発行責任者

日本組織培養学会

会員通信
第 26号

※佐藤温重・※梅田誠※※加納永一※横浜市南区浦畑T 携兵市大・医学部※※京都市東山区山科御凌瀦日葉大昭和 50年 8月 1日発行

dc 第 40回研究会要項決まる
別紙(色頁)の如くに研究会が開催されます。(宿泊 K関しては別項を拳照して下さい〉:*幹事会および総会議事録
6月 19日休:).金沢大・薬・放射薬品化学教室虻おいて幹事会が開かれ，下記の事原について協議された。また.これらの協議事現は翌 20日恰)の第 39回研究会総会 Kおいていづれも可決承認された。
なお.幹事会出席者は野瀬.久米川，佐藤.堀川.黒木.難波，二階堂の新旧各幹事のほか.
会計担当の山田氏.会員通信担当の梅田氏.ピプリオグラフィー担当の乾氏であった o(
1) 幹事長:幹事改選 K伴い. 1975年度の幹事長は東大・医科研・癌細胞の野瀬清氏に決定。

(
2) 新幹事:本年 2月 Kおこなわれた幹事選挙の結果.以下の 4氏が当選され，新幹事となった
治Z(東部) 黒木登志夫氏.武田久雄氏(西部〉

難波正義氏.二階堂修氏

このうち黒木氏が渡欧，武田氏が渡ソされるため，くりあげて

丸野内様氏(三菱主命研) .

佐藤茂秋氏(国立がんセンタ→にお世話を願うこと Kなった o なお，本来なら次点の須田立雄
氏(東京医歯大)がくりあげ当選 Kなるところであるが，今回K限り特殊事情を認めて銘旦を承認した。

(
3) 会計報告: 担当の山田氏より別紙(会計通信第 25号 多 照 )~てもとづき昭和 49 年度会計報告があり，承認された。
なお，種々問題はあるが会費は当面 1000円すえおき Kされたo(4) ピプリオグラフィー:黒木氏の渡欧に伴¥" .ピプリオグラフィー担当乾氏の補佐役として.
新たに佐膝茂秋氏 Kお願L、することになった。(本年度のピプリオグラフィーに関しては別紙多照)(
5) 会員通信:原稿の集りがわるく，会員各位からどしどし原稿を送ってほしい旨担当の梅田氏より要望があった。なお.今後は長期海外出張の会員 K も会員通信を送ること忙なった。
また.会員が住所変更した場合 Kは，学会事務センターに直接連絡するよう要望があった。
(
6) 会員名簿作製: 再三の催促にもかかわらず未だ約 110名の未回答者があり.作製は難守している。未回答者 Kは今後再度催促して今年中 Kは何と b完成にこぎつけたL、旨作製担当の佐藤氏.堀川￨より報告があった。
‑1‑

(
7
) 新入会員:新入会員，新入賛助会員，退会会員(別展重量照)~ついては，すベて承認さ

れた。

(
8
) 次回研究会:下記の要領で開催する。
世話人: 独 協 大 学 医 学 部 山 田 喬 氏
会 期 : 昭和 50年 10月 24日
・ 25日
場 所 : 独協大学医学部構堂

特 Kシ y ポジウム形式のものはやらず，上皮細胞および血液細胞の培養等を中心としたウワ
ークショップを企画中(別紙重量照)。

(9) 次々回研究会: 世 話 人 : 九 州 大 学 医 療 短 大側その他

高木良三郎氏

3年 後 K 日本で国際老年学会( International Congress ofGerontology)が開催されるが培養細胞関係では東大・医科研勝田氏がオーガナイズする o Dr.Hayf1
ickを始めとする外人 5名と日本側から 2名の合計 5名位をシンポジウムのスピーカーに考えている旨勝田氏より報告があった。(別項参照)(文責堀川正克)

ぬ 昭 和 50年 6月

新入会員名簿

所属機関

同住所・

電話

980 仙台市星陵町 2‑1

東北大学医学部

第 5解剖学教室

氏

名

石川

博

専門分野
解剖学

(0222)74‑1111
内総

472
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三楽中オ央

251 藤沢市城南 4・9・1

沖

俊一

徴生物学

誠

皮膚科学

(0466) 34・4176
資生堂研究所

第 12研究室
昭和大学医学部
泌尿器科学教室

222 横 浜 市 制 限 新 邪T
1050(045) 542・1331

宇塚

141 東京都品川区旗の台
1‑5・8(03)784・1151

池内隆夫

257 神奈川県秦野市

渋谷

薬物学
~尿器科学

徹

突然変異

210 川崎市高津区下作延
1604(044)833・5151

中山隆治

生化学

東京慈恵会医科
問郎内科 *‑
企訪

105 東京都港区西新橋

尾林紀雄

内科学

東京大学医学部

113 東京都文京区本郷
7・5・1(03)815‑5411

食品薬品安全センター
秦野研究所
帝国薬臓理器研製究薬郎株会
社
式

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老年病学教室(精尿病)

5・25‑8

4

内線

8344

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老年病学

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東京大学薬学都
生理 化 学 教 室

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名

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専

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113 東京都文京区本郷7‑3‑1(03) 812‑2111

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内藤
東京大学医科学研

細胞生物学

348自
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108 東京都港区白4

究所附属病院舛科
ペーリンガー・イ y腫痕免疫666‑01 兵 庫 県 川 西 市 ￨ 西 川 順 子

グルハイム・日本

矢問守高田 103

薬理学研究所(0727) 93・8585

金沢大学薬学部

920 金沢市宝町 13・1

生物薬品化学教室(0762) 62‑8151
内線￨京都大学医学部
放射能基礎医学

衛生化学教室医 学 部 解 剖j
学437

内 線 4410.

15

内線 4410‑4412

伴内 海

1606 京 都 市 左 駆 F吉I田下(075) 751‑2111 述町

高木

内線
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606 京叡市左
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4537

545 大阪市阿倍野区 h伊了
1‑4‑54(06) 633‑1221

教室

内線

愛媛大学医学部

791‑02 愛媛県温重泉郡信町

第 1解剖j学教室(089964) 3811

鹿児島大学医学部

890 鹿児島市宇宿町
1
1
1
208・1
(0992) 56‑22

病理学教室

組織化学

大西礼子

細胞生物学

2015
高島庸一郎￨解剖学・超徴綱削除

内線一 2112
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同住所・電話I458 名古屋市緑区鳴海町四本木

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梅原澄子

2113

(052) 622・ 5261
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102 東京都千代田区飯田鶴

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イースト・リミテッド(03) 264・2491
成 住 所 変 更

951 新潟市旭町通一番地 757

新潟大学医学郎(0252) 23・6161

病理学教室
北陸大学薬学部.

生理化学教室名古屋保健衛生大学医470・11 豊明市沓掛町田楽ケ僅

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宇都宮市大谷 1228

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武宇都宮鵬官下車(0286) 52‑0524 I

但 し 宿 泊 者 15名以ー切場合
はマイクロパスで東武宇都宮駅まで送りますo

千 隠 荘

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I2β00円 2食付

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1室 1‑2人

東武宇都宮線おもちゃの町
駅下車徒歩 5分

'
‑
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喧
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.

注怠事項
1 会場は来武宇郁也:1B'J!おもちゃの町駅(京武宇都宮K
Rより 15分)下車徒歩 1:，分

2
. 東武宇都宮訳と国鉄宇都宮駅は徒歩 30分位(パス 10分)はなれていますので。
、御注窓下さ L‑5ー￨

*国際老人病会議について
1978年夏第 11聞の会議が日本で開催されます。これは一般演説と各種シンポジウムから成s in Culture "というのが計画っていますが，そのシ Y ポジウムの一つ.‑c:"Aglng of Cel1

されています。演者として日本人 2人，外国人 5人.計 5人が予想されます。つきましては，この日本人演者 2人を公募いたしますので，御希望の方はあと 1年余以内位の培養学会で成果を御発表下さ、、。採否はその成果 K よって判定いたします。

dc 講演会のお知らせ
時所者

日場演

8月 25日 午 前 10時
東大医科研本館

2階会議室

Profo Fo Ho Kasten
LouisianaState Univo l:Iedo Center Dep色。Anatomy"Differentia
もedandundifferentiatedneuroblasもoma
cells in vitro""Mitosis and differontiaも io~ ofmammaliancardiaccells in cultureU
官

演 題

~

第3
9回研究会を終えて
堀川正克(金沢大・薬・放化)

ω

(
金
)
・ 21日 の両日，金沢大学医学郎十全講堂で日本組織培養学会第 39回研究会は 6月 20日
y が学生突習のため，J
年より約 1ヶ月も遅れて研究会は開催され開催された。 Host Universit

た。そのため，今度は雨が心配されたが，幸い前日まで降っていた雨もからりとあがり，会期中
は何とか時天 K恵まれた。研究会が終るやいえEや翌日からまたドシャ降りの雨になったのも皮肉といえば皮肉であるが，世話人側からするとまったく H ついていた H の一語 Kっきる。
さて，地理的条件もあって前回の第 38回研究会陀は及ばないまでも.今回の研究会で登録された参加者は正会員 87名，非会員 102名，学生 25名の計 214名であったから，まあまあの研究会であったと言わざるをえま L、。それにも泊して 39回目 K して初めて北湿の地で研究会が開催されたということ K窓農を見いだしたい。
今回は特に黒木氏(東大・医科研) .黒田氏(国立泣伝研〉の力をおかりして H 崎乳類細胞 K
おける突然変異の機構解析 w と慰するシンポジウムを企直した。しかも一日ぶっ続けのシ y ポジJ
応答のうちに幕切れとなっウムである。当初危倶した質問のマンネリ化もなく，終治活発な質Jたのは両氏の名司会に負うところ大であるが，くわえて各演者 K よって発表された研究内容の深さ，充実さ，東 Kその重要性によるものであったろう。晴乳類細胞を用いての突然変異の研究は細胞の癌化および老化といった重要問題と関連して.今後更にその系の確立，それらを使つての機構解析が精力的 K進められるものと思う。今回のシンポジウムがそのための triggerともなれ‑6‑

ば幸いである。
また.シンポジウム忙関連してか，一般講演の中 K もこれに類する内容のものが多々報告されたが.これも今回の研究会の特徴であったろう。事実，シ Y ポジウムの部に入れるか一般鱗演でやってもらうか.その取捨選択 K悩んだのは舞台患のいつわざる苦労談でもある。ともあれ〆、エ細胞の培養が出 .7=:1ヤ外套臆培養 Kよる真珠倒 1)とまでは行かないまでも.一般錦演 Kも興味ある演題がずらりと並んだのは事実である。これらの研究が今後とも更に一段と発展することを祈って止まない。
騒後 tc.長時間シンポジウムの司会を努めて下さった黒木氏.黒田氏.会をもりたてて下さった各セッションの座長および演者，また遠路をいとわず御多会いただいた出席者諸氏 K この場をかりてお礼申しあげる。

ぬ 海 外 情 報(
1
) アメリカ合衆国では各種の workshop や training course がはやっているが，段丘医c.本年度の組織培養関係の education program が届い九
科研勝目先生の手許 t
日本の本学会会員諸氏 Kは既 K終ってしまったものもあり.出席は不可能 K近いと思われるが.
御善参考迄 K要点を御紹介する。
主催は W. Alton Jones Cell Science Center (Lake Placid ，NY12946)
で培養学会が協賛?の形をとっているようである。

対象は大学院生，大朝淀卒業者.成主充

分な基礎知識を持つ技術者で，費用は培養学会員で $110‑550・会員外でー制高とえEっている o

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工法 C
hromosome Mapping
Ce11 Hybridization adiation Bio1ogy
Ce11u1arRa
The Preuara
もionofMadia forAnima1 Cell
Tissue andOrgan Culture置u1ation
10 For
1L Steri1izationSe'Pt
o 22 2ιÇuantitativ~ 9ptic~1 T
echniques in Ce11 Culture
Septog9 Ocも03 Detection of時 cop1asma均r
cop1asmaandAcbo1eplasmain Biological andOcto6‑7
Medical Problems
Cel1 Culture in Studies of Senescence
Octo16‑17
Octo23 2ラCu1ture ofNervous TissuePr
imaryVertebrate Ce11 Cu1ture
Octo 27・3
1
Clonal Cult
町 e
Novo 3・7
The Bulk CulもureofHeteroploidDiploidand
Novo 10‑1らPrimaryCells
Deco1国ラKaryo1ogy of Cu1turadCe11s
圃回

圃

日本の学会で其似るとなるといろいろの怠味で大変であるが，やはり会社が中 K入り，費用も高くならざるを得ない現状がうががえる o しかし興味のあるのは 0 印をつけた標題もあり，
このような形でも知見交換，正しい培養法の標準化 Kは役立つかも知れないと思われる o(2) これも勝目先生の入手した資料ですが. Annual Meeting of the European Tissue
Cu1ture Society が 5月 5日から 7日迄聞かれた。 Programmeの演題を沿ってみると，

以下のようです。

1 Oncogenic virus and tissue culture (8題 )，I Massculturcs (3思〉
1T
I Gro‑:，
rth characteristics of variOU3 cells in vitro (7題 )rv Special aspects of tis3ue culture (6思 )V Drug action on cclls in vitro (7沼)な 一 般 民 冠 ( 17窓)
彼地の研究の傾向がわかればと{司会考迄にお知らせしましたo(文責梅田〉~編集後記

会長治信玄号をお送りします。
わけありません。

夏休み前に発行すると計図していながら，いつも遅れて巾し

次回研究会での発表の予定陀問[ぐっ守うことを腐っています3

2
4
・事会議事録I'Lもありますが，会員名ほ，ピプリオグラ 7 ィ-~~}・の本三=の事業 K{:1~jl 何協力をお思いします。

私共からのお瓜いとしてはf
斤属変災，海外出混等の変廷がありましたら，これからはすべて学会
事務センタ ‑
tc通知していただくこと Kなりましたので宜段く。

長}本号には川崎医大難波氏の御協力で Dr.Hayflick の競演原稿をのせました。紙面の関係で小さな活字 Kなりましたが御容赦下さい。

次号原稿は 1
1月 15日締切りの予定てす。

‑8ー会員樹影〉御投稿を望んでいます、(u)

1 am sure that you wil1 appreciate that the subjectof aging or gerontology is a very large subject and impossible
HUMAN AGING AT THE CELLULAR LEVEL

to discuss in one hour to say nothing of the fact that 1
must speak very slowly during the 1hour. 50，1will confine
my remarks to one very small area of the cell biology of
aging，with your understanding hopeful1y that 1 am covering

Professor Leonard Hayflick，P
h
.D
.
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s
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just a very narrow area.
1 think that one of the major problems in the field ofgerontology unlike other areas of biology is the definition(Lectured at Kawasaki Medical College
on 5eptember 19，1974)of the subject under study，strangely enough you might besurprised to learn that the gerontologists or biologists who
study aging cannot agree even on the definition of the subject，
that is，what is biological aging. 1 thought that 1would
try to give you the best definition that 1 know by simply
showing you the two extremes of the problem，which appear
on the first slide that 1 have. 50，if 1 can have the firstslide，1will show you，this is the definition of aging，
that is，what biological changes take place in going fromthis condition to this condition.
This is the easiest definition that 1am able to give you.
But 1 am sure you wil1 understand that this i
s a verycomplicated subject that results in these very dramaticchanges.
One of the important points that 1would like to mention
to you is that the study of aging is not necessarily thestudy of the diseases that occur in humans and itis importants a distinction between the diseases
to realize~hat there i‑ 1‑that occur as individua1s age and the bio10gical changes

Here you see very few dea主hs in the ear1y years，then therethat give rise to age changes. And 1 think that itis worthis a p1ateau，and the number of individua1s dying then begins

under1ining this point by showing you some data that revcal

to increase unti1 some individua1s remain at this point

very dramatica11y the question of improvement in hygienic

and 1ive unti1 the maximum period of 1ife. Now the main

conditions and curing of diseases and their impact on the

point that this table of information indicates is that as

human 1ife span. Itis important a1so to recognize thatthe curing of diseases occurs and as better hygienic

there is a very important distinction between the human lifes，what geronto10gist ca11，
conditions occur，then there ispan，which we think is a fixed number of years，rough1y 90

a rectangu1arisation of the surviva1 curve，the surviva1s the number
or 9S years of age，and 1ife expectancy，which i

curves become more and more rectangular unti1 the mostof years that one can expect to 1ive，based on statistical

rectangular curve is produced in this fashion. This wou1ddata as a function of his current age. 50，1would like tos that with the curing
be 90 years. The imp1ication then，ishow you this data that wil1 under1ine this very importantof diseases and the improvement of hygienic conditions.

pOint.

what wi11 happen is that humans wi11 1ive until the stroke

Now，this is what gerontologists ca11 a survival curve.

of midnight of their 90th birthday and then they wi11 dropThis is a fami1y of survival curves obtained from data indead. And this is the imp1ication of work done simp1ywhich a cohort of 100，000 peop1e have been fo110wed fromaddressing onese1f to bettering hygienic and 1iving conditions

birth unti1 death and each year the number of individua1s

and curing diseases.

who have died is noted and then the tabu1ar data i
s compi1ed

The main point is that very 1itt1e work is done on theand represented in the form of this fami1y of curves. 1wi11

question of the bio10gica1 causes of aging，the under1ying

not discuss each curve but just the extremes. The bottom

princip1e bio10gica1 causes of aging which are the physio‑

curve is generated fromaata taken from individua1s in

10gica1 and biochemica1 decrements that resu1t in the age

British India from 1921 to 1930. And as you can see in thechanges that a11 of us are fami1iar with. There are very

first few years of 1ife，there is a dramatic number of deaths

few peop1e who work on this prob1em，and itis importantin the post nata1 period. Then in 1ater years this begins

if there is any merit to extending the 1ife span. Itis

to f1atten out，and there are some individua1s i
n this groupimportant to recognize that very 1itt1e work is being done

who survive up unti1 roughly the 90th year of age. The otheron this question. There is a1so a subsidiary question inextreme curve is from New Zea1and in this period of time.

respect to whether it is important and necessary to do work

‑ 2‑

‑ 3‑

in an arca of biology that might result in the increase ofhuman longevity. This is a very important moral question，and a vcry important ethical question that unfortunately 1
will not have time now to discuss with you. But if theres ofis any mcrit to increasing the human life span，there icourse very little work being done in this particular area.
Now to give you further information on the implicationsof curing the major causes of death in my country and inyour country，1 would like to show you some projections ofdata that makes for some very revealing information. And
that is to ask ourselves the qucstion what would pappen tohuman longevity if we were able by some miracle，tomorrows，heartmorning，to cure the major causes of death，that i
disease and cancer. And the information that 1 want to show
you is on this next slide i
n which it has been determined
what these effects would be. This is the gain in expectationof life at birth and at age 6S due to the elimination ofvarious causes of dcath. Hcre wc have the major causes ofdeath，major cardiovascular renal disease，heart disease，
vascular diseascs;if these diseases could be cured tomorrow，
then people born tomorrow would have a total gain in life
expectation of about 17 or 18 years. People who are age
6S tomorrow would have somewhat less，but not significantly

less，cxpectation of additional life. The next major causeof dcath would bc cancer. And if cancer were to be cured
tomorrow morning，the impact would be，that people born
tomorrow would only have an additional 2・3 years of life

‑ 4 ‑expectation，people 6S tomorrow only 1.2 years of additional
life expectation. And 1 would think that these numbers aremuch lower numbers than you would have guessed，based on theamount of effort and money and energy 6f so many pcople whoare investing their lives in learning more about theseimportant causes of death. That is not to say that the
study of these diseases is not important. Itis certainly
important.

But 1 think that the impact on longevity is notvery great. 1 would suspect that you would have thoughtthat curing cancer will result in increase of life expectancy.
perhaps 20 or 30 or more years. But this simply is not thecase.
Now with this information as background，what 1 would
like to do now is to give you some information in an area ofcel1 biology and 1 think we can have the lights now and theslide off. 1 would like to give you some information in anarea of cell biology in which we and others have been working
for a number of years，that has some important impact on thet will be necessary forstudy of aging. For this purpose，ial1 of you to have a good background in tissuc culture techniques，
because what 1 intend to discuss is based on a knowledge oftissue culture. 8ince 1 have been told that there arecertainly many experts on tissue culture here today，there
are stil1 some people here who are not expert on tissue
culture. 80 1 will spend only S minutes，giving you some
1 be necessary for you
basic background information which wil

to understand the rest of my presentation.

‑ S‑

Many of you of course know that itis possible to takein a chemically defined media. But much of what 1want totissue from any animal and to grow the cells from this

discuss with you this afternoon has to do with the cultivation

tissue in culture. This is done ordinarily by taking theof normal cells. 50 we now have the cells growing in thetissue and exposing it to a chemical preparation or enzyme

cu1ture vesse1 and in a matter of a few days or a week the

preparation called trypsin which results in the separation

cells are growing quite rapidly and they reach a point whereof the individual cel1s from the tissue that you then canthey have now covered entire surface of the glass vesse1.

col1ect in a centrifuge tube and this can be centrifugedThis condition is known as a confluent culture. Itis alsoand cells col1ected as a pellet. The trypsin of course issometimes cal1ed monolayer culture and the most importantthen discarded and the cel1s now can be put into a variety

event that takes place at this time is the fact that whenof g1ass bottles and cu1tured. Generally，the type of culturenormal cells cover the entire surface and touch al1 of theirvesse1 that people uses is something of this shape. This

mitotic activity essential1y stops.
neighboring cel1s，

would be a side view and this a top view. The ce11s will

Abnormal ce11s or cancer cells may continue to grow a little

be introduced into culture vessel，in this manner，and ina

bit under the circumstances and this i
s a very important

period of a few hours ce1ls would begin to stick the glass

area of investigation now，to discover what the signals are

surface and then begin to grow. Now the medium that is usedthat tel1 a cancer cell to contiune to divide under these

for the growth of cel1s in tissue culture is very complicated.

circumstances of conf1uency and a norma1 cel1 does not

1wil1 not say any more about the media than to say that incontinue to divide very much.

general it consists of 13 aminoacids，7 or 8 vitamins and a

If you are interested in obtaining more ce11s，that isseries of inorganic sa1ts，the kind of sa1ts that you wouldtis necessary
more than is contained in this vessel，then i

expect，potassium，calcium etc. This is the chemica11y

to undertake a new or different technique，and that techniquedefined portion of the growth media，but normal cel1s willis known as subcultivation. We wil1 remove the media fromnot grow in a chemica11y defined media. 50 that the media

the cu1ture vesse1，the cel1s wi11 remain behind，trypsin

must include 10¥ of serum and the serum used is genera11yis then introduced to raise the cel1s from the surface，serum from calves or horses. The deve10pment of media has

separate them from their neighbors，and then the ce1ls can

not resulted in any chemica1 definition of al1 of thebe distributed into two daughter bott1es further subcu1tivation.

component~

necessary to grow normal ce11s i
n tissue cu1ture.

Itis possible to grow a number of ahnorma1 cel1 popu1ations‑ 6‑

50 this is ca11ed a subcultivation. It also gocs under theterm "a split" and we refer to do this as "a 1 to 2 split

‑ 7‑

rat1
0
"，，~hich means that ¥"c havc increa~cd surface nrca ofactive cel1 replication，Phase 11
1 which is the death of

thc first hottlc hy a factor of 2
. 50 that thc numbcr ofthe cells i
n tissuc culture. Now to give you somc notion

daughtcr culturcs i
s twicc thc numhcr of thc mother culturc.

of the time paramcter， if you cultu1'e tissue f1'om a embryo，

N O\~

i
ti
s possiblc to do suhcllltivations or 'sp1
its' byand thc typc of ccll that gcnc1'ally g1'ows most well inmaking thrcc hottlcs， 0" fivc， 01
' t
cn 0
1
'・
"hatcvcr numhcr'， from

if you culture
tissue culture，is cal1cd thc fibroblast，thc first，but mathcmatics becomcs a little difficult and. the pe1'iod of time thathuman embryonic tissue in cultU1'e

5incc 1want to kecp simplc， wc will assume that the 'splits'

s something like 1
0 01' 1
2 months. And thethey can g1'ow i

arc donc by the 1 to 2 ratio. although thcoretically every‑

number of population doublings is equivalent to SOtlO.

thing is thc same. 50 at the cnd of thc first wcck wc would

Now this is the usual course of activity of the cultivation

havc two cultllrcs， at thc end of 2nd week we wil1 havc fou1'，

of normal cells in culturc.

and e
i民ht，sixteen，and so f
o1'th. 50 that thc numhcrs of・
cs would incrcase by pOlぜcrs of 2.
cultuT~ow

this thcn isWhen you cultu1'e normal human cells，therc can occurin these cultu1'es ve1'y rarely， and until few yca1's ago underhasical1y thc technology that 1wantcd to introduce you to.

ci1'cumstances that we1'e pa1'ticularly unknown. a ncw cel1

And now Iwould likc to discuss somc of thcoretical

type ~hich can a1'ise f1'om cultures of normal cells. The

implications of cultu1'e in cells i
n tissuc culture hy the

f1'equency that this occurs in human material is less thanmcthod that I havc just outlincd. I
f onc takcs tissue from

0.01% of thc time. Fo1' othe1' anim3l species the f1'cquency

a varicty of sourccs including humans and grows the ccllsof the new ccll arisin~ is much greater. Mouse tissue. fo1'from such tissue in culturc， if you start with a scrap of

examplc，may give rise to this ncw cell typc within 3 f
1'c
quency

tissl
Ic n
nd y
O
l
l plot thc numhcr of cclls that al'C prodllccdof 40% or perhaps cven 50%.

from thc tissuc as a function of time. thcn you wil1 generate

Now 1 would like to point out the major distinctions

datn that will givc you a cu1'vc looking something likc this.

between those two types of cells. The f
i1's
t type of cel1

Thi5 would bc thc primary culturc， the fi1'st cultu1'e. andthat 1 have been discussing we ¥
.
.
ri
l
l cal
1 "cell strain" andaftcr a wcek you wil1 thcn do a suhcultivation so that you

n tissuc cultu1'e we will
the type of cell that a1'ises 1'arcly io1' ahavc two cultu1'cs. fou1'， cight. sixtecn so fo1'th. fcal
1 tla cell linc". Now 1want to cmphasize that 1will be

numhcr of wccks. until you 1'each a point when the cclls stop

defining thcse two terms i
n a ve1'y precise way but thedividing and bcgin to dic. And we havc divided this curve

litc1'atu1'e. the scientific litcrature， is often not ve1'yinto three parts， Phnse 1， Phas~ 1
1， which is the pcriod of

clca1' on thc distinction between thesc two tcrms and you

‑ 8‑

‑ 9‑

must know more about the author's work before you can under‑s in fact what
karyotype of the tissue ori~in ， and that istand whether he is working with this type of cell or withs HeLahappens. The cell line，on the other hand，which i

this type of cell. So we will now contrast the major propcrties

a good example，has a spectrum of chromosome numbers aroundbetween a cell strain and a cell line.

some modal value with somc cells having as few as 50 chromosomes，The first chracteristic that 1 have indicated alreadysome having perhaps as many as 350，the modal value is usually・is the cell strain has finite capacity for replication orin the 70 s somcwhcre，and we refer to this condition as

multiplication in tissue culture. A cell line，on the otherheteroploid.

hand，has an infinite capacity for replication. There are

The third property that distinguishes these two types of cells

a number of these cell lines that are known，perhaps the，p for a number of variahles.
1 will use as a general headinmost popular is the HeLa cells. This is the cell line that is

That is to say，if you look at the following kinds of properties，

derived from human cervical carcinoma in early 1950s by I
:
e
o
r
g
e

staining characteristics，hiochemical properties，or properties

Guy of John Hopkins University and this cell has been growingof the cells they are inoculated into a variety laboratory

in laboratories throughout the world for these past 25 years.

animals，you come to the conclusion that a cell strain has

As 1 indicated that there are approximately 500 or 600 at least

properties of normal cells and that a cell line has propertiesof these cell lines that have been described，another famousof abnormal cells，not only are these properties abnormal，

one is L cell derived from mouse tissue and so forth. The

but in many cases the properties are properties of cancer

cell lines have developed in laboratories throughout world

cells. So that this phenomenon，where a cell line has resulted，under spontaneous circumstances and at very low frequencies

has emerged from a culture of normal cell is a very important

if you are working with human tissues.
The second property that distinguishes these two typesof cells has to do with their karyology，or their chromosomelte name of "transformation".
phenomenon，and goes under tThis phenomena of transformation was recognized by cell
culturists many years ago，hut very little could be done about

configuration. If you look at the chromosomes of a cell

the problem，especially with human tissue because the frequency

strain derived from normal tissue thcn if for example theof transformation was so low that you could not constructtissue came from your forearm or wrist，then you would expect

experiments to answer important questions about this problem.

that these cells would be diploid and for men of course theSince the importance of that was generally recognized that

number of chromosomes would be 46. 50 depending on the tissue

t represented the emergence of cancer
when it occured，ithat one shows you would expect the strain to have thecells in a population of normal cells in culture. This was‑ 10 ‑‑11‑

the 5ituation for a number of years until the middle 1960s，

emergence of a transformed population. Here you can see on

whcn it was discovcrcd that you could transform normal humanthe left the transformed cclls which look very much diffcrcnt

cclls each time if you added to the culture a virus calledfrom the untransformcd or cell strain type of cel1 here on

5V40. This virus has the unique capacity to transform normal

the right. Thcsc cells then have al1 of the properties ofhuman cclls into cancer cells. As far as J know this is thethe type of cell 1 indicated here，the cel1 line type of

only agency by which this can be done with the cxception

cel1. Now if you look at the ccntcr of one of these islandsn my lahoratorythat Dr. Namha and 1，when he was working iof transformation and the transformation occurrs in smal1

have now cvidence that this transformation can he accomplishedislands in the culture，you will see a trcmendous amount ofcalled 4NQO.
hy adding to the culture a chcmical carcinogen，

mitotic activity. When cells divide in tissue culture，they

But aside from these two situations，there is no other way

retract their processes，become spherical，go through thethat one can routinely transform normal human cel1s. There

mitotic cycle and then become flattened out again. And herearc a numher of othcr viruses that can transform a variety

you see for example，a mitotic figure with chromosomes onof othcr ccl1s from othcr animal species，and thcse viruses

the metaphase slate，here are also representations of cells

arc such a~ polioma，and some of the adenoviruses etc.，arein mitosis. 50 one of the features of one of thesc trans.

al1 known as oncogcnic or cancer causing viruses.

formations is thc tremendous increase in mitotic activity.

Now with this information，1 would like now to concentrate

Now this amnion was taken from my daughter at her birth，on the qucstion of thc death of normal cel1s i
n tissue culture，

some 16 years ago，so this culturehas been in，contiunousand what wc currently know about this question in respectcultivation in our lahoratory and in a number of otherto thc most rccent research on this area. But hcfore 1 golaboratories for 16 years.

any further，perhaps we can look at some of the slides thatBut 1 would like now to talk more precisely about normal

I havc that will il1ustratc some of these points that 1 have heen

human fibroblasts which have the greatest capacity formaking. 50me years ago，we were lucky enough to see one ofproliferation in tissue culture. This is a semiconfluent

these spontaneous transformations occurring in a culture ofculture of normal human fibroblast showing one mitotic figure.

normal human amnion cells. These are human amnion cells here，

After a few more days these cells will become more dense

after one month i
n culture，these amnion cells which are

and the confluent condition will exist as you see here，herecasily obtainable from hospitals and represent normal humanagain as one mitotic figure. But this represents then these cultures thecel1s，after about one month，we saw inconfluent culture at which time，if you are interested i

ー

12 ‑

‑ 13 .
.

obtaining additional ce11s，i
s necessary to subcu1tivate

n respcct to somece11s，which is an important question i

this cu1ture and the subcultivations 1 described to you

genetic studies. Now if you take norma1 human ce11s as you

beforc. Comparc this slidc with the next slide which showshave seen in previous slides and expose it.to SV40. andyou the way thcse cells look during phase 1
1
1. The cel1stransform them as 1 have indicated，this is what you see.

now havc complctely lost their morphological characteristics.

This is WI‑38 which is a normal human diploid cel1 population

Therc is a considerable amount of debris in the cu1ture.

that is one of the ce11 popu1ations that we do much of our

Thesc thcn rcprcsent cc11s that are somewhcre at this partwork with. This ce11 of course has a doub1ing potential ofof the curvc. There is sti11 some continuation ofmetabolic

about SO population doublings and then the cells die. But

activity but in gcnera1 over a period of Z or 3 weeks theif you expose the ce11s to SV40，they become a1tered or

cclls stop dividing，bcgin to 100k unhealthy and then begin

transformed. And this is their appearance. and of course

to dic and in a matter of 4 or S or 6 weeks most of the cells

the transformed cel1s have a11 these properties.

arc dead. 50 this is the appearance of cel1s in culture inphase 11
1.

Now，1 wou1d 1ike to give you some further informationon the question of the re1evance of thesc observations to

Onc of the important points that 1wou1d 1ike to remind

aging. One of the most important questions that has been

you of appears in the next slide. Since it forms the basis

raised about the cu1tivation of ce11s with a finite life

for an cxpcriment that 1 would to dcscribe to you. and thattime in tissue cu1ture is the question of whether the death1s simply to rcmind you that itis possib1e to distinguishof these cel1s i
s due to some error in laboratory technique;between male and female fibroblasts in tissue cu1ture by theit is possible of course for cells to die in tissue as a

prcsence of the bar body in nuc1ci of fema1e ce11s，and theresult of faulty media or the presence of viruses or any

absence of the bar body for the inactive exchromosome in a

number of extrinsic factors. There have been many experiments

nuclci of male cel1s. 1wi11 say more about that in a few

that have been done that have essential1y exc1uded thisminutcs. To those of you who might be interested，it is a1so

possibility. But 1would like to describe just one experimentpossible to c10ne norma1 human cel1s. We can iso1ate tissue

but，1 think，gives the most valuable information on thisfrom your forearm. grow the cel1s out，and then iso1ate one

question. If you take female cel1s. female normal humancel1 and from that onc cc11 produce progeny as you see here.

s
cel1s at the 10th population doubling level (PDLl. that i

this is progeny or daughter ce11s from a single cell，giving

somewhere here，and mixed them with an equivalent number ofrise each of these to a colony or a clone of normal humanmale dip10id cel1s at the 40th PDL and keeping unmixed control‑1
4‑‑ lS ‑

cultures separate，after the mixture is made，if you waitthat must be deluded is the smallest molecule which wi11 be

30 more subcultivations and look at the cells that remain，hydrogen，then you ask yourself the question how many hydrogen

you wil1 find that the only cel1s remaining are female ce11s.

molecules must be present in this first ce11，so that you

You wil1 a1so find that the female cel1 control is sti11can get one moleculc in the cell at the 50th population

viab1e. The cel1s are stil1 growing，because 10 + 30 a 4
0.

s at this poing. And if you do the mathema‑doubling，that i

You wil1 find however that several weeks before，you looked

tics，you come to the conclusion that each ce11 initial1ys dead，because
at the mixture，the male cell component imust weigh at least 3 times more than we know they weigh.

it is now somewhere here. Now if the death of the maleand be composed entirely of hydrogen in order for simple acomponent is due to media，bad media or viruses or faulty

dilution to occur so that you end up with one molecule intechnique. then it would be necessary to argue that thiss a1so not aeach cell. 50 that we think this explanation i

prob1em cou1d distinguish between male and female ce11s，

valid exp1anation.

which of course we know is not possible. 50 this kind of

Now this left us several years ago with the difficult

experiment，have led us to helievc that the deat~ ofsituation of being unab1e to explain why norma1 human andnormal cc11s in tissue culture is an intrinsic cel1 phenomenon.

n tissue cuJture. And \~e thought
anima1 cells u1timately die i

There is some kind of clock mechanism within cells which

at first that the suggestion that this was aging at thc lcvel

dictate the number of population doublings that thcy canof the cell，was a rather interesting possibility but probably

undergo apparent1y 担 己 主 ro and as 1wil1 show you later

not true. And we use this as a method to devise subsequentalso in vivo.

cxperiments and 1must say that in subsequent years as at was thought
Whcn we first observed this phenomenon，i

resu1t of work in our laboratory and many other laboratories，

旦
that one other possible exp1anation could be that ce11s 1

it appears that this hypothesis may be a valid hypothesis.

註 盟 might be able to produce some essential metabilites

and 1 would 1ike to give you some of the recent work that has

旦己主旦・
that they are unable to produce 1

And it was argued

been done on this question，to show you the relevance of thisthat possib1y some essential metabolite is being deluded out

observation to some of the variables that we know of in theas the ce11s traverse this course and when they run out thisfield of aging.

metabolite，al1 of the cells wi11 then die. And this hypothesis

One of technological breakthroughs that allowed us tocan be tested by looking at this question from a purely

do some interesting experiments i
n this field was the possibility

mathematica1 standpoint. If you assume that the mo1ecu1eof freezing cells or preserving cells for long periods of‑ 16 ‑‑ 17 ‑

time. As you can appreciate，when you culture normal human

ampu1es gave rise to cultures that deteriorated or reacheddip10id ce11，after 12 months，the ce11s are gone. Itis

Phase 11
1 between 40 as you see here. and between 60. 50

possib1e，however，to take the surp1us cu1tures after eachthe answer to the question is that the ce11s do remember

population doub1ing，for examp1e，you wou1d take these twoat what popu1ation doubling they were when frozen andcultures and freeze them，and the next week you will takecontinue to rep1icate so that the tota1 cumu1ative number

those two cu1tures and freeze them，and the third week theseof population doublings is50. and this is an important

cu1tures and freeze those. 50 that at each population

point.

doub1ing 1eve1 you wou1d have frozen in your bank cell
cultures from each population doubling to that final period.

Now by increasing the number of cultures each week bymultiples of 2
. you can easi1y appreciate that number of

The important question to be asked is when you remove the

popu1ations. the number of cells wi11 increase tremendously.

ce11s from liquid N2 which is where we ordinari1y store suchce11s，what happens to the c10ck? Do the ce11s remember

The number of potential cells that can be produced from a
cu1ture that has popu1ation doubling potentia1 50，is

at what popu1ation doub1ing they were when you reconstituteapproximately 20 million metric tons ofce11s. 50 WI・38

them，and continue on，or do they recyc1e back to the origin，

which was frozen many years ago has been used in laboratories

and then go through，or is there some other possibility thatthroughout world. and we sti11 have p1enty of ce11s left.

happens? And this hypothesis was tested，and 1 wou1d 1ike

because the potential yield is this. This is some of theto show you some of the resu1ts on the next slide. If you

history of WI・38 which was frozen at the 8th population

reconstitute the cells from a variety of population doublings

doubling 1evel. most of it was frozen then. and each time

you generate what looks like a very complicated genealogy

an ampule was reconstituted we gave it a roman numeral as

or family tree. Here is the cu1ture. the original culture

you see here. The number of popu1ation doub1ings thatfrom 0 passage until the 50th passage here. and these

resu1ted from each thawed ampule is indicated here. averaRevertica1 lines indicate ampules that have been reconstituted.

is about 50 and range again between 40 and 60. These areThis is number 5 and that was reconstituted and then grown

numbers of weeks each cu1ture has been preserved and thisin tissue culture unti1 i
t reach Phase 1
1
1 at about 4
7.

is a very old slide. because we now have WI・38 frozen for

There are others you can see here. This one at 1
4，or 13

12 years. which is over 600 weeks. 50 WI・3
8 has been fro:en

was reconstituted. and it died at the 50th population

10nger than any other norma1 human ce11 and. after 12 years.

doub1ing. The important point is that a11 of the reconstituted

the average is sti11 about 50 and the range between 40 and

‑1
8

19 ‑

6
0
. 50 the memory of the cells is very good indeed.
Now if what 1 have been discussing is related to aging.

analysis indicated that there is a decrement of 0.2 of
population doubling per year of life. 50 the older thethen 50 is the number that is important for human embryonicperson，the fewer number of population doublings are ahle

tissue. but we would like now to know the number of population

to be seen.

doublings that normal human adult cells will undergo inNow，if this is true for human material，the next

tissue culture and this data is indicated on the next slide.

question is whcther this i
s a general phenomena that may

Here you see a list of about 12 or 15 human diploid embryonichave some bearing on the life span of other animals，andcultures that have undergone a number of population doublings

one of the most interesting questions is to determine theaverages is50. ranging. between 40 and 60. And here arenumber of population doublings that can be undergone byhuman diploid cell populations from human adults. these areother animal populations. And 1 would like to show you thisthe number of population doublings. the age of eachdonor

slide. This is work done by four different laboratories

and the number of population doublings possible in the casewith chicken embryo fibroblasts. the number of populationis 20 and range between 10 and 30. 50 there is a verydoublings as you see. is indicated here. the average about

dramatic difference between the number of population doublings

23.S. Now 1 should point out at this time that one of thethat adult tissue and embryonic tissue will undergo in culture.

most important experiments done in the tissue culture field

This work was done before we had experience with thethat had a bearing on gerontological or aging questions wasdetails of the knowing how to count these doublings accurately.

done with chicken embryo fibroblasts. Many of you will

but a couple years ago Dr. J. Martin from University ofn the 19205.
recall the work that was done many years ago，iWashington did a very interesting study on 100 human skinby A
. Carrel who claimed to be able to culture chick heart

biopsies taken from individuals who were either embryoniccells for something like 34. or 35 years. Carrel's work wasor up to 90 years of age. And he determined for al1 onevery important. because if itis possible to culture normal

hundred the number of population doublings that the fibro‑

animal cel1s for periods of time. greatly in excess of theirblasts from each culture would undergo and 1would like tomaximum life span. then clearly aging is not a result ofshow you this data. The number of cell doublings is indicateddecrements i
n the function of individual cells but must be

here，the age of the donor indicated here on abscissa. thea result of actions taking place at higher levels in theopen circles indicate the number of the population doublings

single cell. that is tissue level or organ level. 50 thatthat the cultures in each group underwent. And his statisticalthe experiment of Carrel is very important. Becau5e it‑ 20 ・
・一 21

contradicts what has been found recently. We now know thethe donor which is reflected in an inverse way，in respect

explanation for the cultures of Carrel that lived for 34of the age of the donor.

years. And the explanation is the following. The culture

Now 1 would like to mcntion vcry briefly that it has hpen

media that was used at that time was prepared from chickpossible to repeat this kind of work in animals where theembryo extract (CEE) and that CEE was prepared and thensame situations seen in tissue culture has also resulted.

centrifuged in such a way that many cells were still floating

If you take tissue from a laboratory animal，for example，thein supernatant fluid，so that when you put a pipette into

mouse，and this has been done for a variety of tissues fromthe centrifuge tube，you took up centrifuge tube in themice and also other animals and transplant this normal tissue，

pipette new fibroblasts each time you fed the culture，that

and this can be done，then you will find that the numbcr ofwas supposed to be grown for 34 years. And this is generallytransplants that can be effected are finite，4 or 5 or some.

regarded to be the explanation for this contradiction. No

thing of this sort，whereas the continuous transplantation of・

one has ever been able to confirm Carrel s work even tocancer cells of course can occur indefinitely. 50 that there

culture normal chick fibroblasts for 2 or 3 years.

is now担 己 主 evidence for this i旦己旦~ phenomena. 1 would

The next question then is the number of population

like to say in ending here that 1 do not believe that animal's

doublings that ccl1s from othcr species will undergo，andor individual's age，because their cells stop dividing. What

here you see some of this data. For man as 1 have indicatedhas been discovered recently is that as the cells age inthe range is between 40 and 60. For embryonic fibroblasts

tissue culture roughly in this period，there occur a number

the maximum life span about 110 years，let's say，chickenof dramatic biochemical changes resulting in physiologicalmaterial between 15 and 35，the maximum life span about 30

decrements that 1 think are much more important in respect

years，the mouse between 14 and 28 population doublings andto our understanding of aging phenomena than the fact thatthe maximum life span thought to be about 3.5 years，thethe ce11s ultimately stop dividing. 1 don't havc time to

Galapagos tortise，this work was published very recently by

discuss a11 of these biochemical changes but 1 thought 1

Goldstein，the range of population doublings between 90 andwould just show you a list of them which is incomplete list，

125，the maximum life span is thought to be ahout 175 years.

to give you some idea of the amount of work that has been

50 there docs appear to be some relationship b~tween the a~edone in this area and without discussing each of theseof the mean maximum life span which is reflected in a direct

individually，but you can see some of parameters increase，

way in the number of population doublings，and the age ofother decrcase，some do not change.

‑ 22 ‑‑ 23

Finally 1would like to summarize by ~iving you some
information on three of the current theorics of aging，since
thesc current thcorics also are applicable for possiblc
explanations of the Phasc 1
1
1 phcnomenon i
n tissue culture.
Thcrc are many theories of a~ing ・but

1 think the threc mostimportant oncs are as follows; One is that there arc spccificgenes for a~ing.

That isi
n thc dcvelopmental sequcnce inanimals as developmental chan~cs are programed by genetic
information. Somc pcople think that at the very end thcre
arc specific genes that shut down the metaholic machinary
and force the cell to closc down its functional reproductive
capacities. Another possihility is that there is simply as accurate gcnetic infor‑loss of genetic information，that imation，that as developmental 5cquenccs occur，infomation
containing moleculcs may in fact incur errors and as thcsc
crrors accumulate，they may accumulatc for a period of timc，and then thcy reach a point where the whole cel1 stops divid‑
ing or stops functioning and thcsc agc changcs occur. Of
coursc thcrc arc mcthods for rcpairing errors in hiologicalmatcrial hut as wc al1 will appreciate these rcpair mechanism
n themselves，not pcrfect. The final informationarc a150， ithat 1would like to give you is the possihility that whcn
thc [
)
:
¥
A molccule，the rcdandacy of message，may he directly
rclated to thc longevity of a particular species. I
f thesamc message was repeated many times in information contain・
ing moleculcs and if crrors accumulated hcre，such that thisinformation after a period of time hecamc invalid of nonscnse‑2.
1

information，thcn the ncxt 5cquence containing the sam~ bitof information could take ovcr，and this could occur agnin.
50 that the number of times that the rcdandant messugc
appcared could be directly rclatcd to the longcvity of thc
species，and many people feel that the evolution of thes much morc
mnximum life span of~ndividual specics，which i
specific than the life span of individual members of a
species，that this evolution occured following lincs of this50rt.

1975 年 Bibli句 raphy 原 稿 募 集
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ibliograpby 編集の季節になりました。下記の知く原稿を募集致しますので， 1974年の年号のついた倫文の英文拶鳴を漏れなくお送り下さい。

1 〆切

昭和 50年 11月 15日

2
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先

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