动物细胞工程李福利微生物技术国家重点实验室
1 动物细胞特点
无细胞壁
倍增时间长，生长缓慢
需氧量少，对搅拌敏感
聚集体形成
原代细胞培养 50代即开始退化
2 动物细胞培养定义
动物细胞与组织培养是从动物体内取出细胞或者组织，模拟体内的生理环境，在无菌、适温和丰富的营养条件下，使离体细胞或者组织生存、生长并维持结构和功能的一门技术。
是动物细胞工程的基础。
3 发展历史
1907，美国的哈里森使用盖玻片悬滴培养蛙胚神经组织细胞
4 生长特性
贴附生长型如人胚肺细胞,Hela细胞，巨噬细胞，
神经细胞
悬浮生长型细胞如血液白细胞，淋巴组织细胞等
5 体外培养细胞基本技术
体外培养特点
体外培养工具
体外培养条件
体外细胞生长增殖过程
培养方法
大规模培养技术
5.1 体外培养特点
营养条件苛刻
适应性差,敏感
培养时间长，易污染
Animal cells are more difficult to culture
than microorganisms because they require
many more nutrients and typically grow
only when attached to specially coated
surfaces,Despite these difficulties,various
types of animal cells,including both
undifferentiated and differentiated ones,
can be cultured successfully.
5.2 体外培养工具
空心纤维
微球
5.3 体外培养条件
温度,37度
pH,7.2-7.4
气体，氧，二氧化碳，氮气
营养条件需要多种氨基酸，维生素，辅酶，核酸，
嘌呤，嘧啶，激素和生长因子，其中多种成分可以由血清提供
Rich Media Are Required for
Culture of Animal Cells
Nine amino acids,referred to as the essential amino
acids,cannot be synthesized by adult vertebrate animals
and thus must be obtained from their diet,Animal cells
grown in culture also must be supplied with these nine
amino acids,namely,histidine,isoleucine,leucine,lysine,
methionine,phenylalanine,threonine,tryptophan,and
valine,
addition,most cultured cells require cysteine,glutamine,
and tyrosine,
The other essential components of a medium for
culturing animal cells are vitamins,which the cells
cannot make at all or in adequate amounts; various salts;
glucose; and serum,the noncellular part of the blood
Most Cultured Animal Cells
Grow Only on Special Solid
Surfaces
The extracellular matrices in various
animal tissues consist of several common
components,fibrous collagen proteins;
hyaluronan (or hyaluronic acid),a large
mucopolysaccharide; and covalently linked
polysaccharides and proteins in the form
of proteoglycans (mostly carbohydrate)
and glycoproteins (mostly protein),
5.4 体外细胞生长增殖过程
原代培养期
传代培养期
衰退期
5.5 培养方法
悬滴
旋转管
灌注小室
培养瓶
培养板
5.6 大规模培养技术
悬液培养，使用微球载体
6 动物细胞培养应用
1962年开始，用于生物医学研究，生产酶制剂，生长因子，疫苗，单抗等
Vero细胞和狂犬病毒的培养工艺
DMEM培养基胎牛血清其他成分锥形瓶逐级放大培养加碱（碳酸氢钠）
加糖（葡萄糖）
灌注培养液微载体
5L种子罐培养培养液
50L
生物反应器接种狂犬病毒出液口收获病毒
6.1 单克隆抗体
1975年英国科学家 Milstein和 Kohler将产生抗体的淋巴细胞同肿瘤细胞融合，成功建立了单克隆抗体技术，而获得 1984年诺贝尔医学和生理学奖。
每个 B淋巴细胞仅专一地产生、分泌一种针对某种抗原决定簇的特异性抗体，而肿瘤细胞可以无限增殖，因此杂交瘤细胞可在体外培养或移植到体内条件下分泌大量单克隆抗体。
单克隆抗体技术的最主要优点是可以用不纯的抗原分子大量制备纯一的单克隆抗体。
杂交瘤细胞产生单克隆抗体示意图
6.2 动物克隆生物繁殖后代通常是以精、卵细胞结合的有性生殖方式进行。通过营养体细胞繁殖个体的方法称为无性繁殖。克隆是指离体条件下的无性繁殖。 1981年 Illmenses
率先报告用小鼠幼胚细胞核克隆出正常小鼠。随后，
1984年 Willadsen用未成熟羊胚细胞核克隆出一头羊。
英国 PPI生物技术的罗斯林（ Roslin）研究所的维尔穆特 (Wilmut)博士 1997年 2月 27日在世界著名权威杂志
,Nature,宣布的用乳腺细胞的细胞核克隆出一只绵羊
,多莉 (Dolly),的消息。,多莉,的诞生，既说明了体细胞核的遗传全能性，也翻开了人类以体细胞核竞相克隆哺乳动物的新篇章。此项技术因而荣登美国,Science》
周刊评出的 1997 年十大科学发现 的榜首。
绵羊 B
乳腺细胞核易核卵融合卵绵羊 C子宫多莉植入植入生产克隆多莉羊示意图取出未受精卵去核电激体外胚胎发育绵羊 A
多莉 克隆猴
(1)遗传素质完全一致的克隆动物将更有利于开展对动物 (人 )生长,发育,衰老和健康等机理的研究；
(2)有利于大量培养品质优良的家畜 ;
(3) 经转基因的克隆哺乳动物,将能为人类提供源源不断的廉价的药品,保健品以及较易被人体接受的移植器官；
(4)科学家将很快地从目前的同种克隆技术推进到异种克隆,即借腹怀胎的新领域,这无疑将大大促进对濒临灭种的哺乳动物的保护工作。
克隆技术将对 21世纪产生的重大影响
6.3 干细胞工程
干细胞定义现在发现哺乳动物的许多已分化组织和器官中都保留了一些能够分裂形成该组织或器官的未分化原始细胞,这些细胞称为 干细胞 。
干细胞形态特征：
圆形,椭圆型,体积小,核质比大,具有较强的端粒酶活性,因此具有较强增殖能力 。
干细胞增殖特性缓慢性，自稳性
干细胞分类人体干细胞基本分为三种类型：全能干细胞、
多能干细胞和专能干细胞。
·干细胞分化模式
·胚 胎 干 细 胞 (embryo stem
cell),具有分化成多种细胞类型及构建组织的潜能 。
·造血干细胞
·单能干细胞 （ monopotential cell）
体内干细胞的意义 在于源源不断地补充体内一些短命组织的细胞来源，如血液细胞、皮肤细胞以及精巢等，或者组织或器官受到局部损伤时，它们可以再分裂形成新的该组织和器官，是体内的一种自我修复机制。
人类干细胞工程就是利用人体内干细胞的特征来达到体外产生人类所需的产物的探索。主要用于器官修复，基因治疗等。
第三节 植物细胞工程
植物细胞工程,就是进行物种改良，选育优良作物品种，增加植物的优良性状，利用植物生产各种化学制品，还可用于保留濒危灭绝和有重要经济价值的植物物种。
植物细胞工程技术：
一、快速繁殖技术通过植物体细胞胚胎再生成一完整植株。这种技术主要用于花卉、树木、蔬菜、果树、中草药和农作物的快速繁殖。
二、诱导增加或减少植物体内染色体组数的技术有些植株的染色体比正常细胞少一半（单倍体），而有些经诱导后染色体加倍（多倍体）。单倍体对植物品种的改良很有利；多倍体产量高，为不同倍性植物间的杂交提供了遗传保证。
三、体细胞杂交技术利用自然或人工方法使两个工几个不同细胞融合成一个细胞，形成体细胞杂种。
四、植物细胞应用生产技术利用植物细胞培养技术，给植物细胞提供最适宜的生活环境，让它们进行无性繁殖，大量地培养成植株，解决天然资源的不足。
一,植物组织培养外植体 愈伤组织 新植株进行植物组织培养，一般要经历以下五个阶段：
1 外植体的选择及培养 。
2 诱导去分化阶段 。
3 继代增殖阶段 。
4 诱导分化生根成芽阶段 。
5 移栽成活阶段 。
植物细胞培养定义在离体条件下，将愈伤组织活则其他易分散的组织置于液体培养基中，进行振荡培养，得到分散成游离的悬浮细胞，通过继代培养使细胞增殖，从而获得大量的细胞群体的一种技术。
二,植物细胞培养工业化植物细胞培养系统主要有两大类：
悬浮细胞培养系统和固定化细胞培养系统三,植物细胞杂交（细胞融合）
1960年英国诺丁汉大学 Cocking 教授领导的小组率先利用真菌纤维素酶,成功地制备出了大量具有高度活性可再生的番茄幼根细胞原生质体,开辟了原生质体融合研究的新阶段 。
植物细胞杂交的主要过程如下：
1,原生质体的制备 。
2,原生质体的融合 。
3,杂合体的鉴别与筛选 。
细胞育种诱导突变，筛选新品系、新品种次生代谢产物生成从培养的植物细胞中提取所需的代谢产物。
优点
比栽培原料作物更易控制最佳生产条件；
培养物为无菌、无虫材料，能保证产品质量；
工艺操作较为简单，可减少劳动费用，提高生产率。
植物细胞工程的应用
PERSPECTIVES for the Future
Advances in the culture of human cells will allow them to
be applied to the treatment of disease and injury,Even
today,severe burn patients can be treated by removing
some of their undamaged skin,culturing the fibroblasts,
and applying them to areas where the skin has been
destroyed,As specific growth factors for various cell
types are identified and expressed in sufficient quantities
by recombinant DNA techniques,other cell types will be
cultured and used therapeutically.
In the not too distant future,safe and effective viral
vectors will likely be developed to deliver genes
encoding therapeutic proteins or RNAs to the specific
cells where they are needed,Such gene therapy
potentially could successfully treat numerous human
diseases caused by the absence of critical cellular
molecules.
SUMMARY
Growth of vertebrate cells in culture requires rich media containing essential amino acids,
vitamins,and peptide or protein growth factors,frequently provided by serum,Most cultured
vertebrate cells will grow only when attached to a negatively charged substratum that mimics the
extracellular matrix in animal tissues.
Primary cells,which are derived directly from animal tissue,have limited growth potential in culture and may give rise to a cell strain.
Transformed cells,which are derived from animal tumors or arise spontaneously from primary
rodent cells,grow indefinitely in culture (see Figure 6-5b),They usually have an unstable,
aneuploid complement of chromosomes,including abnormal chromosomes,Transformed cells
derived from a single parental cell are called cell lines.
Cultured cells can be induced to fuse into heterokaryons (hybrids) by treatment with certain
viruses or polyethylene glycol,Heterokaryons between cells of different species tend to lose the
chromosomes of one species as they divide.
Panels of hybrid lines prepared from mutant mouse cells and normal human cells,each containing different human chromosomes,can be used to map the gene encoding a specific
human protein to a specific human chromosome.
Fusion of an HGPRT myeloma cell and a single B lymphocyte yields a hybrid cell that can grow
on HAT medium and proliferate indefinitely,forming a clone called a hybridoma (see Figure 6-10),
Since each individual B lymphocyte produces antibodies specific for one antigenic determinant
(epitope),a hybridoma produces only the monoclonal antibody synthesized by its original B-
lymphocyte parental cell.
Procedure for producing a
monoclonal antibody to
protein X,Immortal myeloma cells that lack HGPRT,an enzyme of the purine-salvage pathway (see
Figure 6-9),are fused with normal antibody-
producing spleen cells from an animal that
was immunized with protein X,The spleen
cells can make HGPRT,When plated in HAT
medium,the unfused cells do not grow,the
mutant myeloma cells because they cannot
make purines via the salvage pathway,and
the spleen cells because they have a limited
life span in culture,Thus only fused cells,
formed from a myeloma cell and a spleen cell,
survive on HAT medium,proliferating into
clones called hybridomas,Each hybridoma
produces a single antibody,Once a hybridoma
that produces a desired antibody is identified,
the clone can be cultured to yield large
amounts of that antibody.
Primary Ce ll Cul ture s Are Primary Ce ll Cul ture s Are
Useful,bu t H ave a F inite L ife Useful,bu t H ave a F inite L ife
SpanSpan
Stag e s in th e e st abl is hm e nt o f a ce l l cu ltur e,Stag e s in th e e st abl is hm e nt o f a ce l l cu ltur e,
(a) Wh e n an initial (a) Wh e n an initial e x pl a nte x pl a nt is ma d e o f hu ma n c e ll s,is ma d e o f hu ma n c e ll s,
some ce ll s di e a nd o th e rs (ma inly fib r o bl a sts ) some ce ll s di e a nd o th e rs (ma inly fib r o bl a sts )
sta rt to gr o w; o ver a ll th e g ro wt h ra t e i ncreas e s sta rt to gr o w; o ver a ll th e g ro wt h ra t e i ncreas e s
(p ha se I ),I f th e re ma ining c e ll s are co nt in ua ll y (p ha se I ),I f th e re ma ining c e ll s are co nt in ua ll y
di lut e d,th e c e ll st ra in g ro ws a t a co ns t a nt ra t e fo r di lut e d,th e c e ll st ra in g ro ws a t a co ns t a nt ra t e fo r
a bo ut 5 0 c e ll g e n e r a tio ns ( pha se I I ),af te r whic h a bo ut 5 0 c e ll g e n e r a tio ns ( pha se I I ),af te r whic h
th e g ro wt h ra t e fa ll s rapid ly,Du ri ng t h e e ns ui ng th e g ro wt h ra t e fa ll s rapid ly,Du ri ng t h e e ns ui ng
pe ri o d o f i nc reasing ce ll de a t h ( pha se I I I ),all t he pe ri o d o f i nc reasing ce ll de a t h ( pha se I I I ),all t he
ce ll s i n th e cult ur e e ven tu a ll y d i e,(b ) I n a cul tu r e ce ll s i n th e cult ur e e ven tu a ll y d i e,(b ) I n a cul tu r e
pre pare d from m o u se o r o t he r r o d e n t e mbry o pre pare d from m o u se o r o t he r r o d e n t e mbry o
ce ll s,th e r e is i nitia l ce ll d e a th co u pl e d with t he ce ll s,th e r e is i nitia l ce ll d e a th co u pl e d with t he
e me rge nce o f healt hy gr o wi ng ce ll s,A s th e se a re e me rge nce o f healt hy gr o wi ng ce ll s,A s th e se a re
di lut e d a nd a ll o we d to co nt i nu e g ro wt h,th e y so o n di lut e d a nd a ll o we d to co nt i nu e g ro wt h,th e y so o n
be gi n to lo s e gro wt h p o t e n tial an d mo st ce ll s di e be gi n to lo s e gro wt h p o t e n tial an d mo st ce ll s di e
(th e cul tu re g o e s in to c risis),Ver y ra r e ce ll s d o (th e cul tu re g o e s in to c risis),Ver y ra r e ce ll s d o
not di e bu t c o nt i nu e gro wi n g u nt il t hei r p ro ge ny not di e bu t c o nt i nu e gro wi n g u nt il t hei r p ro ge ny
o verg ro w th e c ultu r e,Th e se ce ll s co ns t itut e a c e ll o verg ro w th e c ultu r e,Th e se ce ll s co ns t itut e a c e ll
li ne,w hich wi ll gr o w for e ver if it is a p pro pri a te ly li ne,w hich wi ll gr o w for e ver if it is a p pro pri a te ly
di lut e d a nd fed wi t h n ut rie n ts,t he ce ll s are di lut e d a nd fed wi t h n ut rie n ts,t he ce ll s are
imm o rta l,imm o rta l,
1 动物细胞特点
无细胞壁
倍增时间长，生长缓慢
需氧量少，对搅拌敏感
聚集体形成
原代细胞培养 50代即开始退化
2 动物细胞培养定义
动物细胞与组织培养是从动物体内取出细胞或者组织，模拟体内的生理环境，在无菌、适温和丰富的营养条件下，使离体细胞或者组织生存、生长并维持结构和功能的一门技术。
是动物细胞工程的基础。
3 发展历史
1907，美国的哈里森使用盖玻片悬滴培养蛙胚神经组织细胞
4 生长特性
贴附生长型如人胚肺细胞,Hela细胞，巨噬细胞，
神经细胞
悬浮生长型细胞如血液白细胞，淋巴组织细胞等
5 体外培养细胞基本技术
体外培养特点
体外培养工具
体外培养条件
体外细胞生长增殖过程
培养方法
大规模培养技术
5.1 体外培养特点
营养条件苛刻
适应性差,敏感
培养时间长，易污染
Animal cells are more difficult to culture
than microorganisms because they require
many more nutrients and typically grow
only when attached to specially coated
surfaces,Despite these difficulties,various
types of animal cells,including both
undifferentiated and differentiated ones,
can be cultured successfully.
5.2 体外培养工具
空心纤维
微球
5.3 体外培养条件
温度,37度
pH,7.2-7.4
气体，氧，二氧化碳，氮气
营养条件需要多种氨基酸，维生素，辅酶，核酸，
嘌呤，嘧啶，激素和生长因子，其中多种成分可以由血清提供
Rich Media Are Required for
Culture of Animal Cells
Nine amino acids,referred to as the essential amino
acids,cannot be synthesized by adult vertebrate animals
and thus must be obtained from their diet,Animal cells
grown in culture also must be supplied with these nine
amino acids,namely,histidine,isoleucine,leucine,lysine,
methionine,phenylalanine,threonine,tryptophan,and
valine,
addition,most cultured cells require cysteine,glutamine,
and tyrosine,
The other essential components of a medium for
culturing animal cells are vitamins,which the cells
cannot make at all or in adequate amounts; various salts;
glucose; and serum,the noncellular part of the blood
Most Cultured Animal Cells
Grow Only on Special Solid
Surfaces
The extracellular matrices in various
animal tissues consist of several common
components,fibrous collagen proteins;
hyaluronan (or hyaluronic acid),a large
mucopolysaccharide; and covalently linked
polysaccharides and proteins in the form
of proteoglycans (mostly carbohydrate)
and glycoproteins (mostly protein),
5.4 体外细胞生长增殖过程
原代培养期
传代培养期
衰退期
5.5 培养方法
悬滴
旋转管
灌注小室
培养瓶
培养板
5.6 大规模培养技术
悬液培养，使用微球载体
6 动物细胞培养应用
1962年开始，用于生物医学研究，生产酶制剂，生长因子，疫苗，单抗等
Vero细胞和狂犬病毒的培养工艺
DMEM培养基胎牛血清其他成分锥形瓶逐级放大培养加碱（碳酸氢钠）
加糖（葡萄糖）
灌注培养液微载体
5L种子罐培养培养液
50L
生物反应器接种狂犬病毒出液口收获病毒
6.1 单克隆抗体
1975年英国科学家 Milstein和 Kohler将产生抗体的淋巴细胞同肿瘤细胞融合，成功建立了单克隆抗体技术，而获得 1984年诺贝尔医学和生理学奖。
每个 B淋巴细胞仅专一地产生、分泌一种针对某种抗原决定簇的特异性抗体，而肿瘤细胞可以无限增殖，因此杂交瘤细胞可在体外培养或移植到体内条件下分泌大量单克隆抗体。
单克隆抗体技术的最主要优点是可以用不纯的抗原分子大量制备纯一的单克隆抗体。
杂交瘤细胞产生单克隆抗体示意图
6.2 动物克隆生物繁殖后代通常是以精、卵细胞结合的有性生殖方式进行。通过营养体细胞繁殖个体的方法称为无性繁殖。克隆是指离体条件下的无性繁殖。 1981年 Illmenses
率先报告用小鼠幼胚细胞核克隆出正常小鼠。随后，
1984年 Willadsen用未成熟羊胚细胞核克隆出一头羊。
英国 PPI生物技术的罗斯林（ Roslin）研究所的维尔穆特 (Wilmut)博士 1997年 2月 27日在世界著名权威杂志
,Nature,宣布的用乳腺细胞的细胞核克隆出一只绵羊
,多莉 (Dolly),的消息。,多莉,的诞生，既说明了体细胞核的遗传全能性，也翻开了人类以体细胞核竞相克隆哺乳动物的新篇章。此项技术因而荣登美国,Science》
周刊评出的 1997 年十大科学发现 的榜首。
绵羊 B
乳腺细胞核易核卵融合卵绵羊 C子宫多莉植入植入生产克隆多莉羊示意图取出未受精卵去核电激体外胚胎发育绵羊 A
多莉 克隆猴
(1)遗传素质完全一致的克隆动物将更有利于开展对动物 (人 )生长,发育,衰老和健康等机理的研究；
(2)有利于大量培养品质优良的家畜 ;
(3) 经转基因的克隆哺乳动物,将能为人类提供源源不断的廉价的药品,保健品以及较易被人体接受的移植器官；
(4)科学家将很快地从目前的同种克隆技术推进到异种克隆,即借腹怀胎的新领域,这无疑将大大促进对濒临灭种的哺乳动物的保护工作。
克隆技术将对 21世纪产生的重大影响
6.3 干细胞工程
干细胞定义现在发现哺乳动物的许多已分化组织和器官中都保留了一些能够分裂形成该组织或器官的未分化原始细胞,这些细胞称为 干细胞 。
干细胞形态特征：
圆形,椭圆型,体积小,核质比大,具有较强的端粒酶活性,因此具有较强增殖能力 。
干细胞增殖特性缓慢性，自稳性
干细胞分类人体干细胞基本分为三种类型：全能干细胞、
多能干细胞和专能干细胞。
·干细胞分化模式
·胚 胎 干 细 胞 (embryo stem
cell),具有分化成多种细胞类型及构建组织的潜能 。
·造血干细胞
·单能干细胞 （ monopotential cell）
体内干细胞的意义 在于源源不断地补充体内一些短命组织的细胞来源，如血液细胞、皮肤细胞以及精巢等，或者组织或器官受到局部损伤时，它们可以再分裂形成新的该组织和器官，是体内的一种自我修复机制。
人类干细胞工程就是利用人体内干细胞的特征来达到体外产生人类所需的产物的探索。主要用于器官修复，基因治疗等。
第三节 植物细胞工程
植物细胞工程,就是进行物种改良，选育优良作物品种，增加植物的优良性状，利用植物生产各种化学制品，还可用于保留濒危灭绝和有重要经济价值的植物物种。
植物细胞工程技术：
一、快速繁殖技术通过植物体细胞胚胎再生成一完整植株。这种技术主要用于花卉、树木、蔬菜、果树、中草药和农作物的快速繁殖。
二、诱导增加或减少植物体内染色体组数的技术有些植株的染色体比正常细胞少一半（单倍体），而有些经诱导后染色体加倍（多倍体）。单倍体对植物品种的改良很有利；多倍体产量高，为不同倍性植物间的杂交提供了遗传保证。
三、体细胞杂交技术利用自然或人工方法使两个工几个不同细胞融合成一个细胞，形成体细胞杂种。
四、植物细胞应用生产技术利用植物细胞培养技术，给植物细胞提供最适宜的生活环境，让它们进行无性繁殖，大量地培养成植株，解决天然资源的不足。
一,植物组织培养外植体 愈伤组织 新植株进行植物组织培养，一般要经历以下五个阶段：
1 外植体的选择及培养 。
2 诱导去分化阶段 。
3 继代增殖阶段 。
4 诱导分化生根成芽阶段 。
5 移栽成活阶段 。
植物细胞培养定义在离体条件下，将愈伤组织活则其他易分散的组织置于液体培养基中，进行振荡培养，得到分散成游离的悬浮细胞，通过继代培养使细胞增殖，从而获得大量的细胞群体的一种技术。
二,植物细胞培养工业化植物细胞培养系统主要有两大类：
悬浮细胞培养系统和固定化细胞培养系统三,植物细胞杂交（细胞融合）
1960年英国诺丁汉大学 Cocking 教授领导的小组率先利用真菌纤维素酶,成功地制备出了大量具有高度活性可再生的番茄幼根细胞原生质体,开辟了原生质体融合研究的新阶段 。
植物细胞杂交的主要过程如下：
1,原生质体的制备 。
2,原生质体的融合 。
3,杂合体的鉴别与筛选 。
细胞育种诱导突变，筛选新品系、新品种次生代谢产物生成从培养的植物细胞中提取所需的代谢产物。
优点
比栽培原料作物更易控制最佳生产条件；
培养物为无菌、无虫材料，能保证产品质量；
工艺操作较为简单，可减少劳动费用，提高生产率。
植物细胞工程的应用
PERSPECTIVES for the Future
Advances in the culture of human cells will allow them to
be applied to the treatment of disease and injury,Even
today,severe burn patients can be treated by removing
some of their undamaged skin,culturing the fibroblasts,
and applying them to areas where the skin has been
destroyed,As specific growth factors for various cell
types are identified and expressed in sufficient quantities
by recombinant DNA techniques,other cell types will be
cultured and used therapeutically.
In the not too distant future,safe and effective viral
vectors will likely be developed to deliver genes
encoding therapeutic proteins or RNAs to the specific
cells where they are needed,Such gene therapy
potentially could successfully treat numerous human
diseases caused by the absence of critical cellular
molecules.
SUMMARY
Growth of vertebrate cells in culture requires rich media containing essential amino acids,
vitamins,and peptide or protein growth factors,frequently provided by serum,Most cultured
vertebrate cells will grow only when attached to a negatively charged substratum that mimics the
extracellular matrix in animal tissues.
Primary cells,which are derived directly from animal tissue,have limited growth potential in culture and may give rise to a cell strain.
Transformed cells,which are derived from animal tumors or arise spontaneously from primary
rodent cells,grow indefinitely in culture (see Figure 6-5b),They usually have an unstable,
aneuploid complement of chromosomes,including abnormal chromosomes,Transformed cells
derived from a single parental cell are called cell lines.
Cultured cells can be induced to fuse into heterokaryons (hybrids) by treatment with certain
viruses or polyethylene glycol,Heterokaryons between cells of different species tend to lose the
chromosomes of one species as they divide.
Panels of hybrid lines prepared from mutant mouse cells and normal human cells,each containing different human chromosomes,can be used to map the gene encoding a specific
human protein to a specific human chromosome.
Fusion of an HGPRT myeloma cell and a single B lymphocyte yields a hybrid cell that can grow
on HAT medium and proliferate indefinitely,forming a clone called a hybridoma (see Figure 6-10),
Since each individual B lymphocyte produces antibodies specific for one antigenic determinant
(epitope),a hybridoma produces only the monoclonal antibody synthesized by its original B-
lymphocyte parental cell.
Procedure for producing a
monoclonal antibody to
protein X,Immortal myeloma cells that lack HGPRT,an enzyme of the purine-salvage pathway (see
Figure 6-9),are fused with normal antibody-
producing spleen cells from an animal that
was immunized with protein X,The spleen
cells can make HGPRT,When plated in HAT
medium,the unfused cells do not grow,the
mutant myeloma cells because they cannot
make purines via the salvage pathway,and
the spleen cells because they have a limited
life span in culture,Thus only fused cells,
formed from a myeloma cell and a spleen cell,
survive on HAT medium,proliferating into
clones called hybridomas,Each hybridoma
produces a single antibody,Once a hybridoma
that produces a desired antibody is identified,
the clone can be cultured to yield large
amounts of that antibody.
Primary Ce ll Cul ture s Are Primary Ce ll Cul ture s Are
Useful,bu t H ave a F inite L ife Useful,bu t H ave a F inite L ife
SpanSpan
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some ce ll s di e a nd o th e rs (ma inly fib r o bl a sts ) some ce ll s di e a nd o th e rs (ma inly fib r o bl a sts )
sta rt to gr o w; o ver a ll th e g ro wt h ra t e i ncreas e s sta rt to gr o w; o ver a ll th e g ro wt h ra t e i ncreas e s
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di lut e d,th e c e ll st ra in g ro ws a t a co ns t a nt ra t e fo r di lut e d,th e c e ll st ra in g ro ws a t a co ns t a nt ra t e fo r
a bo ut 5 0 c e ll g e n e r a tio ns ( pha se I I ),af te r whic h a bo ut 5 0 c e ll g e n e r a tio ns ( pha se I I ),af te r whic h
th e g ro wt h ra t e fa ll s rapid ly,Du ri ng t h e e ns ui ng th e g ro wt h ra t e fa ll s rapid ly,Du ri ng t h e e ns ui ng
pe ri o d o f i nc reasing ce ll de a t h ( pha se I I I ),all t he pe ri o d o f i nc reasing ce ll de a t h ( pha se I I I ),all t he
ce ll s i n th e cult ur e e ven tu a ll y d i e,(b ) I n a cul tu r e ce ll s i n th e cult ur e e ven tu a ll y d i e,(b ) I n a cul tu r e
pre pare d from m o u se o r o t he r r o d e n t e mbry o pre pare d from m o u se o r o t he r r o d e n t e mbry o
ce ll s,th e r e is i nitia l ce ll d e a th co u pl e d with t he ce ll s,th e r e is i nitia l ce ll d e a th co u pl e d with t he
e me rge nce o f healt hy gr o wi ng ce ll s,A s th e se a re e me rge nce o f healt hy gr o wi ng ce ll s,A s th e se a re
di lut e d a nd a ll o we d to co nt i nu e g ro wt h,th e y so o n di lut e d a nd a ll o we d to co nt i nu e g ro wt h,th e y so o n
be gi n to lo s e gro wt h p o t e n tial an d mo st ce ll s di e be gi n to lo s e gro wt h p o t e n tial an d mo st ce ll s di e
(th e cul tu re g o e s in to c risis),Ver y ra r e ce ll s d o (th e cul tu re g o e s in to c risis),Ver y ra r e ce ll s d o
not di e bu t c o nt i nu e gro wi n g u nt il t hei r p ro ge ny not di e bu t c o nt i nu e gro wi n g u nt il t hei r p ro ge ny
o verg ro w th e c ultu r e,Th e se ce ll s co ns t itut e a c e ll o verg ro w th e c ultu r e,Th e se ce ll s co ns t itut e a c e ll
li ne,w hich wi ll gr o w for e ver if it is a p pro pri a te ly li ne,w hich wi ll gr o w for e ver if it is a p pro pri a te ly
di lut e d a nd fed wi t h n ut rie n ts,t he ce ll s are di lut e d a nd fed wi t h n ut rie n ts,t he ce ll s are
imm o rta l,imm o rta l,
