JPH05192135A - Removing method for and transplantation of animal cell from carrier - Google Patents
Removing method for and transplantation of animal cell from carrierInfo
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- JPH05192135A JPH05192135A JP4008393A JP839392A JPH05192135A JP H05192135 A JPH05192135 A JP H05192135A JP 4008393 A JP4008393 A JP 4008393A JP 839392 A JP839392 A JP 839392A JP H05192135 A JPH05192135 A JP H05192135A
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- Prior art keywords
- carrier
- cells
- adherent animal
- animal cells
- cell
- Prior art date
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Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、動物細胞の担体からの
剥離及び移植方法に係り、特に接着性の動物細胞の担体
からの剥離及び新たな担体への移植方法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for exfoliating animal cells from a carrier and transplanting the same, and more particularly to a method for exfoliating adherent animal cells from a carrier and transplanting them to a new carrier.
【０００２】[0002]
【従来の技術】最近、担体に付着して増殖する動物細胞
いわゆる接着性動物細胞を液体培地中で培養し、各種の
有用な生理活性蛋白を生産しようとする動きが活発化し
ている。培養方法としては、直径数１00μｍの担体粒子
の表面に培養細胞を接着させて培養する、いわゆるマイ
クロキャリア法が一般的である。代表的な公知技術とし
て以下のものがある。2. Description of the Related Art Recently, animal cells that adhere to a carrier and proliferate, so-called adherent animal cells, have been cultivated in a liquid medium to produce various useful bioactive proteins. As a culturing method, a so-called microcarrier method is generally used in which cultured cells are adhered to the surface of carrier particles having a diameter of several hundreds of μm for culturing. The following are typical known techniques.
【０００３】（１) ジー・ケー・ジャコブソン，エス・
オー・ジョリイ，バイオテクノロジー，第７巻ビー，ジ
ーン・テクノロジー，第３ビー章，マンマリアン・セル
・カルチュア，ページ 143〜164, 1984年, ブイ・シー
・エイツ・フェルラーグス・ゲセルシャフト・エム・ビ
ー・エイチ社 (G. K. Jacbson and S.O. Jolly, Biotec
h-nolgy Vol.76, p143-164, 1989年, VCH Verlags gese
llshaft mbH)。 （２) 特開昭52-7479 号公報（公開昭52年１月20日)
「動物細胞の培養方法及びその装置」 （３) 特開昭56-51981号公報（公開昭56年５月９日)
「細胞の培養方法」 （４) 特開昭62-4117 号公報 (公開昭62年１月28日)
「インターフェロン産生方法」(1) GK Jacobson, S.
Oh Jolly, Biotechnology, Volume 7 Bee, Gene Technology, Chapter 3 Bee, Mammalian Cell Culture, pp. 143-164, 1984, V.C.Eights Verlags Gesellschaft M.B.・ H (GK Jacbson and SO Jolly, Biotec
h-nolgy Vol.76, p143-164, 1989, VCH Verlags gese
llshaft mbH). (2) Japanese Patent Laid-Open No. 52-7479 (Published January 20, 1982)
"Method for culturing animal cells and apparatus therefor" (3) JP-A-56-51981 (published May 9, 1981)
"Method of culturing cells" (4) JP-A-62-4117 (published on January 28, 1987)
"Interferon production method"
【０００４】[0004]
【発明が解決しようとする課題】これら付着性細胞は担
体表面に沿って一重に表面を飽和に達するか、飽和に近
づくと、細胞の増殖が停止し、培地を交換しても培養を
持続できなくなる。したがって、飽和する前にもしくは
飽和時点で担体に結合している細胞を損傷しないように
剥離し、これをバージンの担体に移植する必要がある。
上記の先行技術に示される従来の方法は、細胞と担体及
び細胞相互の結合を解合するため、キレート剤存在下で
高濃度の蛋白分解酵素であるトリプシンと接触させ、細
胞膜を加水分解する方法が取られてきた。しかし、トリ
プシンにより細胞が損傷を受けるため担体からの分離収
率が高くならない段階で反応を停止せざるを得ないた
め、生きた細胞の収率が低い欠点を有していた。さら
に、本反応の進行状態を継時的にサンプリングして顕微
鏡下で監視することが通常行われるが、細胞の損傷が少
なくかつ剥離した細胞を収率よく得るために、どの時点
で反応を停止すべきかを、顕微鏡下で監視しつつ適切に
判断するには高度な熟練を必要とする欠点を有してい
た。These adherent cells do not reach cell surface saturation along the surface of the carrier, or when they approach saturation, cell growth stops and the culture can be continued even if the medium is replaced. Disappear. Therefore, before or at the time of saturation, it is necessary to detach the cells bound to the carrier so as not to damage them, and transplant them into the virgin carrier.
The conventional method shown in the above-mentioned prior art is a method of hydrolyzing a cell membrane by contacting with trypsin, which is a proteolytic enzyme at a high concentration in the presence of a chelating agent, in order to break the bond between cells and carriers and cells. Has been taken. However, since the cells are damaged by trypsin, the reaction has to be stopped at a stage where the yield of separation from the carrier does not increase, and thus the yield of live cells is low. Furthermore, the progress of this reaction is usually sampled continuously and monitored under a microscope.However, the reaction is stopped at any point in order to obtain less detached cells and less damage to the cells. It had a drawback that it required a high degree of skill to properly judge whether or not it should be done under a microscope.
【０００５】本発明の目的は、上述した従来の欠点を解
決し、担体に結合している接着性細胞を収率よく解合す
ること、及び解合分離した細胞をバージン担体に移植
し、増殖する方法を提供するにある。The object of the present invention is to solve the above-mentioned conventional drawbacks, to dissociate adherent cells bound to a carrier in a high yield, and to transplant the dissociated cells to a virgin carrier to proliferate. To provide a way to do it.
【０００６】[0006]
【課題を解決するための手段】本発明者らは、担体と細
胞との結合を切断するのに、従来のように蛋白分解酵素
を使って細胞側を分解することによらず、担体側を優先
的に分解する酵素を用いて担体からの細胞の分離及び移
植方法について鋭意実験研究を行い、その方法が担体に
結合している接着性細胞を収率よく解合分離し、バージ
ン担体に移植しうることを知見し、本発明を完成するに
いたった。[Means for Solving the Problems] The present inventors have proposed a method for cleaving the carrier side without breaking the cell side with a proteolytic enzyme to cleave the bond between the carrier and cells. We carried out an intensive experimental research on the method of separating cells from the carrier and transplanting them using an enzyme that preferentially decomposes, and by this method, the adherent cells bound to the carriers were dissociated and separated in good yield and transplanted to a virgin carrier. The inventors have found that they can do so and have completed the present invention.
【０００７】すなわち、本発明は、接着性動物細胞の担
体からの剥離方法であって、細胞側の接合部には作用せ
ず、非蛋白性の担体側の結合部にのみ作用する非蛋白性
高分子加水分解酵素を剥離時に作用させることを特徴と
する、接着性動物細胞の担体からの剥離方法を開示す
る。さらに、本発明は、少なくとも (１) 培養液中か
ら、細胞が接着した非蛋白性の有機性材料からなる担体
を分離する工程、及び、 (２) 該担体を、担体構成材成
分を基質とする加水分解酵素を含有する液と接触させ、
該担体を分解して溶解させることにより、細胞を液中に
分散する工程、とを包含する接着性動物細胞の担体から
の剥離方法をも開示する。That is, the present invention relates to a method for detaching an adhesive animal cell from a carrier, which does not act on the cell-side junction, but acts on the non-protein carrier-side binding site. Disclosed is a method for separating adherent animal cells from a carrier, which is characterized in that a high molecular hydrolase is allowed to act upon the separation. Furthermore, the present invention comprises at least (1) a step of separating a carrier composed of a non-protein organic material to which cells are adhered from the culture solution, and (2) using the carrier as a carrier component component as a substrate. Contact with a liquid containing a hydrolase,
Also disclosed is a method for separating adherent animal cells from a carrier, which comprises a step of dispersing cells in a liquid by decomposing and dissolving the carrier.
【０００８】本発明は、上記の２つの工程に加え、さら
に (３) 該分散した細胞を液から分離する工程、 (４)
該分離した細胞を未使用の担体と接触させ、生理的条件
下で一定時間維持して、該担体に該分離した細胞を接着
する工程、及び、 (５) 第４工程で得られる担体に接着
した細胞を培養する工程、とを包含する接着性動物細胞
の移植方法をも開示する。In addition to the above two steps, the present invention further comprises (3) a step of separating the dispersed cells from a liquid, (4)
A step of contacting the separated cells with an unused carrier, maintaining the physiological cells for a certain period of time to adhere the separated cells to the carrier; and (5) adhering to the carrier obtained in the fourth step. And a method of transplanting adherent animal cells, the method comprising culturing the cells.
【０００９】図１は、上記の本願各発明の概要を一連の
流れとして示したプロセスフロー図であり、培養液中か
ら細胞を接着した非蛋白性の有機性担体を分離し
（ａ）、担体構成成分を基質とする加水分解酵素と接着
させて、担体側を分解して液中に溶解させることによ
り、細胞を液中に分散する（ｂ）。分離した細胞の状態
が分散状態にある場合にはそのまま、集密状態にある場
合は蛋白質分解酵素により細胞相互を分散させた後
（ｃ）、該分散した細胞を液から分離し（ｄ）、分離し
た細胞を未使用の担体と接触させ（ｅ）、生理的条件下
で一定時間維持して該担体に細胞を接着させ培養する
（ｆ）。FIG. 1 is a process flow chart showing the outline of each invention of the present application as a series of flows. A non-protein organic carrier to which cells are adhered is separated from a culture solution (a), and the carrier is separated. The cells are dispersed in the liquid by allowing the components to adhere to a hydrolase that uses the substrate as a substrate, decomposing the carrier side and dissolving in the liquid (b). When the separated cells are in a dispersed state, they are as they are, and when they are in a confluent state, the cells are dispersed with a proteolytic enzyme (c), and then the dispersed cells are separated from the liquid (d), The separated cells are contacted with an unused carrier (e), and the cells are allowed to adhere to the carrier for a certain period of time under physiological conditions to be cultured (f).
【００１０】上記から容易に理解されるように、本発明
の方法を用いることにより、接着性動物細胞の培養がき
わめて容易にかつ高効率のものとなる。従って、本発明
は、接着性動物細胞の培養方法として表現することもで
きる。本発明に適用できる細胞は、接着性を有する動物
細胞であれば特に限定されない。担体は非蛋白性の有機
性材質、例えばセルロース、ヘミセルロース、架橋デキ
ストランやこれらの誘導体等を有効に用いることができ
る。酵素は担体材質に対応する加水分解酵素であればよ
い。例えば、セルロースに対しセルラーゼ、ヘミセルロ
ースに対するヘミセルラーゼ、デキストランに対してデ
キストラナーゼ等が用いられる。これらの酵素は細胞の
生理的条件下で効率的に作用できるものが好ましい。例
えば、セルラーゼでも作用pHが８以上、５以下で作用す
るものが存在するが、動物細胞の培養条件を考慮すると
これらのものは必ずしも好適な酵素とはいえないもので
ある。As will be readily understood from the above, the use of the method of the present invention makes the culture of adherent animal cells extremely easy and highly efficient. Therefore, the present invention can also be expressed as a method for culturing adherent animal cells. The cells applicable to the present invention are not particularly limited as long as they are animal cells having adhesiveness. As the carrier, a non-protein organic material such as cellulose, hemicellulose, crosslinked dextran, or a derivative thereof can be effectively used. The enzyme may be a hydrolase corresponding to the carrier material. For example, cellulase for cellulose, hemicellulase for hemicellulose, and dextranase for dextran are used. It is preferable that these enzymes can efficiently act under physiological conditions of cells. For example, there are cellulases that act at an action pH of 8 or more and 5 or less, but these are not necessarily suitable enzymes in consideration of the culture conditions of animal cells.
【００１１】反応条件及び反応液組織は、細胞に重大な
阻害を与えない範囲で、対応する酵素の活性を適合する
条件と組成を適宜選択して用いることができる。上記の
ように、担体分解酵素で担体を溶解した際、細胞が集塊
を形成した状態で分散している場合には、担体の溶解に
続いて稀薄な蛋白分解酵素の溶液を軽度に作用させ、細
胞相互の分散をはかることが好ましい。蛋白分解酵素と
しては、従来、担体から直接的に細胞から剥離させると
きに用いるトリプシンやデスパーゼも十分使用できる。The reaction conditions and the reaction solution tissue can be used by appropriately selecting the conditions and composition that are compatible with the activity of the corresponding enzyme, as long as they do not seriously inhibit the cells. As described above, when the carrier is lysed with the carrier-degrading enzyme and the cells are dispersed in the state of forming an agglomerate, the solution of the dilute proteolytic enzyme is allowed to act gently after the lysis of the carrier. It is preferable to disperse the cells. As the proteolytic enzyme, trypsin or despase, which has been conventionally used for detaching cells directly from a carrier, can be sufficiently used.
【００１２】担体形状も特に限定されないが、本発明を
最も適用しやすいものはマイクロキャリアである。細胞
の接着したマイクロキャリアを培養液から分離する手段
も特に限定されないが、重力沈降分離、濾過、遠心沈降
分離が適している。分散後の細胞の回収も沈降分離以外
に濾過あるいは遠心分離でも対応できるが、遠心分離が
最も適している。The shape of the carrier is not particularly limited, but the one to which the present invention is most applicable is a microcarrier. The means for separating the microcarriers to which the cells are attached from the culture solution is not particularly limited, but gravity sedimentation, filtration, and centrifugal sedimentation are suitable. After the dispersion, cells can be recovered by filtration or centrifugation in addition to sedimentation, but centrifugation is most suitable.
【００１３】[0013]
【作用】担体と細胞との結合を切断するのに、従来のよ
うに蛋白分解酵素を用いると、蛋白分解酵素は細胞膜を
も分解する。このため、細胞は担体から剥離しても細胞
が化学的に損傷を受ける。損傷度が高い場合には細胞の
活性低下や細胞の脆弱化をひきおこし、ひいては細胞の
壊死、崩壊を引きおこす。本発明においては、高分子加
水分解酵素すなわち担体構成成分を基質とする加水分解
酵素を、細胞を接着した担体に作用させることにより、
細胞膜には全く作用せず、担体を分解して溶解させるた
め、細胞の無傷で担体から剥離することができる。When a proteolytic enzyme is conventionally used to cleave the bond between the carrier and the cell, the proteolytic enzyme also decomposes the cell membrane. Therefore, even if the cells are detached from the carrier, the cells are chemically damaged. When the degree of damage is high, it causes a decrease in cell activity and cell fragility, which in turn causes cell necrosis and collapse. In the present invention, a polymer hydrolase, that is, a hydrolase having a carrier component as a substrate, is caused to act on a carrier to which cells are adhered,
Since it does not act on the cell membrane at all and decomposes and dissolves the carrier, the cells can be peeled from the carrier without damage.
【００１４】また、担体に接着していた細胞が細胞の種
類や培養条件により細胞が互いに比較的強固に結合して
いる場合がある。このような場合には、担体を加水分解
酵素で溶解した後、細胞が集塊として液中に分散する場
合が生じる。集塊で分散した細胞懸濁液を種細胞として
そのまま未使用の担体に接着しようとすると、単細胞の
状態に分散している時に比べ、細胞は担体上に不均一に
付着し、接着の効率が低下する。この場合には、担体分
解後、前述のように細胞相互間の結合を切断するに足る
穏和な反応条件下での蛋白分解酵素処理を行い、例え
ば、従来の 1/5〜1/10の低い酵素濃度で、短時間反応さ
せることにより、細胞を損傷せずに単細胞に効率的に分
散することができる。In some cases, the cells adhered to the carrier are relatively tightly bound to each other depending on the cell type and culture conditions. In such a case, after the carrier is dissolved with a hydrolase, the cells may be aggregated and dispersed in the liquid. When attempting to adhere the cell suspension dispersed by agglutination as seed cells to an unused carrier as it is, the cells adhere more unevenly on the carrier than when dispersed in a single cell state, and the efficiency of adhesion is improved. descend. In this case, after the carrier is decomposed, a proteolytic enzyme treatment is performed under mild reaction conditions sufficient to cleave the bonds between the cells as described above, and for example, it is as low as 1/5 to 1/10 that of the conventional method. By reacting at an enzyme concentration for a short time, cells can be efficiently dispersed in single cells without damage.
【００１５】[0015]
【実施例】次に実施例を示し本発明をさらに詳しく説明
する。 〔実施例１〕１Ｌローラボトルに、セルロースを材質と
する球状多孔質マイクロキャリア (直径１00〜２00μ
ｍ) を湿潤容量で５mlと10％牛血清添加ダルベコ変法イ
ーグル培地90mlを入れ、接着性のＣＨＯ細胞の懸濁液を
培養液あたり１×103 cells/mlになるように接種して37
℃で培養した。培養５日目に増殖速度が低下しボトル内
の培養液１mlあたり１×105 cells の細胞濃度に達した
後、培養液を無菌雰囲気下で濾過し、細胞付着マイクロ
キャリアを回収した。EXAMPLES The present invention will be described in more detail with reference to the following examples. [Embodiment 1] A spherical porous microcarrier made of cellulose (diameter: 100 to 200 μm) was placed in a 1 L roller bottle.
m) in a wet volume of 5 ml and 90 ml of Dulbecco's modified Eagle medium supplemented with 10% bovine serum, and inoculated with a suspension of adherent CHO cells at a concentration of 1 × 10 3 cells / ml per culture medium.
Cultured at ° C. After 5 days of culturing, the growth rate decreased and reached a cell concentration of 1 × 10 5 cells per ml of the culture medium in the bottle, and then the culture medium was filtered under a sterile atmosphere to recover cell-attached microcarriers.
【００１６】回収した細胞付着マイクロキャリアの0.５
mlを試験管に取り、ゆるやかに攪拌しつつ、トリコデル
マ属菌起源のセルラーゼ（市販品）1000国際単位を溶解
した10％牛血清添加ダルベコ変法イーグル培地３mlを加
え、37℃で５分間インキュベートしてマイクロキャリア
を溶解した。マイクロキャリアの溶解後に残留する細胞
分散液を振動式攪拌機で２秒間処理して、細胞の50％以
上が単細胞に分散していることを確認した。得られた細
胞分散液を500rpmで３分間遠心分離して細胞を回収し
た。0.5 of the collected cell-attached microcarriers
Take 3 ml in a test tube, add 3 ml of Dulbecco's modified Eagle medium containing 10% bovine serum containing 1000 international units of cellulase (commercially available product) derived from Trichoderma spp, and gently incubate at 37 ° C for 5 minutes. To dissolve the microcarriers. The cell dispersion remaining after the dissolution of the microcarriers was treated with a vibrating stirrer for 2 seconds, and it was confirmed that 50% or more of the cells were dispersed in single cells. The obtained cell dispersion liquid was centrifuged at 500 rpm for 3 minutes to collect cells.
【００１７】次いで、得られた細胞ペレットを10％牛血
清添加ダルベコ変法イーグル培地 950mlに懸濁し、バー
ジンマイクロキャリアを50ml加えて、１Ｌローラボトル
10本に１00mlづつ分注した。これを37℃で５時間インキ
ュベート後、細胞がキャリア表面に付着したことを確認
した。さらにローラボトルを37℃で５日間培養した結
果、細胞濃度は1.２×105 cells/ml-培養液に達した。 〔実施例２〕実施例１の濾過で回収した細胞付着マイク
ロキャリアの0.５mlを試験管に取り、ゆるやかに攪拌し
つつ、実施例１と同じ要領でセルラーゼによりマイクロ
キャリアを溶解して得た細胞分散液3.５mlを得た。本液
を同様に振動式攪拌機で処理して、細胞の50％が単細胞
に分散していることを確認後、牛膵臓起源のトリプシン
を50国際単位を含むリン酸緩衝液 (pH7.０) 0.１mlを加
え、37℃で１分間処理して細胞の90％が単細胞状態に分
散した。本液を500rpmで３分間遠心分離して細胞を回収
した。Then, the obtained cell pellet was suspended in 950 ml of Dulbecco's modified Eagle medium supplemented with 10% bovine serum, 50 ml of virgin microcarrier was added, and 1 L roller bottle was added.
Dispense 100 ml into 10 bottles. After incubating this at 37 ° C. for 5 hours, it was confirmed that the cells adhered to the carrier surface. As a result of further culturing the roller bottle at 37 ° C. for 5 days, the cell concentration reached 1.2 × 10 5 cells / ml-culture medium. [Example 2] Obtained by dissolving 0.5 ml of the cell-attached microcarriers collected by filtration in Example 1 into a test tube and dissolving the microcarriers with cellulase in the same manner as in Example 1 while gently stirring. 3.5 ml of cell dispersion was obtained. This solution was similarly treated with a vibrating stirrer, and after confirming that 50% of the cells were dispersed in single cells, a phosphate buffer solution (pH 7.0) containing 50 international units of trypsin derived from bovine pancreas was prepared. 0.1 ml was added and treated at 37 ° C. for 1 minute to disperse 90% of the cells in a single cell state. This solution was centrifuged at 500 rpm for 3 minutes to collect cells.
【００１８】次いで、得られた細胞のペレットを実施例
１の要領でバージンマイクロキャリアに接着させ、１Ｌ
ローラボトル10本で37℃で培養した。その結果４日目で
細胞濃度は最大の1.５×105 cells/ml-培養液に達し
た。 〔比較例１〕従来のトリプシンによる剥離を行った。す
なわち、実施例１の濾過で回収した細胞付着マイクロキ
ャリアの0.５mlを試験管に取り、ゆるやかに攪拌しつ
つ、牛膵臓起源のトリプシンを1000国際単位及びＥＤＴ
Ａ10mgを含むリン酸緩衝液 (pH7.0)を0.１ml加え、37℃
でインキュベートした。適宜、反応の途中でサンプリン
グし顕微鏡にて細胞のマイクロキャリアからの剥離の進
行を追跡し、1.５分後に細胞の90％が単細胞状態に剥離
していることを確認した。次いで10％牛血清を含むダル
ベコ変法イーグル培地３mlを加えて反応を停止した。本
液を500rpmで３分間遠心分離して細胞を回収した。得ら
れた細胞のペレットを実施例１の要領でバージンマイク
ロキャリアに接着させ、１Ｌのローラボトル10本で37℃
で培養した。その結果、８日目で細胞濃度は最大の0.５
×105 cells/ml-培養液に達した。Then, the obtained cell pellet was adhered to a virgin microcarrier as in Example 1 to give 1 L.
It was cultured at 37 ° C in 10 roller bottles. As a result, on day 4, the cell concentration reached the maximum of 1.5 × 10 5 cells / ml-culture medium. [Comparative Example 1] Conventional stripping was performed using trypsin. That is, 0.5 ml of the cell-attached microcarriers collected by the filtration of Example 1 was placed in a test tube and gently mixed with 1000 international units of trypsin derived from bovine pancreas and EDT.
0.1 ml of phosphate buffer (pH 7.0) containing 10 mg of A was added, and the temperature was 37 ° C.
Incubated at. The progress of detachment of cells from the microcarriers was appropriately followed by sampling during the reaction, and it was confirmed that 90% of the cells detached in a single cell state after 1.5 minutes. Then, the reaction was stopped by adding 3 ml of Dulbecco's modified Eagle medium containing 10% bovine serum. This solution was centrifuged at 500 rpm for 3 minutes to collect cells. The obtained cell pellet was adhered to a virgin microcarrier in the same manner as in Example 1, and 10 1L roller bottles were used at 37 ° C.
It was cultured in. As a result, the maximum cell concentration was 0.5 on the 8th day.
× 10 5 cells / ml-culture medium was reached.
【００１９】本比較例によれば、担体と細胞間の結合を
反応で切断する際に細胞が損傷を受け、実施例１、２に
くらべ増殖速度が低下し、かつ到達細胞濃度も低いレベ
ルにとどまっていることが分かる。また、実施例２は、
実施例１でのセルラーゼによる担体溶解で残留する細胞
集塊をさらに担体−細胞間の結合よりも弱いトリプシン
処理を行うことにより細胞を効果的に単細胞に分散して
いる。このため実施例１に比べ、バージンの担体に細胞
を均一に接着でき、さらに増殖速度と到達細胞濃度を向
上できることが分かる。 〔実施例３〕１00mlのスピンナーフラスコに、架橋化デ
キストランを材質とする球状マイクロキャリア (平均直
径 150μｍ) の湿潤容量で１mlと８％牛血清添加イーグ
ル培地45mlを入れ、接着性のＣＨＯ細胞の懸濁液を培養
液あたり0.８×103cells/mlになる様に接種して37℃、5
0rpm で培養した。培養４日目に細胞濃度が最大値であ
る0.７×105 cells/mlに達した。攪拌を停止し、５分間
静置して細胞付着マイクロキャアリを沈降分離した。According to this comparative example, the cells were damaged when the bond between the carrier and the cells was cleaved by the reaction, the growth rate was lower than in Examples 1 and 2, and the ultimate cell concentration was also low. You can see that it stays. In addition, in Example 2,
By further treating the cell clumps remaining after lysis of the carrier with cellulase in Example 1 with trypsin, which is weaker than the carrier-cell bond, the cells are effectively dispersed into single cells. Therefore, it can be seen that the cells can be uniformly adhered to the virgin carrier and the growth rate and the ultimate cell concentration can be improved as compared with Example 1. Example 3 In a spinner flask of 100 ml, 1 ml of spherical microcarriers (average diameter of 150 μm) made of cross-linked dextran and 45 ml of Eagle medium supplemented with 8% bovine serum were placed to suspend adherent CHO cells. The suspension was inoculated to give 0.8 × 10 3 cells / ml per culture, and the mixture was incubated at 37 ℃ for 5
Cultured at 0 rpm. On the 4th day of culture, the cell concentration reached the maximum value of 0.7 × 10 5 cells / ml. Stirring was stopped, and the mixture was allowed to stand for 5 minutes to sediment and separate the cell-attached microcaary.
【００２０】上清をデカンテーションにより除いて得た
細胞付着マイクロキャリアにペニシリウム属菌起源のデ
キストラナーゼ（市販品）３00国際単位を含む８％牛血
清添加イーグル培地10mlを添加し、37℃、３分間インキ
ュベートした。マイクロキャリアの溶解を確認後、上清
をデカンテーションで除き、分散した細胞に８％牛血清
添加イーグル培地を１00ml添加した本液中の細胞の60％
以上が単細胞に分散していることを確認した。To the cell-attached microcarriers obtained by removing the supernatant by decantation, 10 ml of 8% bovine serum-added Eagle medium containing 300 international units of dextranase of Penicillium spp. Incubated for 3 minutes. After confirming the dissolution of the microcarriers, the supernatant was removed by decantation, and 100% of the dispersed cells were supplemented with 100 ml of Eagle medium supplemented with 8% bovine serum.
It was confirmed that the above was dispersed in single cells.
【００２１】得られた細胞分散液を37℃、50rpm で培養
した。細胞濃度は培養４日目に1.２×105 cell/ml-培養
液に達した。The obtained cell dispersion was cultured at 37 ° C. and 50 rpm. The cell concentration reached 1.2 × 10 5 cells / ml-culture medium on day 4 of culture.
【００２２】[0022]
【発明の効果】本発明により、接着性細胞を担体から損
傷することなく剥離して水中に分散することができ、こ
れによりバージン担体に均一に接着させ、担体上で効率
よく、かつ高い密度に細胞を増殖させることが可能とな
る。EFFECTS OF THE INVENTION According to the present invention, adherent cells can be detached from a carrier without being damaged and dispersed in water, whereby the adherent cells can be uniformly adhered to a virgin carrier, and efficiently and with high density can be obtained on the carrier. It is possible to grow cells.
【図１】本発明のプロセスフローの概略を示す図であ
る。FIG. 1 is a diagram showing an outline of a process flow of the present invention.
Claims (16)
あって、細胞側の接合部には作用せず、担体側の結合部
にのみ作用する高分子加水分解酵素を剥離時に作用させ
ることを特徴とする、接着性動物細胞の担体からの剥離
方法。1. A method for detaching an adhesive animal cell from a carrier, wherein a polymeric hydrolase that does not act on the cell-side junction but acts only on the carrier-side joint is acted upon detachment. A method for peeling an adherent animal cell from a carrier, which comprises:
特徴とする接着性動物細胞の担体からの剥離方法。(１)
培養液中から、細胞が接着した非蛋白性の有機性材料
からなる担体を分離する工程、及び、(２) 該担体を、
担体構成材成分を基質とする加水分解酵素を含有する液
と接触させ、該担体を分解して溶解させることにより、
細胞を液中に分散する工程。2. A method for separating adherent animal cells from a carrier, which comprises at least the following steps. (1)
A step of separating a carrier composed of a non-proteinaceous organic material to which cells adhere from the culture solution, and (2) the carrier
By contacting with a liquid containing a hydrolase having a carrier component material as a substrate and decomposing and dissolving the carrier,
Dispersing cells in a liquid.
もしくは濾過により行うことを特徴とする、請求項２記
載の接着性動物細胞の剥離方法。3. The method for detaching adherent animal cells according to claim 2, wherein the carrier is separated from the culture solution by sedimentation separation or filtration.
徴とする、請求項２又は３記載の接着性動物細胞の剥離
方法。4. The method for detaching adherent animal cells according to claim 2, wherein each step is performed in the same culture tank.
特徴とする、請求項２又は３記載の接着性動物細胞の剥
離方法。5. The method for detaching adherent animal cells according to claim 2 or 3, wherein each step is performed in a place other than the culture tank.
体であることを特徴とする、請求項２ないし５いずれか
記載の接着性動物細胞の剥離方法。6. The method for detaching adherent animal cells according to claim 2, wherein the carrier is cellulose or a cellulose derivative.
特徴とする、請求項６記載の接着性動物細胞の剥離方
法。7. The method for detaching adherent animal cells according to claim 6, wherein the carrier is a microcarrier.
る、請求項６又は７記載の接着性動物細胞の剥離方法。8. The method for exfoliating the adherent animal cell according to claim 6, wherein the carrier is porous.
特徴とする接着性動物細胞の移植方法。(１) 培養液中
から、細胞が接着した非蛋白性の有機性材料からなる担
体を分離する工程、(２) 該担体を、担体構成材成分を
基質とする加水分解酵素を含有する液と接触させ、該担
体を分解して溶解させることにより、細胞を液中に分散
する工程、(３) 該分散した細胞を液から分離する工
程、(４) 該分離した細胞を未使用の担体と接触させ、
生理的条件下で一定時間維持して、該担体に該分離した
細胞を接着する工程、及び、(５) 第４工程で得られる
担体に接着した細胞を培養する工程。9. A method for transplanting adherent animal cells, which comprises at least the following steps. (1) a step of separating a carrier made of a non-protein organic material to which cells adhere from a culture solution, (2) a carrier containing a hydrolase having a carrier component component as a substrate A step of dispersing the cells in a liquid by bringing them into contact with each other to decompose and lyse the carrier; (3) a step of separating the dispersed cells from the liquid; (4) a separation of the separated cells with an unused carrier. Contact
A step of adhering the separated cells to the carrier while maintaining them under physiological conditions for a certain period of time, and (5) a step of culturing the cells adhered to the carrier obtained in the fourth step.
しくは濾過により行うことを特徴とする、請求項９記載
の接着性動物細胞の移植方法。10. The method for transplanting adherent animal cells according to claim 9, wherein the carrier is separated from the culture solution by sedimentation separation or filtration.
徴とする、請求項９又は10記載の接着性動物細胞の移植
方法。11. The method for transplanting adherent animal cells according to claim 9, wherein each step is performed in the same culture tank.
特徴とする、請求項９又は10記載の接着性動物細胞の移
植方法。12. The method for transplanting adherent animal cells according to claim 9, wherein each step is performed in a place other than the culture tank.
体であることを特徴とする、請求項９ないし12いずれか
記載の接着性動物細胞の移植方法。13. The method for transplanting adherent animal cells according to claim 9, wherein the carrier is cellulose or a cellulose derivative.
特徴とする、請求項13記載の接着性動物細胞の移植方
法。14. The method for transplanting adherent animal cells according to claim 13, wherein the carrier is a microcarrier.
であることを特徴とする、請求項13又は14記載の接着性
動物細胞の移植方法。15. The method for transplanting adherent animal cells according to claim 13, wherein the carrier is porous having a pore size larger than that of cells.
分解酵素と接触させ、その後に第３の工程を行うことを
特徴とする、請求項９ないし15いずれか記載の接着性動
物細胞の移植方法。16. The adhesive animal cell according to claim 9, wherein the dispersed cells are contacted with a proteolytic enzyme after the second step, and then the third step is performed. How to port.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4008393A JPH05192135A (en) | 1992-01-21 | 1992-01-21 | Removing method for and transplantation of animal cell from carrier |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP4008393A JPH05192135A (en) | 1992-01-21 | 1992-01-21 | Removing method for and transplantation of animal cell from carrier |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH05192135A true JPH05192135A (en) | 1993-08-03 |
Family
ID=11691952
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP4008393A Pending JPH05192135A (en) | 1992-01-21 | 1992-01-21 | Removing method for and transplantation of animal cell from carrier |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH05192135A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2015097481A (en) * | 2013-11-18 | 2015-05-28 | 大日本印刷株式会社 | Method for producing cell laminated body |
| JP2018078906A (en) * | 2018-01-17 | 2018-05-24 | 大日本印刷株式会社 | Cell laminate production method |
| JP2018143211A (en) * | 2017-03-09 | 2018-09-20 | 三洋化成工業株式会社 | Cell culture substrate, cell production method and cell culture kit |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01215281A (en) * | 1988-02-24 | 1989-08-29 | Hitachi Ltd | Method for immobilized culture of animal cell included in gel and apparatus therefor |
-
1992
- 1992-01-21 JP JP4008393A patent/JPH05192135A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01215281A (en) * | 1988-02-24 | 1989-08-29 | Hitachi Ltd | Method for immobilized culture of animal cell included in gel and apparatus therefor |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2015097481A (en) * | 2013-11-18 | 2015-05-28 | 大日本印刷株式会社 | Method for producing cell laminated body |
| JP2018143211A (en) * | 2017-03-09 | 2018-09-20 | 三洋化成工業株式会社 | Cell culture substrate, cell production method and cell culture kit |
| JP2018078906A (en) * | 2018-01-17 | 2018-05-24 | 大日本印刷株式会社 | Cell laminate production method |
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