JPS5951791A - Cell fusion - Google Patents

Cell fusion

Info

Publication number
JPS5951791A
JPS5951791A JP16082882A JP16082882A JPS5951791A JP S5951791 A JPS5951791 A JP S5951791A JP 16082882 A JP16082882 A JP 16082882A JP 16082882 A JP16082882 A JP 16082882A JP S5951791 A JPS5951791 A JP S5951791A
Authority
JP
Japan
Prior art keywords
cell
cells
strain
fusion
hybrid
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
Application number
JP16082882A
Other languages
Japanese (ja)
Inventor
Tsutomu Abe
力 阿部
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Asahi Kasei Corp
Asahi Chemical Industry Co Ltd
Original Assignee
Asahi Chemical Industry Co Ltd
Asahi Kasei Kogyo KK
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Asahi Chemical Industry Co Ltd, Asahi Kasei Kogyo KK filed Critical Asahi Chemical Industry Co Ltd
Priority to JP16082882A priority Critical patent/JPS5951791A/en
Publication of JPS5951791A publication Critical patent/JPS5951791A/en
Pending legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10Cells modified by introduction of foreign genetic material
    • C12N5/12Fused cells, e.g. hybridomas

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)

Abstract

PURPOSE:To prepare a hybrid cell strain in high efficiency, by synchronizing each period of at least one cell strain with G1 period after cell division, carrying out cell fusion. CONSTITUTION:In preparing a hybrid cell strain by cell fusion between cell strains or from a cell strain and a normal cell, at least one cell strain is cultivated previously in the presence of an inhibitor for cell growth such as indomethacin, etc. only for a time required for one cell division, the inhibitor for cell growth is then removed, the cultivation is continued, an agent for cell fusion is added to the cell strain after 1-3 cell divisions, preferably at G1 period after one cell division, and cell fusion is carried out.

Description

【発明の詳細な説明】 本発明は細胞融合方法に関し、特に高い効率で雑種細胞
株を得る方法に関する。
DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a cell fusion method, and particularly to a method for obtaining hybrid cell lines with high efficiency.

近年細胞融合技術が急速に進歩し、動植物の種種の籠種
細胞株の作成が可能となった。
Cell fusion technology has progressed rapidly in recent years, and it has become possible to create caged cell lines for a variety of plants and animals.

特に細胞融合の促進剤としてポリエチレングリコールは
、極めて用い易く植物のみならず動物細胞のh;東金が
容易に実施しうるまでになった。
In particular, polyethylene glycol is extremely easy to use as a promoter of cell fusion, and has come to the point where it can be easily carried out not only in plants but also in animal cells.

しかし細胞融合による雑種細胞株の形成率は、極めて低
く、より高い効率で屑+: Ili x″III ni
1株を獲得する技術が望まれている。そこで本発明者は
、細胞1捗合の確率を高めよ多多くの雑種細胞株を得る
ことを目標とし、細胞の***周期と細胞融合による雑種
細胞株の形成率との関係に鋭意検討を施し、本発明を完
成するに至った。
However, the rate of formation of hybrid cell lines by cell fusion is extremely low, and the higher efficiency is
Technology to acquire one share is desired. Therefore, the present inventors aimed to increase the probability of cell 1 rate and obtain a large number of hybrid cell lines, and conducted intensive studies on the relationship between the cell division cycle and the rate of formation of hybrid cell lines by cell fusion. The present invention has now been completed.

すなわち本発明は、細1抱林間もしくは細j旭株と正常
細胞とから細胞融合法によシ雑種細1112株を形成す
るに際して、小くとも一方の、71(目1ル株をあらか
じめ細胞増殖の阻害剤存在Fで培養せしめた後、次いで
細胞増殖の阻害剤を除去して培養せしめて工ないし3回
の細胞***をせしめた後の01期に於て細胞融合剤を添
加して細胞融合せしめることからなる雑種細胞株を形成
する方法である。
In other words, the present invention provides that, when forming the hybrid Hosei 1112 strain from Hosho 1 Horinkan or Hosij Asahi strain and normal cells by cell fusion method, at least one of the 71 (eyes 1 Le strain) is subjected to cell proliferation in advance. After culturing in the presence of inhibitor F, the inhibitor of cell proliferation was removed and the cells were cultured to undergo cell division three times. In the 01 stage, a cell fusion agent was added to perform cell fusion. A method of forming a hybrid cell line that consists of incubating a cell line.

本発明いう細胞株とは、動物もしくは植物細胞が試験管
内で無限の増殖をしうる状態になったものである。正常
細胞とは、2倍体の染色体本数を有し試験管内では有限
の増殖もしくは全く増殖しえない細胞である。
The cell line referred to in the present invention is an animal or plant cell that has reached a state where it can proliferate indefinitely in a test tube. A normal cell is a cell that has a diploid number of chromosomes and can only proliferate to a limited extent or cannot proliferate at all in vitro.

細胞融合法は、ポリエチレングリコールな′どの水溶性
高分子もしくは、パラインフルエンザウィルス群のセン
ダイウィルスなどの細胞融合能を有するウィルスを用い
て、複数の細胞を人為的に融合せしめ雑種細胞を形成す
る技術である。
Cell fusion method is a technology in which multiple cells are artificially fused to form hybrid cells using water-soluble polymers such as polyethylene glycol or viruses with cell fusion ability such as Sendai virus of the parainfluenza virus group. It is.

融合せしめる細胞が二種の細胞株である場合は、雑種細
胞株として、二種の細胞株の形質をあわせ持つ雑種細胞
株を獲得しうる。
When the cells to be fused are two types of cell lines, a hybrid cell line that has both the characteristics of the two types of cell lines can be obtained.

また融合せしめる細胞が細胞株と正常細胞である場合は
、雑種細胞株として特定の正常細胞の形質を発現する雑
種細胞株全獲得することができる。
Furthermore, when the cells to be fused are a cell line and normal cells, it is possible to obtain all hybrid cell lines that express specific characteristics of normal cells.

特に後者の細胞株と正常細胞とから雑種細胞株を形成し
、正常細胞の持つ多様な形質を個々に保持した雑種細胞
を獲得できる意義は極めて大きい。
In particular, it is of great significance that hybrid cell lines can be formed from the latter cell line and normal cells, and hybrid cells that individually retain the various characteristics of normal cells can be obtained.

通常細胞融合法を行って雑種細胞株のみを選択する為に
は、耐性株と云われる酸素欠損株を作成し融合に供する
方法、もしくはマイトマイシンCなどの制癌剤で細胞株
を前処理し、増殖能をなくした状態で細胞融合を行い、
細胞融合によシ移入された無傷の増殖機能により雑種細
胞株を選択的本発明を実施する場合特に制限的ではない
が、前者の耐性株を用いる方法は確実に雑種細胞株を獲
得できるので使用し易い。
Normally, in order to select only hybrid cell lines using the cell fusion method, it is necessary to create an oxygen-deficient strain called a resistant strain and use it for fusion, or to pre-treat the cell line with an anticancer drug such as mitomycin C to increase its proliferation ability. Cell fusion is carried out in the absence of
Selective hybrid cell lines due to intact proliferation function transferred by cell fusion Although not particularly limited, the former method using resistant strains can be used because hybrid cell lines can be reliably obtained. Easy to do.

以下耐性株を用いる背景および作成方法を示す。The background and production method for using resistant strains are shown below.

細胞の増殖には核酸合成が必須であるが、大別して核酸
合成の様式には、アミノ酸などから生合成されるデノボ
回路、および核酸塩基の骨格が再利用されるサルベージ
回路がある。
Nucleic acid synthesis is essential for cell proliferation, and the modes of nucleic acid synthesis can be roughly divided into de novo circuits in which biosynthesis is performed from amino acids and the like, and salvage circuits in which nucleobase skeletons are reused.

デノボ回路は、アミノプテリンにょシ阻害を受けること
が知られている。またサルベージ回路中には、ヒボキサ
ンチン・グアニン・ホスホリボシルトランスフェラーゼ
(以下HGPRT  と略す)、チミジン°カイネース
(以下TKと略す)などの皓素が関与している。8−ア
ザグアニンもしくは6−チオグアニンは嗟素HGP几T
によp取り込凍れ、細胞を死滅させる。5−ブロモデオ
キシウリジンはr轟素TKによって取シ込まれ細胞を死
滅させる。
The de novo circuit is known to be inhibited by aminopterin. Further, in the salvage circuit, melons such as hyboxanthin guanine phosphoribosyltransferase (hereinafter abbreviated as HGPRT) and thymidine kinase (hereinafter abbreviated as TK) are involved. 8-Azaguanine or 6-thioguanine is HGP
It freezes up and kills cells. 5-bromodeoxyuridine is taken up by R-TK and kills cells.

細胞株を1〜6ケ月の長期間培養し、8−アザグアニン
もしくは、5−ブロモデオキシウリジンの濃度を序々に
高めてゆくと、自然突然変異により、酵素HGPRTや
TKの欠損した細胞株(以下耐性株という)を得ること
ができる。これらの細胞株はサルベージ回路の一部に欠
陥がある為、デノポ回路のみで核酸合成がなされており
、アミノプテリンを含む培地中では生存しえない。
When cell lines are cultured for a long period of 1 to 6 months and the concentration of 8-azaguanine or 5-bromodeoxyuridine is gradually increased, spontaneous mutations occur in cell lines lacking the enzymes HGPRT and TK (hereinafter referred to as resistant). Stocks) can be obtained. Since these cell lines have a defect in a part of the salvage circuit, nucleic acid synthesis is carried out only through the denovo circuit, and they cannot survive in a medium containing aminopterin.

耐性株の作成方法は、用いる細胞株にょシ薬剤感受性が
異なり、−概に云えないが、黒田行昭著「動物給紙培養
法」共立出版(1974)などの成書を参照し、対象と
する細胞株の薬剤感受性を検訓した後、低濃度から徐々
に薬剤濃度を高めれば良い。もしも剛性様が得にくい場
合には、上述の成書に記載のある、突然変異誘起物質を
用いることによる方法を用いても良い。
The method for creating resistant strains varies depending on the cell line used and their drug susceptibility - although it cannot be generalized, refer to books such as "Animal Paper-Fed Culture Method" by Yukiaki Kuroda (Kyoritsu Shuppan (1974)). After testing the drug sensitivity of the cell line, the drug concentration can be gradually increased from a low concentration. If it is difficult to obtain a rigidity, a method using a mutagenic substance described in the above-mentioned book may be used.

細胞融合に除して、細胞株間の場合は、一方の細胞をH
GPR,T欠損株としておき、もう一方をTK欠損株と
しておき、細胞融合操作を施し、アミノプテリン存在下
で培養することにょジ雑種細胞株の選択は可能である。
In addition to cell fusion, in cases between cell lines, one cell is
It is possible to select a hybrid cell line by using one strain as a GPR, T-deficient strain and the other as a TK-deficient strain, performing cell fusion, and culturing in the presence of aminopterin.

細胞株と正常細胞の場合には、正常細胞は有限の増殖能
を有する為、 1rli4性化された#+++胞株を用
いれば融合したml胞を長期にアミノプテリン存在下で
培養することにより雑種細胞のみが選択されてくる。
In the case of cell lines and normal cells, since normal cells have a limited proliferation ability, if a 1rli4 sexed #+++ cell line is used, the fused ml cells can be cultured for a long period in the presence of aminopterin to generate hybrids. Only cells will be selected.

しかし一般に雑種細胞株が形成される確率は極めて低く
、方法的改良が望まれている。
However, the probability of forming a hybrid cell line is generally extremely low, and methodological improvements are desired.

ところで細胞の***増殖過程における増殖周期は、Di
NA 合成Mj期’(Gt all)、D N A 合
成JtJ] (8期)、DNA合成後期(02期)およ
び***期(M期)に分類されており1通常の培養層II
胞株もしくは正常二倍体細胞は、これら4期のA111
胞の混合状態で存在する。
By the way, the proliferation cycle in the process of cell division and proliferation is Di
It is classified into NA synthesis Mj phase' (Gt all), DNA synthesis JtJ] (8 phase), late DNA synthesis phase (02 phase), and division phase (M phase).1 Normal culture layer II
Cell lines or normal diploid cells are A111 in these four stages.
Exists in a mixed state of cells.

種々な組合せの細胞融合の系があるが、本発明において
はいずれの系に於ても少くとも一方の細胞株の各々の周
期を細胞***後のG□期に同調させることによ〃、雑椋
細胞形成率を有意に向上させることができるとい2知見
を基にしている。この場合他方の細胞株もしくは正常缶
体A411カ包にも、同調操作を施して+i:III胞
***後のG□期として用いても良い。
There are various combinations of cell fusion systems, but in the present invention, by synchronizing the cycles of at least one of the cell lines to the G□ phase after cell division, it is possible to This is based on two findings that it can significantly improve the rate of muku cell formation. In this case, the other cell line or normal A411 cells may also be subjected to a synchronizing operation and used as the G□ phase after +i:III cell division.

牟 細胞の同調する為には細胞増殖の阻害剤存在下で細胞を
培養することが必要であり、代謝阻害剤処理法、チミジ
ンやアデノシン々どの正常な核酸塩基を過剰に培地中に
添加する方法、酸性消炎剤を添加する方法がある。なか
でも酸性消炎剤の使用は好丑しく、具体的にはアスピリ
ンなどのザリチル酸系、フェニルブタシンなどのピラゾ
リジン系、インドメサシンなどのインドール系、ツル7
X−)−ム叡などのアントラニル酸系および、フェニル
酢酸、フェニルプロピオン酸系などがある。
In order to synchronize the murine cells, it is necessary to culture the cells in the presence of cell growth inhibitors, such as metabolic inhibitor treatment methods and methods of adding excessive amounts of normal nucleobases such as thymidine and adenosine to the culture medium. There is a method of adding an acidic anti-inflammatory agent. Among them, it is preferable to use acidic anti-inflammatory agents, such as salicylic acid-based drugs such as aspirin, pyrazolidine-based drugs such as phenylbutacin, indole-based drugs such as indomethacin, and
Examples include anthranilic acids such as

特にインドール系は好捷しく、中でもインドメサシンは
、面・1性株に用いた場合細胞障害性が低く用いやすい
Indoles are particularly preferred, and among them, indomethacin has low cytotoxicity and is easy to use when used against monosexual strains.

インドメサシンの濃度は、細胞培養用の牛脂児穐清(以
下FC8と略す)を10%含むイーグルのMEMもしく
は、R,PMI−1’640などのそれぞれの細胞株に
適した培地に0.05〜1mMに調整し用いられる。
The concentration of indomethacin is 0.05 to 0.05 to 0.05 to 0.05 or more in a medium suitable for each cell line, such as Eagle's MEM containing 10% FC8 for cell culture, or R, PMI-1'640. It is adjusted to 1mM and used.

細胞株を該薬剤入り培地で培養するが、このとき用いる
細胞株の1回の***に要する時間をあらかじめ既知とし
おき、その50〜200チの時間培養ずれば良い。好1
しくは1回の***に要する時間だけ、細胞株を培養すれ
ば良い。
The cell line is cultured in the drug-containing medium, and the time required for one division of the cell line used at this time is known in advance, and the culture may be delayed for 50 to 200 hours. Good 1
Alternatively, the cell line may be cultured for only the time required for one division.

続いて薬剤を除去し、通常の培地で培養を絖けA径時的
に細胞数を計測することにより、一定時間後に同調した
***現象が藏(められる。この***が生じた後に該細胞
株は、G1期に移行する。この状態の細胞株を細胞融合
に供する。
Next, by removing the drug and culturing in a normal medium and counting the number of cells over time, a synchronous division phenomenon can be detected after a certain period of time.After this division occurs, the cell line The cell line enters the G1 phase.The cell line in this state is subjected to cell fusion.

インドメサシンは、11111胞を01期にて停止させ
ることが知られているが、インドメサシン除去直後に細
胞融合に供しても、著量な麗種細胞の形成促進効果はな
い。これは、インドメサシンによる毒性の効果であろう
と考えられ、インドメサシン除去後1ないし3回、好ま
しくは1回の11日胞***の後の01期細胞を用いるこ
とが良い。また薬剤処理の後あまり多数回の細胞***後
の、細胞株を細胞融合に供しても徐々に通常の培養条件
の細胞株に近すいてしまい、効果は低減していく。
Indomethacin is known to arrest 11111 cells at the 01 stage, but even if the cells are subjected to cell fusion immediately after indomethacin removal, there is no significant effect of promoting the formation of Reishin cells. This is considered to be a toxic effect of indomethacin, and it is preferable to use 01 stage cells after one to three, preferably one 11-day cell divisions after removal of indomethacin. Furthermore, even if a cell line that has undergone cell division too many times after drug treatment is subjected to cell fusion, it will gradually become closer to the cell line under normal culture conditions, and the effect will be reduced.

G1期細胞が細胞融合に適していることの理論的説明は
未だ充分になしえないが、恐(DNA合成期(8期)以
前の染色体構造が寄与しているのであろう。
Although there is still no satisfactory theoretical explanation for why G1 phase cells are suitable for cell fusion, the chromosomal structure before the DNA synthesis phase (8th phase) probably contributes.

次に抗体を分泌しないマウスミエローマの8−アザグア
ニン耐性株P3−U1株と、ヒト末梢血リンパ球との細
胞融合による雑種細胞コロニー形成方法を例にとυ1本
発明の実施方法を具体的に示す。
Next, a method for implementing the present invention will be specifically described using a method for forming a hybrid cell colony by cell fusion of a mouse myeloma 8-azaguanine-resistant strain P3-U1, which does not secrete antibodies, and human peripheral blood lymphocytes. .

P3−U1株は、10%FC8を含むRPMI −16
40培地では、1***に要する時間は16〜18時間で
ある。
P3-U1 strain is RPMI-16 containing 10% FC8.
40 medium, the time required for one division is 16-18 hours.

P3−U1株I X 107個を0.4mMのインドメ
サシンおよび10チのFe2を含むR,PMI −16
40培地20−に分散せしめ24時間培養する。絖いて
細胞を遠心分離し、培養上清を除去し、10%のFe2
を含む几PMI −1640培地にて24時間培養する
P3-U1 strain I x 107 cells were treated with R, PMI-16 containing 0.4mM indomethacin and 10% Fe2.
40 medium and cultured for 24 hours. Centrifuge the cells, remove the culture supernatant, and add 10% Fe2.
The cells were cultured for 24 hours in a PMI-1640 medium containing PMI-1640.

この操作によシ同調処理後、1回の細胞***をへた01
期細胞が得られる。(以下この状態のP3−U1株を処
理細胞株と呼ぶ) 一方健常者末梢血を採取し、ヘパリン添加した後、フィ
コール・コンレイ液を用いた比重遠心法を施して2間層
細胞を得る(以下これをPBLと呼ぶ)。
By this operation, one cell division was completed after the synchronization treatment.
Phase cells are obtained. (Hereinafter, the P3-U1 strain in this state will be referred to as the treated cell line.) On the other hand, peripheral blood from a healthy individual is collected, heparin is added, and then specific gravity centrifugation is performed using Ficoll-Conray solution to obtain bilayer cells ( (hereinafter referred to as PBL).

3.3 X 106個ずつの両細胞を・・ンクス処方生
理食塩水(以下これを)・ンクス液と呼ぶ)にて2回洗
浄し、良く培地中の蛋白成分を除去しておく。
3.3 × 106 cells of both cells were washed twice with NX-prescribed physiological saline (hereinafter referred to as NX solution) to thoroughly remove protein components in the medium.

一方2グのポリエチレングリコールを2.8 meのハ
ンクス液に溶解し、pHを8.1〜8.5に調整した後
除菌フィルターに濾過静閑しておく。(以下これをPE
G溶液と呼ぶ) 処理細胞株とPBLを遠心処理し、ペレット化したもの
に、1mlのPEG溶液を2分間で、徐々に滴下する。
On the other hand, 2g of polyethylene glycol was dissolved in 2.8me of Hank's solution, and after adjusting the pH to 8.1 to 8.5, it was filtered through a sterilizing filter and left to stand still. (Hereinafter, this will be referred to as PE
(referred to as G solution) The treated cell line and PBL are centrifuged and pelleted, and 1 ml of PEG solution is gradually added dropwise over 2 minutes.

次いで、5rn1.の培地RPMI −16401f 
2分間で、徐々に添加し、PEG溶液を希釈する。続い
て15−の培地RPMI −1640を2分間をかけ添
加し遠心分離してPEGを除去する。
Then, 5rn1. Medium RPMI-16401f
Add slowly over 2 minutes to dilute the PEG solution. Subsequently, a 15-ml medium RPMI-1640 was added for 2 minutes and centrifuged to remove PEG.

選択培地として20チのFe2 、 1o ’Mのヒポ
キサンチン、4X10”Mのアミノプテリンおよび1.
5X10’Mのチミジンを含む培地JもPMI−164
0を107!調製し、PEG処理後細胞に添加し細胞を
分散せしめる。
As selection medium 20 t Fe2, 10'M hypoxanthine, 4X10'M aminopterin and 1.
Medium J containing 5 x 10'M thymidine was also used as PMI-164.
0 to 107! It is prepared and added to cells after PEG treatment to disperse the cells.

次いて96ウ工ル組織培養用プレートの半分の穴に20
0μtずつ該細胞浮遊液を分注し、3週間程度培養を継
続し、2〜5日毎に培地交換を行うことにより雑種細胞
のコロニーを獲得しうる。
Then fill the half holes of a 96-well tissue culture plate with 20
Colonies of hybrid cells can be obtained by dispensing 0 μt of the cell suspension, continuing culturing for about 3 weeks, and replacing the medium every 2 to 5 days.

このコロニーを引きつづき培養することにょシ異種動物
間雑種細胞株を獲得することが可能である。
By continuously culturing this colony, it is possible to obtain a hybrid cell line between different species of animals.

この方法は、P3−U1株に限らず、いずれの細胞株を
用いた場合でも雑種細胞株の形成確率を向上することが
可能である。
This method can improve the probability of forming a hybrid cell line when using any cell line, not just the P3-U1 strain.

実施例1 (P3−U1株のインドメサシン処理)P3−U1株1
0?個を0.4 mMのインドメサシンおよび10%の
Fe2を含む20rntの培地RPMI−1640にて
24時間培養した後、インドメサシンを遠心分離し10
チのFe2を含む2o−のRPMI −1640にて2
4時間培養する。生細胞比率をトリハンプルー色素排除
試験により求めた後、生細胞3.3X10’個 を以下
の細胞融合に供した1、(健常者末梢血単核球(PBL
)の調製)健常者末梢血201dを、抗凝固剤としてヘ
パリンヲ用いて採血し、フィコール・コンレイ液(比重
1.077)  を用いた比重遠心法により、新鮮リン
パ球を含むPBL 2X107個を獲得した。
Example 1 (Treatment of P3-U1 strain with indomethacin) P3-U1 strain 1
0? After culturing the cells for 24 hours in 20rnt medium RPMI-1640 containing 0.4mM indomethacin and 10% Fe2, indomethacin was centrifuged and 10rnt
2 at RPMI-1640 of 2o- containing Fe2 of
Incubate for 4 hours. After determining the viable cell ratio by Trihampe blue dye exclusion test, 3.3 x 10' living cells were subjected to the following cell fusion (1) (healthy peripheral blood mononuclear cells (PBL)).
) Preparation) 201d of peripheral blood from a healthy individual was collected using heparin as an anticoagulant, and 2×107 PBLs containing fresh lymphocytes were obtained by specific gravity centrifugation using Ficoll-Conray solution (specific gravity 1.077). .

このうち3.3 X 106個の細胞を以下の細胞融合
に供した。
Of these, 3.3 x 106 cells were subjected to the following cell fusion.

(ポリエチレングリコール(PEG)液の作成)2tの
ポリエチレングリコールを2.8mlのハンクス液に溶
解し、pHを8.1〜8.5に調整し、0.22ミクロ
ン孔径の除菌フィルターにてp過除菌を施しておいた。
(Preparation of polyethylene glycol (PEG) liquid) Dissolve 2 tons of polyethylene glycol in 2.8 ml of Hank's solution, adjust the pH to 8.1 to 8.5, and filter through a 0.22 micron sterilization filter. Excessive sterilization was applied.

(細胞融合操作) インドメサシン処理した細胞株およびPBLを各、3.
3X10’個を50−の滅菌した遠心管に入れ、ハンク
ス液にて2回洗浄を施し、細胞をペレット化した。続い
てペレットを良く叩きほぐし、umのPEG溶液を徐々
に滴下し、細胞と良く混合した。この操作には2分間を
要した。
(Cell fusion operation) Indomethacin-treated cell lines and PBL were each treated with 3.
3×10′ cells were placed in a sterile 50-centrifuge tube, washed twice with Hank's solution, and the cells were pelleted. Subsequently, the pellet was well beaten, and the um PEG solution was gradually added dropwise to the pellet and mixed well with the cells. This operation required 2 minutes.

次に5−のRPMI−1640を滴下しPEGを徐々に
希釈した。この操作には2分間を要した。続いて15d
のRPMI−1640にてPEGを希釈した。
Next, 5-RPMI-1640 was added dropwise to gradually dilute the PEG. This operation required 2 minutes. followed by 15d
PEG was diluted in RPMI-1640.

この操作には2分間を要した。This operation required 2 minutes.

次に600回転5分間遠心分離し、上清中のPEGを充
分に除去した。そこに20チのFe2.10−’Mのヒ
ポキサンチン、4X10”−’Mのアミノプテリンおよ
び1.5X10’Mのチミジンを含む培地を10−添加
し、細胞を分散せしめ、96ウ工ル組織培養用プレート
の半分に200μtずつ分注したO1O1oCO2培養
器にて培養した後、生き残ったio個以上の細胞からな
る雑種細胞コロニー数を算出したところ33であった。
Next, the mixture was centrifuged at 600 rpm for 5 minutes to sufficiently remove PEG in the supernatant. A medium containing 20 pieces of Fe2.10'M hypoxanthine, 4 x 10''-'M aminopterin and 1.5 x 10'M thymidine was added thereto, the cells were dispersed, and 96 wells of tissue were prepared. After culturing in an O1O1oCO2 incubator in which 200 μt each was dispensed into half of a culture plate, the number of hybrid cell colonies consisting of io or more surviving cells was calculated to be 33.

このコロニーを培培養しつづけたとと18のコロニーか
らヒト免疫グロブリンの産生が認められた。
When these colonies were continued to be cultured, production of human immunoglobulin was observed from 18 colonies.

(比較例1) 実施例1で行ったP3−U1株のインドメサシン処理操
作を施さなかった以外、全く同様の細胞の組合せおよび
手法で細胞融合を実施した。得られた雑種細胞コロニー
数を実施例1と同様の方法で算出したところ、17であ
った。このコロニーを培養しつづけたところ、8のコロ
ニーからヒト免疫グロブリンの産生が認められた。
(Comparative Example 1) Cell fusion was carried out using the same combination of cells and method, except that the P3-U1 strain was not treated with indomethacin as in Example 1. The number of hybrid cell colonies obtained was calculated in the same manner as in Example 1, and was found to be 17. When these colonies were continued to be cultured, production of human immunoglobulin was observed from 8 colonies.

実施例2 ヒトTれ1胞株CCRF −CEM (ATCCNo 
、CCL−119)を87ザグアニン存在下で培養をつ
づけ、耐性株を作成した。8−アザグアニンの濃度は最
終的に上20μ?24艷で培養をつづけ、4X10−’
Mのアミノプテリンを含む培地にて該8一アザグアニン
剛性株が死滅することを確認して以下の実験に供した。
Example 2 Human Tle1 cell line CCRF-CEM (ATCC No.
, CCL-119) was continued to be cultured in the presence of 87 zaguanine to create a resistant strain. 8- Is the final concentration of azaguanine above 20μ? Continue culturing in 24 cells, 4X10-'
It was confirmed that the 8-azaguanine rigid strain was killed in a medium containing aminopterin of M, and was used in the following experiment.

該耐性株10’個を、1mMのアスピリンおよび10%
のFe2を含む培地RPMI −1640Y(て24時
間培養した後、インドメサシンを除去し、30時間培養
しインドメサシン処理劇性株を作成した。
10' of the resistant strains were treated with 1mM aspirin and 10%
After culturing for 24 hours in medium RPMI-1640Y (containing Fe2), indomethacin was removed and cultured for 30 hours to create an indomethacin-treated virulent strain.

以下用いた細胞株以外は実施例1と同一の操作を行い、
ヒト−ヒト細胞融合を試み、実施例1と同様の方法で雑
種細胞コロニー数を算出口たところ、22のコロニーが
検出された。
The same operations as in Example 1 were performed except for the cell lines used below.
When human-human cell fusion was attempted and the number of hybrid cell colonies was calculated in the same manner as in Example 1, 22 colonies were detected.

特許出願人 旭化成工業株式会社Patent applicant: Asahi Kasei Industries, Ltd.

Claims (1)

【特許請求の範囲】[Claims] 細胞株間もしくは細胞株と正常細胞とから細胞融合法に
より雑種細胞株を作成するに際して、少くとも一方の細
胞株をあらかじめ細胞増殖の阻害剤存在下で培養せしめ
た後、次いで細胞増殖の阻害剤を除去して培養せしめて
1ないし3回の細胞***をせしめた後の01期に於て細
胞融合剤を添加して細胞融合せしめることからなる雑種
細胞株を作成することを特徴とする細胞融合方法
When creating a hybrid cell line by cell fusion between cell lines or between a cell line and normal cells, at least one cell line is cultured in the presence of a cell proliferation inhibitor, and then the cell proliferation inhibitor is injected. A cell fusion method characterized by creating a hybrid cell line by adding a cell fusion agent in the 01 stage after culturing and cell division 1 to 3 times to cause cell fusion.
JP16082882A 1982-09-17 1982-09-17 Cell fusion Pending JPS5951791A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP16082882A JPS5951791A (en) 1982-09-17 1982-09-17 Cell fusion

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP16082882A JPS5951791A (en) 1982-09-17 1982-09-17 Cell fusion

Publications (1)

Publication Number Publication Date
JPS5951791A true JPS5951791A (en) 1984-03-26

Family

ID=15723283

Family Applications (1)

Application Number Title Priority Date Filing Date
JP16082882A Pending JPS5951791A (en) 1982-09-17 1982-09-17 Cell fusion

Country Status (1)

Country Link
JP (1) JPS5951791A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008046033A3 (en) * 2006-10-13 2008-08-28 Centocor Inc Enhancement of hybridoma fusion efficiencies through cell synchronization

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008046033A3 (en) * 2006-10-13 2008-08-28 Centocor Inc Enhancement of hybridoma fusion efficiencies through cell synchronization
JP2010511379A (en) * 2006-10-13 2010-04-15 セントコア・オーソ・バイオテック・インコーポレイテッド Improvement of hybridoma fusion efficiency through cell synchronization
US8216842B2 (en) 2006-10-13 2012-07-10 Centocor Ortho Biotech Inc. Enhancement of hybridoma fusion efficiencies through cell synchronization

Similar Documents

Publication Publication Date Title
CN109735497B (en) Method for improving expression level of fetal hemoglobin
Littlefield The use of drug-resistant markers to study the hybridization of mouse fibroblasts
Uchida et al. Different in vivo repopulating activities of purified hematopoietic stem cells before and after being stimulated to divide in vitro with the same kinetics
Holliday Ustilago maydis
Coleman et al. A clonal study of the reversible inhibition of muscle differentiation by the halogenated thymidine analog 5-bromodeoxyuridine
Roberts et al. Evidence for the inactivation and repair of the mammalian DNA template after alkylation by mustard gas and half mustard gas
GB2556276A (en) Engineered crispr-CAS9 compositions and methods of use
Hadwiger et al. Mode of pisatin induction: Increased template activity and dye-binding capacity of chromatin isolated from polypeptide-treated pea pods
DE69333669T2 (en) MUTAGENESIS AND SELECTION METHODS IN THE SAME HOST CELLS
Brachet Nucleocytoplasmic interactions in unicellular organisms
WO2023011638A1 (en) Fusion protein and use method thereof
De Mars et al. A method of selecting for auxotrophic mutants of HeLa cells
Ephrussi et al. Hybrid somatic cells
Limberg et al. Variants of soybean cells which can grow in suspension with maltose as a carbon-energy source
JPS5951791A (en) Cell fusion
Kiang et al. Cyclic biological expression in mouse mammary tumors
JPS58216125A (en) Preparation of human antibody
Gibson et al. The action of ribonuclease and 8-azaguanine on mate-killer paramecia
Thomas Experimental imitation of tumour conditions
CN106754714A (en) The method that cord blood sample dilution, kit and treatment Cord blood obtain stem cell
Wang et al. Establishing cGMP manufacturing of CRISPR/Cas9-edited human CAR T cells
JPH0253490A (en) Arginine deiminase gene
Kuriya et al. Direct visualization of DNA replication dynamics in zebrafish cells
LUEKEN A marine Euplotes (Ciliophora, Hypotrichida) with reduced number of prezygotic micronuclear divisions
Thekkumkara et al. Ochratoxin a decreases the activity of phosphoenolpyruvate carboxykinase and its mRNA content in primary cultures of rat kidney proximal convoluted tubule cells