JPH01296986A - Culture of adherent animal cell - Google Patents
Culture of adherent animal cellInfo
- Publication number
- JPH01296986A JPH01296986A JP12566488A JP12566488A JPH01296986A JP H01296986 A JPH01296986 A JP H01296986A JP 12566488 A JP12566488 A JP 12566488A JP 12566488 A JP12566488 A JP 12566488A JP H01296986 A JPH01296986 A JP H01296986A
- Authority
- JP
- Japan
- Prior art keywords
- culture
- cells
- cell
- medium
- animal cell
- 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.)
- Pending
Links
- 210000004102 animal cell Anatomy 0.000 title claims abstract description 13
- 230000001464 adherent effect Effects 0.000 title abstract description 12
- 210000004027 cell Anatomy 0.000 claims abstract description 39
- 230000010412 perfusion Effects 0.000 claims abstract description 7
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 14
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 claims description 9
- 229910001424 calcium ion Inorganic materials 0.000 claims description 9
- 238000012136 culture method Methods 0.000 claims description 8
- 238000012258 culturing Methods 0.000 claims description 7
- 238000004220 aggregation Methods 0.000 claims description 6
- 230000002776 aggregation Effects 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 5
- 239000000853 adhesive Substances 0.000 claims description 3
- 230000001070 adhesive effect Effects 0.000 claims description 3
- 210000003292 kidney cell Anatomy 0.000 claims description 3
- 239000012679 serum free medium Substances 0.000 claims description 3
- 238000004113 cell culture Methods 0.000 claims description 2
- 239000000126 substance Substances 0.000 abstract description 4
- 210000003754 fetus Anatomy 0.000 abstract 1
- 230000002463 transducing effect Effects 0.000 abstract 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 8
- 239000001301 oxygen Substances 0.000 description 8
- 229910052760 oxygen Inorganic materials 0.000 description 8
- 239000001963 growth medium Substances 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- 230000007774 longterm Effects 0.000 description 3
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 2
- ZCCIPPOKBCJFDN-UHFFFAOYSA-N calcium nitrate Chemical compound [Ca+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ZCCIPPOKBCJFDN-UHFFFAOYSA-N 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- NBVXSUQYWXRMNV-UHFFFAOYSA-N fluoromethane Chemical compound FC NBVXSUQYWXRMNV-UHFFFAOYSA-N 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000017074 necrotic cell death Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 239000004809 Teflon Substances 0.000 description 1
- 229920006362 Teflon® Polymers 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 239000012736 aqueous medium Substances 0.000 description 1
- 239000002473 artificial blood Substances 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000010409 thin film Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
（ａ）産業上の利用分野
本発明は接着性動物細胞の培養方法に関するものである
。ざらに詳しくは有用物質を産生ずる接着性動物細胞を
、細胞同志の凝集を抑えて浮遊細胞と同様の状態で培養
する方法に関するものである。DETAILED DESCRIPTION OF THE INVENTION (a) Field of Industrial Application The present invention relates to a method for culturing adherent animal cells. More specifically, the present invention relates to a method for culturing adherent animal cells that produce useful substances in a state similar to that of floating cells by suppressing cell aggregation.
（ｂ）従来技術
工業的規模による細胞大量培養は、例えばウィルス、ワ
クチン、インターフェロンなどの抗ウィルス剤、あるい
はホルモンなどの生物薬品の製造に必須である。これら
の生理活性物質の生産に用いられる動物細胞は接着性の
ものが多く、その効率的培養技術の開発は工業的に重要
な課題である。(b) Prior Art Mass culture of cells on an industrial scale is essential for the production of, for example, viruses, vaccines, antiviral agents such as interferon, or biological drugs such as hormones. Many of the animal cells used in the production of these physiologically active substances are adhesive, and the development of efficient culture techniques is an industrially important issue.
従来、接着制細胞の大量培養方法及びそのための装置と
していくつかの提案がなされている。しかしその多くは
固体表面に細胞を接着させて増殖させるものであり、ス
ケールアップや操作性、長期安定性などに問題が残って
いる。例えばｖａｎＷｅｚｅｌ らによって開発された
マイクロキャリアー培養法はタンク培養が可能な方法と
して有用であるが、長期間培養していると細胞か担体か
らはがれ落ち、るという問題がある。史にＵＳＰ ４，
０５９，４８５号明細出には血清培地で接着性細胞の浮
遊培養を試みた例が記載されているが、細胞は大きな凝
集塊をつくってしまい内部の細胞の壊死という問題や長
期安定性がないという問題点か残っている。Conventionally, several proposals have been made as methods for mass culturing adherent cells and devices for the same. However, most of these methods allow cells to grow by adhering to a solid surface, and there remain problems with scale-up, operability, and long-term stability. For example, the microcarrier culture method developed by vanWezel et al. is useful as a method that allows tank culture, but there is a problem that cells may peel off from the carrier during long-term culture. History USP 4,
No. 059,485 describes an example in which floating culture of adherent cells was attempted in a serum medium, but the cells formed large aggregates, resulting in problems of internal cell necrosis and lack of long-term stability. There remains a problem.
（Ｃ）発明の目的
そこで、本発明者らは、接着性動物細胞を担体を用いる
ことなく浮遊状態で長期間安定に培養することを目的と
して研究を進めたところ、培養系中のカルシウムイオン
濃度を低く抑えることにより、細胞凝集が抑制されるこ
とを見出し本発明に到達した。(C) Purpose of the Invention The present inventors conducted research with the aim of stably culturing adherent animal cells in suspension for a long period of time without using a carrier, and found that the concentration of calcium ions in the culture system The present invention was achieved by discovering that cell aggregation can be suppressed by suppressing cell aggregation.
（ｄ）発明の構成
すなわら、本発明は、接着性動物細胞を、それ白身を浮
遊させた状態において培養するに当って、培養系中のカ
ルシウムイオン濃度を０．０１ｍＭ以上０、３ｍＭ未満
に維持することを特徴とする接着性動物細胞の培養方法
である。(d) According to the present invention, when culturing adherent animal cells in a suspended state, the calcium ion concentration in the culture system is set to 0.01 mM or more and less than 0.3 mM. This is a method for culturing adherent animal cells, which is characterized by maintaining the cells at a constant temperature.
以下、本発明について詳細に説明する。The present invention will be explained in detail below.
本発明においで用いられる接着性動物細胞は、通常の静
置培養において接着、伸展性を示すものであり、天然の
正常細胞のみならず、人為的あるいは遺伝子操作により
変性された細胞であってもよい。また、本発明は特に有
用な生理活性物質を産生する接る性動物細胞の培養にお
いて、生理活ｔ’ｉｔ＋貿を高い濃度で得る目的のため
に適している。The adherent animal cells used in the present invention exhibit adhesion and spreading properties in normal static culture, and can be applied not only to natural normal cells but also to cells denatured by artificial or genetic manipulation. good. Furthermore, the present invention is particularly suitable for the purpose of obtaining a high concentration of physiologically active t'it+trade in the culture of sex animal cells that produce useful physiologically active substances.
具体的にはヒト胎児腎細胞由来２９３株、ＢＨＫ。Specifically, human embryonic kidney cell-derived 293 strain, BHK.
ＣＨＯまたはそれらに遺伝子を導入したトランスフ号−
マント等が挙げられるが、トランスフ４−マントである
ことが好ましい。CHO or a transgenic gene introduced into them
Although examples include cloaks, transfer 4-mantos are preferred.
本発明の培養方法によれば、担体等を使用することなく
、細胞それ自体を浮遊させた状態で細胞同志の過度の凝
集を抑えて培養することかできる。According to the culture method of the present invention, cells can be cultured in a suspended state without using a carrier or the like while suppressing excessive aggregation of cells among themselves.
また、本発明の培養方法は回分培養であっても潅流培養
であってもよいが、潅流培養であることが好ましい。潅
流培養するに当って重要なことの１つは、培養液中の生
細胞と前記古い培養液とを効率よく分離し、古い培養液
を培養槽外へ取り出し、培養槽内の細胞の生育環境を最
適条件下に維持することである。また、ここで培養液中
から分離され、培養槽外へ取り出された古い培養液は、
膜による分離法、おるいは吸着による分離法などにより
、その中に含まれる生育阻害物質を除去し、更に必要に
よる産生された有用物質を分画された後、培養に必要な
添加成分を新たに加えることにより、新しい培養液とし
て再使用することかできる。Further, the culture method of the present invention may be a batch culture or a perfusion culture, but a perfusion culture is preferable. One of the important things in perfusion culture is to efficiently separate the living cells in the culture solution from the old culture solution, take out the old culture solution outside the culture tank, and improve the growth environment for cells in the culture tank. is to maintain it under optimal conditions. In addition, the old culture solution that is separated from the culture solution and taken out of the culture tank is
After removing the growth-inhibiting substances contained therein using membrane separation methods, or adsorption separation methods, and fractionating the useful substances produced as needed, we add additional ingredients necessary for culture. It can be reused as a new culture medium by adding it to the culture medium.
本発明の培養方法では、培養しようとする細胞それ自身
が培養液中に浮遊した状態で培養される。In the culture method of the present invention, cells to be cultured are cultured in a suspended state in a culture solution.
培養液は実質的に水によりなる水性媒体に、種々の無機
塩、ビタミン類、補酵素、ブドウ糖、アミノ酸、抗生物
質などの通常細胞ｊ８養に使用される添加成分が加えら
れている。また培養液には血清を加えることもできるし
、血清を用いない所謂無血清培地を培養液として使用す
ることもできるが、本発明は特に無血清培地において優
れた効果を示す。いずれにしても培養液中のカルシウム
イオン濃度は一般には０．０１ｍ）１以上０．３ｎ＋Ｈ
未満とし、培養液中のカルシウムイオン濃度をコントロ
ールするだめに添加するカルシウム塩としては塩化カル
シウム、硝酸カルシウムのいずれか、あるいは両者の混
合物などが挙げられる。また血清中のカルシウムイオン
によりコントロールしてもよい。The culture solution is an aqueous medium consisting essentially of water, to which are added various inorganic salts, vitamins, coenzymes, glucose, amino acids, antibiotics, and other additives commonly used for cell culture. Further, serum can be added to the culture solution, or a so-called serum-free medium that does not use serum can be used as the culture solution, but the present invention shows particularly excellent effects in a serum-free medium. In any case, the calcium ion concentration in the culture solution is generally 0.01m) or more than 0.3n+H
Calcium salts added to control the calcium ion concentration in the culture solution include calcium chloride, calcium nitrate, or a mixture of both. It may also be controlled by calcium ions in serum.
本発明の培養系中のカルシウムイオン濃度は０．０１ｍ
Ｍ以上０．３ｍＭ未満、好ましくは０．０５ｍＭ以上０
、２５ｍＭ以下、特に好ましくは０．１ｍ）ｉ以上０．
２ｍＮ以下である。培養系中のカルシウム濃Ｉ身を０．
０１ｌｌｌ）Ｉ以１１０、３ｍＭ未満とすることにより
培養系中に生じる細胞塊は１１５％１当りの平均細胞数
が１０個程度となり細胞塊内部の細胞が壊死することな
く長期的に安定に培養することが可能となる。The calcium ion concentration in the culture system of the present invention is 0.01 m
M or more and less than 0.3mM, preferably 0.05mM or more and 0
, 25mM or less, particularly preferably 0.1m) i or more 0.
It is 2 mN or less. Calcium concentrate in the culture system was 0.
01lll) By setting I to less than 110, 3mM, the cell clusters generated in the culture system will be 115%.The average number of cells per cell will be about 10, and the cells inside the cell clusters will be stably cultured for a long period of time without necrosis. becomes possible.
本発明の培養方法において、培養液中の酸素濃度を一定
に維持するために、酸素を供給下［る方法としては、サ
スペンション液中へ酸素または醗索含ｈガスを直接供給
してもよい。他の供給手段としては、例えば酸素キトリ
アーを用いる方法かある。酸素キＸ／リアーとじては、
水と実質的に混合しないで酸素を溶解し得る液状の化合
物が使用され、その例としては、人工血液の素材として
使用されるような種々のフルオロカーボンが挙げられる
。かようなフルオロカーボンを酸素供給手段として使用
する場合には、酸素を溶解させたフルオロカーボンをサ
スペンション液中の上部から液滴状または薄膜状で添加
すればよい。また、酸素透過性のテフロンやシリコンの
デユープを培養槽内に取り付けることによる供給方法も
ある。In the culture method of the present invention, in order to maintain a constant oxygen concentration in the culture solution, oxygen or a hydrogen-containing gas may be directly supplied into the suspension solution. Other supply means include, for example, a method using oxygen chytrier. As for Oxygen Ki-X/Rear,
Liquid compounds that can dissolve oxygen without substantially mixing with water are used, examples of which include various fluorocarbons such as those used as materials for artificial blood. When such a fluorocarbon is used as an oxygen supply means, the fluorocarbon in which oxygen is dissolved may be added from the top of the suspension liquid in the form of droplets or a thin film. There is also a supply method by attaching an oxygen-permeable Teflon or silicone duplex inside the culture tank.
（ｅ）実施例 以下、実施例を掲げて本発明を詳述する。(e) Examples The present invention will be described in detail below with reference to Examples.
実施例
１）培養装置
添付図に示す培養システムを使用した。培養槽は図のよ
うに外壁の内側に隔壁によって仕切られたセトリングゾ
ーンが設けられ、その上部には培養液の排出口を有して
おり、正味培養容積は約１８０ ｄである。Example 1) Culture device A culture system shown in the attached diagram was used. As shown in the figure, the culture tank has a settling zone partitioned by partition walls on the inside of the outer wall, and has a culture solution outlet at the top thereof, and the net culture volume is about 180 d.
？）培養液
基礎培地として、ＲＰ）ｌＩｉ６４０培地、ハム［１２
培地及びダルベツコ変法イーグル培地を２：１：１で混
合したものにさらにブドウ糖、アミノ酸等を加えたもの
（以下ｅＲＤ Ｆと称する）のカルシウムイオンａ度を
通常の０．７４ｍ）ｌから０．１５ｍＨに低減したもの
を用い、増殖因子としてインスリン、トランスフェリン
、エタノールアミン。? ) Culture solution basal medium, RP) IIi640 medium, Ham [12
A 2:1:1 mixture of culture medium and Dulbecco's modified Eagle's medium was added with glucose, amino acids, etc. (hereinafter referred to as eRDF), and the calcium ion concentration was increased from the usual 0.74ml to 0. Insulin, transferrin, and ethanolamine were used as growth factors.
亜しレン酸ナトリウムを加えた。添加濃度はそれぞれ９
μＣＩ ／ｍ、 １０μｇ／ｄ、１０μ）１．２０μＨ
である。Sodium lenite was added. The concentration of each addition was 9
μCI/m, 10μg/d, 10μ) 1.20μH
It is.
３）培養方法及び結果
あらかじめオートクレーブ滅菌した前記培養槽に正味培
養容積が約１８０ ｍｌになるように培養液を送入し、
これにＡＴＣＣから入手したヒト胎児腎細胞由来の２９
３株を０．８ ｘ１０６ ｃｅｌｌｓ ／ｄとなるよう
に播種した。3) Culture method and results: Pour the culture solution into the culture tank, which had been sterilized in advance by autoclaving, so that the net culture volume is about 180 ml.
In addition to this, 29 cells derived from human fetal kidney cells obtained from ATCC
Three strains were seeded at 0.8 x 106 cells/d.
培養槽では炭酸ガス５％を含む酸素ガス（溶存酸素か３
ｐｐｍとなるように）吹込ノズル（８）を通して自動
的にコントロールされた送入されている。培養槽中の培
養液は３７℃に保持されている。培養槽中にはマリン型
攪拌買が取付けられており攪拌速度は４０ｒｌ）ｍであ
った。In the culture tank, oxygen gas containing 5% carbon dioxide (dissolved oxygen or 3%
ppm) through the blowing nozzle (8) in an automatically controlled manner. The culture solution in the culture tank is maintained at 37°C. A marine type agitator was installed in the culture tank, and the agitation speed was 40 ml).
播種後１日間は回分培養を行い、この後潅流を開始した
。潅流の尺度として正味培養容積の１日当りの置換率と
して表わし実験結果を併記する。Batch culture was performed for one day after seeding, and then perfusion was started. The experimental results are expressed as a daily replacement rate of the net culture volume as a measure of perfusion.
すなわち、ポンプＰを駆動し培養槽内で細胞と分離され
た培養液をライン（Ｄ）から扱き取り、その吊と同じ量
の新培地をライン（Ａ）から連続的に送入した。時間の
経過とともに細胞密度が上昇し、６日日には１５ｘ１０
６ ｃｅｌｌｓ ／１ａｌｌに達した。この後細胞の増
殖は定常状態となり、３０日間培養を継続した。その結
果を第１表に示す。That is, by driving the pump P, the culture solution separated from the cells in the culture tank was taken out from the line (D), and the same amount of new culture medium as the suspension was continuously fed from the line (A). The cell density increased over time, and on the 6th day, the cell density increased to 15 x 10
It reached 6 cells/1all. After this, the cell proliferation reached a steady state, and the culture was continued for 30 days. The results are shown in Table 1.
第１表 実施例実験結果
細胞 ２９３株
培地 ＩＴＥＳ−ｅＲＤＦ
２ ０．８ ２．０４ ０．
８ ４．８６ ０．８
１５
８ １．６ ２２
１０ １．６ ２３
１２ １．６ １８
１４ １．６ １８
１６ １．６ ２０
１８ １．６ １７
２０ １．６ １５
２２ １．６ １８
２４ １．６ ２１
２６ １．６ ２３
２８ １．６ ２３
比較例
培養液は通常のカルシウムイオン）農度（０，７４ｎ＋
Ｈ）のＩＴＥＳ−ｅＲＤＦを用いた。他の実験方法は全
て実施例と同様に行った。結果を第２表に示す。Table 1 Example Experiment Results Cell 293 strain medium ITES-eRDF 2 0.8 2.04 0.
8 4.86 0.8
15 8 1.6 22 10 1.6 23 12 1.6 18 14 1.6 18 16 1.6 20 18 1.6 17 20 1.6 15 22 1.6 18 24 1.6 21 26 1.6 23 28 1.6 23 Comparative example culture solution has normal calcium ion) agricultural rate (0.74n+
H) ITES-eRDF was used. All other experimental methods were performed in the same manner as in the examples. The results are shown in Table 2.
第２表 比較例実験結果
細胞 ２９３株
培地 １丁ＥＳ−ｅＲＤＦ
０ ０ ０．６
２ ０．８ １．３４ １．
６ ５．０＆ １．６
１３
８ １．６ １７
１０ １．６ ２３
１２ １．６ ２４
１４ １．６ １８
１６ １．６ １８
１８ １．６ １０
２０ １．６ ８
２２ １．６ ５
２４ １．６ ３
２６ １．６ １．３２８
１．６ ０．５（ｆ）発明の効果
本発明方法によれば、接着陛動物細胞を、それ自身を浮
遊させた状態において、細胞同志の過度の凝集を抑えて
、長期間安定に、かつ高密度で成育させることが可能と
なった。Table 2 Comparative Example Experimental Results Cell 293 strains Medium 1 tube ES-eRDF 0 0 0.6 2 0.8 1.34 1.
6 5.0 & 1.6
13 8 1.6 17 10 1.6 23 12 1.6 24 14 1.6 18 16 1.6 18 18 1.6 10 20 1.6 8 22 1.6 5 24 1.6 3 26 1.6 1.328
1.6 0.5(f) Effects of the Invention According to the method of the present invention, adherent animal cells can be stably maintained for a long period of time by suppressing excessive aggregation of cells in a suspended state. It became possible to grow at high density.
添付図面は、本発明の培養方法を実施り−るために適し
た培養槽の概略図を示したものでおる。
特訂出願人 帝人株式会社The accompanying drawings show a schematic diagram of a culture tank suitable for carrying out the culture method of the present invention. Special applicant Teijin Ltd.
Claims (5)
おいて培養するに当って、培養系中のカルシウムイオン
濃度を０．０１ｍＭ以上０．３ｍＭ未満に維持すること
を特徴とする接着制動物細胞の培養方法。(1) An adhesive animal characterized by maintaining the calcium ion concentration in the culture system at 0.01 mM or more and less than 0.3 mM when culturing adhesive animal cells in a suspended state. Cell culture method.
遺伝子を導入したトランスフォーマントである請求項１
記載の方法。(4) Claim 1 wherein the cells are human embryonic kidney cell-derived 293 strain or a transformant into which a gene has been introduced.
Method described.
塊１個当りの平均細胞数が１０個以下である請求項１記
載の方法。(5) The method according to claim 1, wherein the average number of cells per cell cluster formed by aggregation of cells during culture is 10 or less.
Priority Applications (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12566488A JPH01296986A (en) | 1988-05-25 | 1988-05-25 | Culture of adherent animal cell |
| EP19890109395 EP0343635B1 (en) | 1988-05-25 | 1989-05-24 | Process for continuously culturing adherent animal cells |
| DE1989617642 DE68917642T2 (en) | 1988-05-25 | 1989-05-24 | Process for the continuous cultivation of adherent animal cells. |
| US07/730,386 US5219752A (en) | 1988-05-25 | 1991-07-15 | Process for continuously culturing adherent animal cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12566488A JPH01296986A (en) | 1988-05-25 | 1988-05-25 | Culture of adherent animal cell |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH01296986A true JPH01296986A (en) | 1989-11-30 |
Family
ID=14915604
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP12566488A Pending JPH01296986A (en) | 1988-05-25 | 1988-05-25 | Culture of adherent animal cell |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH01296986A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021145320A1 (en) * | 2020-01-14 | 2021-07-22 | 味の素株式会社 | Cell culture method |
-
1988
- 1988-05-25 JP JP12566488A patent/JPH01296986A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| J.INVEST.DERM=1983 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021145320A1 (en) * | 2020-01-14 | 2021-07-22 | 味の素株式会社 | Cell culture method |
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