDoセンサ(導入側)を、(4)はDo
センサ(排出側)を、(5)はpHセンサ(導入側)を
、(6)はpHセンサ(排出側)を、(7)はリザーブ
タンクを、(8)は新鮮培養タンクを、(9)は消費培
養液タンクを、(10)は生産物回収タンクを、(11
)はホロファイバー培養槽(カラム)をそれぞれ示す。In each figure, (1) shows the circulation pump, (2) shows the gas exchanger, (3) shows the Do sensor (introduction side), and (4) shows the Do sensor.
Sensor (discharge side), (5) pH sensor (introduction side), (6) pH sensor (discharge side), (7) reserve tank, (8) fresh culture tank, (9) ) is the spent culture medium tank, (10) is the product recovery tank, (11
) indicates the holofiber culture tank (column), respectively.
上記図に示した装置の利用による本発明方法における灌
流培養の条件、培養方法、培養環境の制御等は、利用す
る細胞等に応じて適宜決定でき、特に限定されるもので
はないが、例えば後記する実施例に示した抗体産生細胞
の培養を例にとり詳述すれば以下の如くである。The perfusion culture conditions, culture method, control of culture environment, etc. in the method of the present invention using the apparatus shown in the above figure can be determined as appropriate depending on the cells to be used, etc., and are not particularly limited, but for example, as described below. The following is a detailed description of the culture of antibody-producing cells shown in Examples.
即ち、培養温度としては一般に37℃を採用でき、これ
は培養期間中一定とすることができる。That is, the culture temperature can generally be 37°C, which can be kept constant during the culture period.
培養循環流量は約2011/分[培養初期からり。The culture circulation flow rate is approximately 2011/min [from the initial stage of culture].
(導入側) −DO(排出側)>50111mHgとな
るまでの間]とすればよく、DO(導入側) −D。(introduction side) - until DO (discharge side) > 50111 mHg], and DO (introduction side) -D.
(排出側)>50mmHgとなる度に循環流量を10x
l!/分ずつ増加させるのが望ましい。培地交換は1時
間毎に上記第1図に示す循環培養系(C1osed C
u1ture)と第2図に示すIC3循環培養+新鮮培
地供給系(IC5Overflow Cu1ture)
とを交互に行ない、該IC8循環培養+新鮮培地供給系
において培地交換量を順次増大させるのが望ましい。p
H制御は、PIDコントローラーの設定値をpH=7.
2として該コントローラーにて制御するのがよい。尚、
培養中期においてガス交換器への送気CO2量(PID
コントローラーによる演算値)が0(ゼロ)になってか
ら後は、細胞の成育並びに物質代謝の危険水域であるp
)l=6.8以下にはならないように維持するが、培地
中の積算NaHCO3量が基準培地の3倍濃度になるよ
うであれば、NaHCOs添加によるpH維持をやめて
、−時的にポンプ循環流量を上げて全培養液を交換する
のが望ましい。また、Do制御はPIDコントローラー
の設定値をDO=150mmHgとし、該コントローラ
ーにて制御し、培地循環流量の変更によりDo(排出側
)〉100mmHgを維持させるのがよい。(Discharge side) Every time >50mmHg, increase the circulation flow rate by 10x.
l! It is preferable to increase the time by /minute. The medium was replaced every hour using the circulating culture system (C1osed C) shown in Figure 1 above.
IC3 circulation culture + fresh medium supply system (IC5Overflow Culture) shown in Figure 2
It is desirable to alternately perform these steps, and gradually increase the amount of medium exchanged in the IC8 circulation culture + fresh medium supply system. p
For H control, the set value of the PID controller is set to pH=7.
2, preferably controlled by the controller. still,
During the middle stage of culture, the amount of CO2 supplied to the gas exchanger (PID
After the value calculated by the controller) reaches 0, there is a dangerous zone for cell growth and material metabolism.
)L = 6.8 or less, but if the cumulative amount of NaHCO3 in the medium becomes 3 times the concentration of the reference medium, stop maintaining the pH by adding NaHCOs and periodically pump circulation. It is desirable to increase the flow rate and exchange the entire culture medium. Further, Do control is preferably performed by setting the set value of the PID controller to DO=150 mmHg, and maintaining Do (discharge side) > 100 mmHg by changing the culture medium circulation flow rate.
上記温度制御は潅流回路全体をHEPAフィルターを使
用したクリーンな循環恒温槽中に置くことによって回路
中の培養液温度が一定に維持され得る。The above-mentioned temperature control can maintain a constant temperature of the culture solution in the circuit by placing the entire perfusion circuit in a clean circulation constant temperature bath using a HEPA filter.
上記pH制御はpHセンサー(導入側)の検出器として
ホロファイバー型のガス交換器に通気するN2 02
CO2の3種混合ガス中のCO2組成比率を専用のP
IDコントローラーによって逐次決定送気することによ
り実施できる。更に回路中の新鮮培養タンク(8)を、
複数個並列に接続して、その少なくとも一つよりpH緩
衝液、例えば培地の500倍濃のNaHCO3−PBS
溶液を、pHが随時添加することで培養の進行に伴われ
るpHの低下を防ぐことができる。The above pH control uses N2 02 vented to a hollow fiber type gas exchanger as a detector for the pH sensor (introduction side).
The CO2 composition ratio in the three types of CO2 mixed gas is measured using a dedicated P.
This can be carried out by sequentially determining and supplying air using an ID controller. Furthermore, the fresh culture tank (8) in the circuit,
A plurality of them are connected in parallel, and at least one of them is supplied with a pH buffer, for example, NaHCO3-PBS which is 500 times more concentrated than the medium.
By adding the solution whenever the pH value changes, it is possible to prevent the pH from decreasing as the culture progresses.
上記Do制御はDo(導入側)の検出器としてホロファ
イバー型のガス交換器に通気するN2−O2CO2の3
種混合ガス中の02組成比率を専用のPIDコントロー
ラーによって逐次決定送気することにより実施できる。The above Do control uses 3 of the N2-O2CO2 vented to the holofiber type gas exchanger as a Do (introduction side) detector.
This can be carried out by sequentially determining the 02 composition ratio in the seed mixed gas using a dedicated PID controller and supplying air.
また培地循環流量の制御は培地循環ポンプ(これはべり
スタリックポンプやシリンジポンプやベローズポンプ等
であってもよい)の駆動モーターの回転をコントロール
することによって変化させ得る。Further, the control of the medium circulation flow rate can be changed by controlling the rotation of the drive motor of the medium circulation pump (which may be a besteric pump, a syringe pump, a bellows pump, etc.).
上記各図に示した装置の利用による本発明方法につき更
に詳述すれば、各図に示す装置は、ホロファイバーの外
側空間を細胞存在空間とし、該空間へ培養中連続的に培
養用媒質を供給する回路と、上記空間から供給された培
養用媒質をホロファイバーの内側空間へ連続的に排出し
続けるための回路とを主構成回路としている。To explain in more detail the method of the present invention using the apparatus shown in each of the above figures, the apparatus shown in each figure uses the outer space of the holofiber as a space for cells to exist, and the culture medium is continuously fed into this space during culture. The main constituent circuits are a supply circuit and a circuit for continuously discharging the culture medium supplied from the space into the inner space of the holofiber.
また本発明方法の実施に適した装置は、第2図に示すよ
うに、新鮮な培養用媒質を貯蔵したタンク(8)より、
上記主構成回路に新鮮な培養用媒質を供給し且つ老廃物
を含む使用済みの媒質を上記主回路からリザーブタンク
(7)を経て消費培養液タンク(9)に排出する副構成
回路(以下「回路構成(1)」という)を含んでいても
よく、更に第3図に示すように、上記主回路における細
胞存在空間から細胞非存在空間に至る培養用媒質の主要
な流れ以外に、培養の進行につれて細胞存在空間中に蓄
積されるおそれのある老廃物や死細胞、更に分解死細胞
より漏出される蛋白質分解酵素(プロテアーゼ等)の生
細胞の増殖阻害因子や目的産生物の分解因子等を定期的
にカラム外へ強制排出するための副構成回路(以下これ
を「回路構成(3)」という)を供えていてもよい。Further, as shown in FIG. 2, an apparatus suitable for carrying out the method of the present invention is such that a tank (8) containing fresh culture medium is
A sub-component circuit (hereinafter referred to as " Further, as shown in Figure 3, in addition to the main flow of the culture medium from the cell-existing space to the cell-free space in the main circuit, Waste products and dead cells that may accumulate in the cell space as the cell progresses, as well as proteolytic enzymes (proteases, etc.) leaked from decomposed dead cells, growth inhibitory factors for living cells, and decomposition factors for target products, etc. A sub-configuration circuit (hereinafter referred to as "circuit configuration (3)") for periodically forcibly discharging to the outside of the column may be provided.
上記各図に示す装置を用いた本発明方法は、例えばまず
予め培養回路の構成部品を、高圧蒸気滅菌(オートクレ
ーブ使用)、エチレンオキサイドガス滅菌、放射線滅菌
等の適当な滅菌操作により滅菌した後、クリーンベンチ
等の無菌環境下に回路全体を組み立て、以下の培養潅流
回路の初期化を行ない、次に細胞の播種及び培養を実施
する。In the method of the present invention using the apparatus shown in each of the figures above, for example, first, the components of the culture circuit are sterilized in advance by an appropriate sterilization operation such as high pressure steam sterilization (using an autoclave), ethylene oxide gas sterilization, radiation sterilization, etc. The entire circuit is assembled in a sterile environment such as a clean bench, the culture perfusion circuit is initialized as described below, and then cells are seeded and cultured.
上記初期化は、例えば上記回路を37℃に維持された恒
温槽(該恒温槽はより好ましくはHEPAフィルターを
使用した通風循環システムを伴うクリーン恒温槽である
のがよい)内に設置し、10%FBS (牛胎児血清)
を含むRPMI−1640培地等の適当な培地を培養潅
流回路中に循環させることにより実施できる。上記にお
いてガス交換器には、酸素ガス、炭酸ガス、窒素ガスの
3種のガスを供給できる。酸素ガス供給量はDo調節計
により、炭酸ガスはpH調節計により、それぞれPID
制御出力により、それぞれ酸素ガス専用マスフロー調節
計、炭酸ガス専用マスフロー調節計で調節でき、また窒
素ガス供給量はコンピューターによる演算処理により、
全ガス供給量が所定の一定値になるよう決定され、窒素
ガス専用マスフロー調節計により調節できる。かくして
、pHを7,2、Do(導入側)を150mmHgに維
持するようにガス供給を制御して48時間培養液を潅流
させて、初期化を完了する。The above initialization can be carried out, for example, by installing the circuit in a constant temperature bath maintained at 37°C (the constant temperature bath is preferably a clean constant temperature bath with a ventilation circulation system using a HEPA filter), and %FBS (fetal bovine serum)
This can be carried out by circulating a suitable medium such as RPMI-1640 medium containing . In the above, three types of gases, oxygen gas, carbon dioxide gas, and nitrogen gas, can be supplied to the gas exchanger. The amount of oxygen gas supplied is determined by a Do controller, and the amount of carbon dioxide is determined by a pH controller.
Depending on the control output, the mass flow controller for oxygen gas and mass flow controller for carbon dioxide gas can be adjusted, and the nitrogen gas supply amount can be adjusted by computer processing.
The total gas supply amount is determined to be a predetermined constant value, and can be adjusted by a mass flow controller dedicated to nitrogen gas. In this way, the gas supply is controlled to maintain the pH at 7.2 and the Do (introduction side) at 150 mmHg, and the culture solution is perfused for 48 hours to complete the initialization.
上記初期化に引き続(細胞の播種は、例えば所定の細胞
密度、RPMI−8226株の場合は好ましくは4X1
06個/ 7A’程度、に調節した細胞浮遊液5011
を針なしシリンジに取り、空になっているホロファイバ
ーの外側空間(もしくは内側空間)にポートより注入す
ることにより実施でき、これによりカラム当り約lX1
0B個のRPMI−8226を細胞を播種させるのが適
当である。Following the above initialization (cell seeding is carried out, for example, at a predetermined cell density, preferably 4X1 in the case of the RPMI-8226 strain)
Cell suspension 5011 adjusted to approximately 06 cells/7A'
This can be carried out by taking a needleless syringe and injecting it into the empty outer space (or inner space) of the hollow fiber through the port.
It is appropriate to seed the cells with 0B of RPMI-8226.
更に上記で播種された細胞の培養は、例えば約1時間毎
に数回ホロファイバーの向きを変えてカラム内で細胞が
極在しないように調節した後、数時間培養液の循環速度
を111/分で培養液を前記主構成回路に循環させ、D
o(導入側)とり。Furthermore, to culture the cells seeded above, for example, after changing the orientation of the holofiber several times approximately every hour to adjust the cells so that they are not localized within the column, the circulation rate of the culture medium is adjusted to 111/1/2 for several hours. The culture solution is circulated through the main component circuit for 1 minute, and D
o (introduction side).
(排出側)の差が播種時の差に比べて減少しないようで
あれば、培養用媒質の循環速度を20zl!/分に上昇
させる。リザーブタンク(7)の容量を約21とし、培
養開始48時間後より、第1図に示す循環培養系(C1
osed Cu1ture)と第2図ニ示すIC3循環
培養+新鮮培地供給系(IC5Overflow Cu
1ture)とを交互に切り替工且つ時間比率を調整す
ることによって、培養1週間まで、500zl/日の交
換比率で、消費培養用媒質を新鮮培養液と連続的に交換
しながら培養を続ける。If the difference (on the discharge side) does not seem to decrease compared to the difference at the time of seeding, increase the circulation speed of the culture medium by 20zl! /min. The capacity of the reserve tank (7) was set to about 21, and from 48 hours after the start of culture, the circulating culture system (C1
osed culture) and the IC3 circulation culture + fresh medium supply system (IC5Overflow Cu) shown in Figure 2D.
The culture is continued by continuously exchanging the spent culture medium with fresh culture medium at an exchange rate of 500 zl/day for up to one week of culture by alternating between 1 ture and 500 zl/day and adjusting the time ratio.
播種7日より培養用媒質の交換比率を1000xi1日
に上昇させると共に、2日毎に1回副構成回路2の機能
を使用して、細胞非存在空間側の培養液を10Ilずつ
回収する。pH値が6.8以下になると自動的にp)(
緩衝液(5%NaHCO3含有リン酸緩衝溶液(PBS
)=培養液の50倍濃度溶液を、第2図に示す構成回路
(1)以外の部分(以下これを「構成回路(2)」とい
う)を゛使用して、培養潅流回路中に1回当り6xl添
加する。但しこの添加操作は6時間以内に2回以上行な
ってはならないように自動的にセットする。From the 7th day of seeding, the exchange rate of the culture medium is increased to 1000 x 1 day, and the function of the sub-component circuit 2 is used to collect 10 Il of the culture medium in the cell-free space once every 2 days. When the pH value becomes 6.8 or less, p)(
Buffer solution (phosphate buffer solution containing 5% NaHCO3 (PBS)
) = A 50 times concentrated solution of the culture solution was added once during the culture perfusion circuit using a part other than the component circuit (1) shown in Figure 2 (hereinafter referred to as "the component circuit (2)"). Add 6xl per serving. However, this addition operation is automatically set so that it cannot be performed more than once within 6 hours.
添加されたpH緩衝液が潅流回路中で充分均一に拡散さ
れるまでに時間を必要とするので、pH緩衝液が添加さ
れてから2時間以降で且つ6時間以内に、灌流培養液の
pH値が6.8より低下するようであれば、改めて新鮮
培養液を1000//2時間の高速で消費培養液と交換
する。この時構成回路(2)を使用する。Since it takes time for the added pH buffer to be sufficiently and uniformly diffused in the perfusion circuit, the pH value of the perfused culture solution should be adjusted after 2 hours and within 6 hours after the addition of the pH buffer. If it seems to be lower than 6.8, replace the spent culture medium with fresh culture medium again at a high speed of 1000//2 hours. At this time, the configuration circuit (2) is used.
次いで1〜2日に1回、構成回路(2)を使用して、2
xlの灌流培養液を回収し、グルコース濃度を測定し、
50■/l以下の場合は、上記と同様の培養液交換を実
施する。Then once every 1 to 2 days, using component circuit (2),
xl perfusion culture was collected, the glucose concentration was measured,
If it is less than 50 μ/l, replace the culture medium in the same way as above.
培養14日1より、新鮮培養液と消費培養液との交換比
率を2000z//日に上昇させると共に、EC8側の
培養液を2日毎に2511づつ回収する。From day 1 on day 14 of culture, the exchange ratio between fresh culture solution and spent culture solution was increased to 2000z/day, and 2511 culture solutions on the EC8 side were collected every 2 days.
かくして、本発明方法によれば、ホロファイバーの分画
分子量より大きい分子量をもつ高分子物質を常時培養細
胞に供給でき、増殖細胞によって産生された老廃物を培
養期間中常時強制排出でき、新鮮な培養環境を常時維持
でき、それ故、細胞培養維持期間を延長して、最大到達
細胞密度を向上させ得ると共に、長期間に亘って、高収
率で目的産生物の生産を可能とする。Thus, according to the method of the present invention, it is possible to constantly supply cultured cells with a polymeric substance having a molecular weight larger than the molecular weight cut-off of the holofiber, and to forcefully discharge waste products produced by proliferating cells at all times during the culture period. The culture environment can be maintained at all times, therefore, the cell culture maintenance period can be extended, the maximum cell density can be improved, and the target product can be produced at high yield over a long period of time.
実 施 例
以下、本発明を更に詳しく説明するため実施例を挙げる
。EXAMPLES Examples will be given below to explain the present invention in more detail.
実施例 1
この例は下記ホロファイバー培養槽利用の培養装置(添
付図面参照)を用い、以下の基本条件下に、次の通り実
施された。Example 1 This example was carried out as follows under the following basic conditions using a culture apparatus using the following holofiber culture tank (see attached drawings).
(1)対象細胞:RPMr−8226細胞(モノクロー
ナル抗体産生株)
(2) 目的産生物質:モノクローナル抗体(IgG
L−λ鎖)
(3)使用培養液:RPMI−1640培地十MEM培
地(1: 1) +
10%FBS
(4)検出法:IgGL−λ産生量の検出は、EIA法
による。(1) Target cells: RPMr-8226 cells (monoclonal antibody producing strain) (2) Target production substance: monoclonal antibody (IgG
(L-λ chain) (3) Culture solution used: RPMI-1640 medium + MEM medium (1:1) + 10% FBS (4) Detection method: IgG-λ production amount was detected by EIA method.
(5)ホロファイバー培養槽:
アミコン社製ヴイタファイバー■
(分画分子量=30000)ホロ
ファイバー(ポリサルフォン製)
使用
(6)播種細胞数:lX10B細胞
(播種密度=4 X 106細胞/I)(7)培養日数
=34日
(8)細胞存在側空間体積: 25cm3(xi)(9
)最終到達細胞数:5X109細胞(10) 生産物
量:430■(rgG L−λ)即ち、予め培養回路
の構成部品を、高圧蒸気滅菌(オートクレーブ使用)、
エチレンオキサイドガス滅菌、放射線滅菌等の適当な滅
菌操作により滅菌した後、クリーンベンチ等の無菌環境
下に回路全体を組み立て、以下の培養潅流回路の初期化
を行ない、次に細胞の播種及び培養を実施した。(5) Holofiber culture tank: Vitafiber made by Amicon (molecular weight cutoff = 30000) Holofiber (made of polysulfone) used (6) Number of seeded cells: 1 x 10B cells (seeding density = 4 x 106 cells/I) ( 7) Number of culture days = 34 days (8) Volume of space on cell side: 25 cm3 (xi) (9
) Final number of cells reached: 5 x 109 cells (10) Product amount: 430 ■ (rgG L-λ) In other words, the components of the culture circuit were sterilized with high pressure steam (using an autoclave),
After sterilization using an appropriate sterilization operation such as ethylene oxide gas sterilization or radiation sterilization, assemble the entire circuit in a sterile environment such as a clean bench, initialize the culture perfusion circuit as described below, and then seed and culture the cells. carried out.
上記初期化は、例えば上記回路を37℃に維持された恒
温槽(該恒温槽は好ましくはHEPAフィルターを使用
した通風循環システムを伴うクリーン恒温槽であるのが
よい)内に設置し、10%FBS (牛胎児血清)を含
むRPMI−1640+MEM (1: 1)培地を、
培養潅流回路中に循環させることにより実施した。上記
においてガス交換器には、酸素ガス、炭酸ガス、窒素ガ
スの3種のガスを供給し、酸素ガス供給量はDo調節計
により、炭酸ガスはpH調節計により、それぞれPID
制御出力により、それぞれ酸素ガス専用マスフロー調節
計、炭酸ガス専用マスフロー調節計で調節でき、また窒
素ガス供給量はコンピューターによる演算処理により、
全ガス供給量が所定の一定値になるように決定し、窒素
ガス専用マスフロー調節計により調節した。かくして、
pHを7.2、Do(導入側)を150mmHgに維持
するようにガス供給を制御して48時間培養液を潅流さ
せ、初期化を完了した。The above initialization can be performed, for example, by installing the circuit in a constant temperature bath maintained at 37°C (the constant temperature bath is preferably a clean constant temperature bath with a ventilation circulation system using a HEPA filter), and RPMI-1640+MEM (1:1) medium containing FBS (fetal bovine serum)
This was performed by circulating in a culture perfusion circuit. In the above, three types of gases, oxygen gas, carbon dioxide gas, and nitrogen gas, are supplied to the gas exchanger.
Depending on the control output, the mass flow controller for oxygen gas and mass flow controller for carbon dioxide gas can be adjusted, and the nitrogen gas supply amount can be adjusted by computer processing.
The total gas supply amount was determined to be a predetermined constant value, and adjusted using a mass flow controller exclusively for nitrogen gas. Thus,
Gas supply was controlled to maintain pH at 7.2 and Do (introduction side) at 150 mmHg, and culture solution was perfused for 48 hours to complete initialization.
上記に引き続く細胞の播種は、4X106個/11程度
に調節したRPMI−8226株の細胞浮遊液5011
を、針なしシリンジに取り、空になっているホロファイ
バーの外側空間にポートより注入して行ない、かくして
カラム当り約lX10B個のRPMI−8226を細胞
を播種した。Following the above, cell seeding was carried out using a cell suspension of RPMI-8226 strain adjusted to about 4 x 106 cells/11 cells.
was taken into a needleless syringe and injected into the empty outer space of the hollow fiber through the port, thus seeding about 1×10 B cells of RPMI-8226 per column.
上記播種細胞の培養は、約1時間毎に数回ホロファイバ
ーの向きを変えてカラム内で細胞が極在しないように調
節後、数時間培養液の循環速度を1zl/分で培養液を
主構成回路に循環させ、D。The above-mentioned seeded cells were cultured by changing the direction of the holofiber several times approximately every hour so that the cells were not concentrated in the column, and then the culture medium was mainly circulated at a circulation rate of 1 zl/min for several hours. circulate through the constituent circuits,D.
(導入側)とDo(排出側)の差が播種時の差に比べて
減少しないようであれば、培養用媒質の循環速度を20
Il1分に上昇させて行なった。リザーブタンク(7)
の容量を約21とし、培養開始48時間後より第1図に
示す循環培養系(C1osedCulture)と第2
図に示すIC8循環培養十新鮮培地供給系(IC3Ov
erflow Cu1ture)とを交互に切り替え且
つ時間比率を調製することによって、培養1週間まで、
500z//日の交換比率で、消費培養用媒質を新鮮培
養液と連続的に交換しながら培養を続けた。If the difference between Do (introduction side) and Do (output side) does not decrease compared to the difference at the time of seeding, increase the circulation speed of the culture medium to 20%.
The temperature was increased to 1 minute. Reserve tank (7)
48 hours after the start of culture, the circulating culture system (C1osedCulture) shown in Figure 1 and the second
The IC8 circulating culture fresh medium supply system (IC3Ov) shown in the figure
By alternating the erflow culture and adjusting the time ratio, the culture can be carried out for up to one week.
Cultures were continued with continuous exchange of spent culture medium with fresh culture medium at an exchange rate of 500 z/day.
播種7日より培養用媒質の交換比率を1000xi1日
に上昇させると共に、2日毎に1回副構成回路2の機能
を使用して、細胞非存在空間側の培養液を10Ilずつ
回収した。pH値が6.8以下になると自動的にpH緩
衝液(5%NaHCO3含有PBS=培養液の50倍濃
度)溶液を構成回路2を使用して、培養潅流回路中に1
回当り61/添加した。但しこの添加操作は6時間以内
に2回以上行なってはならないように自動的にセットし
た。From the 7th day of seeding, the exchange rate of the culture medium was increased to 1000 x 1 day, and 10 Il of the culture medium in the cell-free space was collected once every 2 days using the function of sub-component circuit 2. When the pH value is below 6.8, the pH buffer (PBS containing 5% NaHCO3 = 50 times the concentration of the culture medium) solution is automatically added to the culture perfusion circuit using configuration circuit 2.
Added 61/ml per serving. However, this addition operation was automatically set so as not to be performed more than once within 6 hours.
添加されたpH緩衝液が潅流回路中で充分均一に拡散さ
れるまでに時間を必要とするので、pH緩衝液が添加さ
れてから2時間以降で且つ6時間以内に、灌流培養液の
pH値が6.8より低下するようであれば、改めて新鮮
培養液を1000z//2時間の高速で消費培養液と交
換した。この時構成回路(2)を使用した。Since it takes time for the added pH buffer to be sufficiently and uniformly diffused in the perfusion circuit, the pH value of the perfused culture solution should be adjusted after 2 hours and within 6 hours after the addition of the pH buffer. If the value decreased below 6.8, fresh culture solution was replaced with the spent culture solution at a high speed of 1000z//2 hours. At this time, configuration circuit (2) was used.
次いで1〜2日に1回、構成回路(2)を使用して、2
1A’の灌流培養液を回収し、グルコース濃度を測定し
、50■/l以下の場合は、上記と同様の培養液交換を
実施した。Then once every 1 to 2 days, using component circuit (2),
1 A' of perfusion culture solution was collected and the glucose concentration was measured, and if it was less than 50 µ/l, the culture solution was replaced in the same manner as above.
培養14日目より、新鮮培養液と消費培養液との交換比
率を20003F//日に上昇させると共に、EC8側
の培養液を2日毎に25yJづつ回収した。From the 14th day of culture, the exchange ratio between fresh culture solution and spent culture solution was increased to 20003F/day, and 25 yJ of the culture solution on the EC8 side was collected every 2 days.
かくして、目的産生物質を得た。In this way, the target product was obtained.
上記に従う細胞培養結果を第4図に示す。The results of the cell culture according to the above are shown in FIG.
図において、横軸は培養時間(Hr)を、縦軸は目的1
gG L−λ産生量(■)[曲線(1)として図示す
るコ及び酸素消費量(0,C,R,) (μモル/分
)[曲線(2)として図示するコを示す。In the figure, the horizontal axis represents the culture time (Hr), and the vertical axis represents the purpose 1.
gGL L-λ production (■) [shown as curve (1)] and oxygen consumption (0, C, R,) (μmol/min) [shown as curve (2)].
また第4図には上記酸素消費量(0・C,R,)に基づ
く算出細胞数(推定、個)を、該酸素消費量の横に表示
した。Further, in FIG. 4, the calculated cell number (estimated, cells) based on the above oxygen consumption amount (0·C,R,) is displayed next to the oxygen consumption amount.
該図より、本発明方法によれば、細胞培養維持期間を延
長して、最大到達細胞密度を向上させ得ると共に、長期
間に亘って、高収率で目的産生物が生産できることが明
らかである。From the figure, it is clear that according to the method of the present invention, the cell culture maintenance period can be extended, the maximum cell density can be improved, and the target product can be produced with high yield over a long period of time. .
第1図乃至第3図は、本発明方法の実施に適したホロフ
ァイバー灌流培養装置の一例を示す概略図であり、第4
図は本発明方法の実施による細胞培養の結果を示すグラ
フである。
(以 上)1 to 3 are schematic diagrams showing an example of a holofiber perfusion culture device suitable for carrying out the method of the present invention, and FIG.
The figure is a graph showing the results of cell culture according to the method of the present invention. (that's all)
Claims (1)
当り、該ホロファイバーの細胞存在空間側より細胞非存
在空間側へ細胞培養用媒質を流すことを特徴とする潅流
培養方法。(2)灌流培養期間中の任意の時期に、細胞
存在空間に蓄積される老廃物、死細胞、生細胞の増殖阻
止因子及び目的生産物の分解因子の少なくとも1種を細
胞存在空間外に強制的に排出する請求項1記載の方法。(1) A perfusion culture method characterized by flowing a cell culture medium from the cell presence space side to the cell non-existence space side of the holofiber in perfusion culture of cells using a holofiber. (2) At any time during the perfusion culture period, at least one of waste products, dead cells, growth-inhibiting factors for living cells, and decomposition factors for target products, which are accumulated in the cell space, is forced out of the cell space. 2. The method according to claim 1, wherein the waste is discharged in a vacuum.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP27198790A JPH04148676A (en) | 1990-10-09 | 1990-10-09 | Perfusion culture |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP27198790A JPH04148676A (en) | 1990-10-09 | 1990-10-09 | Perfusion culture |
Publications (1)
Publication Number | Publication Date |
---|---|
JPH04148676A true JPH04148676A (en) | 1992-05-21 |
Family
ID=17507573
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP27198790A Pending JPH04148676A (en) | 1990-10-09 | 1990-10-09 | Perfusion culture |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPH04148676A (en) |
Cited By (3)
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WO2016140213A1 (en) * | 2015-03-05 | 2016-09-09 | 東洋紡株式会社 | Cell culture method using hollow fiber module |
JP2020188691A (en) * | 2019-05-20 | 2020-11-26 | 株式会社Ihi | Cell culture device |
WO2024116408A1 (en) * | 2022-12-02 | 2024-06-06 | 日本電信電話株式会社 | Apparatus and method for adding substance produced by cultured organism |
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JPS63188386A (en) * | 1987-01-29 | 1988-08-03 | Shimadzu Corp | Bioreactor |
JPS63276481A (en) * | 1987-05-06 | 1988-11-14 | Hitachi Ltd | Cultivation unit |
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WO2016140213A1 (en) * | 2015-03-05 | 2016-09-09 | 東洋紡株式会社 | Cell culture method using hollow fiber module |
JP2020188691A (en) * | 2019-05-20 | 2020-11-26 | 株式会社Ihi | Cell culture device |
WO2024116408A1 (en) * | 2022-12-02 | 2024-06-06 | 日本電信電話株式会社 | Apparatus and method for adding substance produced by cultured organism |
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