Bài giảng Nhập môn công nghệ sinh học - Animal culture

Animal culture 
Kỹ thuật nuôi cấy tế bào động vật 
Cell culture 	 
Tissue 
Tripsin 
Suspended cell/medium 
Adhere to bottom of a flask and form a monolayer 
Proliferation then stop 
subculture 
Treat with EDTA/ tripsin 
(take out from flask) 
Secondary culture 
Group of cell strain 
t 0 
proliferation 
2 main categories of animal cell cultures. 
Primary culture 
Continuous cell line 
Primary Cultures 
Taken from fresh tissue 
Limited life span in culture 
Treated by proteolytic enzyme ( Trypsin ) 
Separate into single cells 
	-epithelial cells 
	-fibroblasts 
Continous cell lines 
Not any primary cell and its generation are die after proliferating.However , some cells continue growing. They have different morphology, grow faster. Their generation exist unlimited –called cell line 
Through transfer subculture they may be changed their property. EX: increasing group of cell irregularity ( neoplastic---tế bào ung thư ) 
Cell + glycerin, dimethylsulphosid kept Cool preservation to prevent cold crystal then placed into LN 
Continuous Cell Line 
Derived from humans 
Been transformed 
	-lose sensitivity to factors associated with growth control 
Produce immortalised cell lines 
Cell lines are neoplastic 
Often lose their anchorage-dependence 
	-associated with an altered xsome pattern 
More easily cultured 
Cell 
line 
Species of origin 
Tissue of origin 
Cell morphology 
Growth in suspension? 
3T3 
Mouse 
Connective 
Fibroblast 
No 
CHO 
Chinese 
Hamster 
Ovary 
Epithelial 
Yes 
BHK21 
Syrian 
Hamster 
Kidney 
Fibroblast 
Yes 
HeLa 
Human 
Cervical 
Carcinoma 
Epithelial 
Yes 
Medium 
Enough nutrition for cell growing 
Ph = 7.0-7.3 (need buffer) 
Same isotonic with cytoplasm 
Sterility 
It should have : 
Inorganic ion 
Precise osmotic 
Ph 
Energy (glucose) 
pH Indicator (phenol) 
AA ( Arg , Cys , Tryp , tyr,Meth , Glu , Isoleu , Leu , Lys , phe ,) 
Vit (acid Floric , Inositol , Nicotinic, B6, B2, B3) 
Buffer ( Bicarbonatnatri ) 
Antibiotic 
Serum (5-10%) 
Water ion- free 
Standard for cultural medium 
Application of animal cell culture 
In vitro toxicity testing 
 Produce virus vaccines 
Chicken embryo/ cell culture 
Animal cell culture 
(kidney of monkey or checken embryo) 
Infection with virus 
Collect virus after replication 
Purification virus by filter/condense 
Suspended virus +stably material to prevent decease activity – keep under cold condition 
Produce Hormones 
What Are Stem Cells? 
Stem cells are the raw material from which all of the body’s mature, differentiated cells are made. Stem cells give rise to brain cells, nerve cells, heart cells, pancreatic cells, etc. 
How do cells remain 
Self renewing and 
unspecialized? 
What are the signal???? 
Hormones 
Growth factors 
Small proteins 
Definition: is able to proliferation continuously and develop into differentiation cell 
Stem cell 
Totipotency – The capacity of a cell (or a group of cells) to give rise to an entire organism. 
Germ cell: capacity to divide into one or definite special cell. 
EX: blood germ cell develop into different blood cells (red blood, leukocyte, lymphocyte) 
STEM CELL BASICS 
 Where do stem cells come from? 
Sources of stem cells during development 
Teratomas ” (benign); 
“ Teratocarcinomas ” 
(malignant 
[P] = pluripotent 
[M] = multipotent 
[T] = totipotent 
Origin of Human Embryonic Stem Cells 
Isolation of Human Embryonic Stem Cells 
First done by Dr. 
James Thompson 
and colleagues at 
the University of 
Wisconsin (1998 
Properties of Human Embryonic Stem Cells in culture 
• Pluripotent – able to form any of ~200 different types of cells of the body 
• Self renewing in vitro – can propagate or proliferate indefinitely in the undifferentiated state 
• Express the enzyme telomerase (required to maintain the ends of chromosomes) and Oct 4 (a master regulator of ESC pluripotency ) 
• Maintain normal chromosome structure and complement even after long periods in culture (unlike many other tissue culture cell lines) 
What’s So Special About Stem Cells? 
They have the potential to replace cell tissue that has been damaged or destroyed by severe illnesses. 
They can replicate themselves over and over for a very long time. 
Understanding how stem cells develop into healthy and diseased cells will assist the search for cures. 
Two Kinds of Stem Cells 
 Embryonic (also called “pluripotent”) stem cells are capable of developing into all the cell types of the body.  
 Adult stem cells aer less versatile and more difficult to identify, isolate, and purify. 
Embryonic Stem Cells 
Researchers extract stem cells from a 5-7 days old blastocyst .  
Stem cells can divide in culture to form more of their own kind, 
thereby creating a stem cell line. 
The research aims to induce these cells to generate healthy tissue needed by patients. 
Two Kinds of Stem Cells 
 Embryonic (also called “pluripotent”) stem cells are capable of developing into all the cell types of the body.  
 Adult stem cells are less versatile and more difficult to identify, isolate, and purify. 
Two Sources of Embryonic Stem Cells 
1 Excess fertilized eggs from IVF (in-vitro fertilization) clinics 
2 . Therapeutic cloning (somatic cell nuclear transfer) 
Tens of thousands of frozen embryos are routinely destroyed when couples finish their treatment. 
These surplus embryos can be used to produce stem cells. 
 Regenerative medical research aims to develop these cells into new, healthy tissue to heal severe illnesses. 
Somatic Cell Nuclear Transfer 
 The nucleus of a donated egg is removed and replaced with the nucleus of a mature, "somatic cell" (a skin cell, for example). 
 No sperm is involved in this process, and no embryo is created to be implanted in a woman’s womb. 
The resulting stem cells can potentially develop into specialized cells that are useful for treating severe illnesses. 
Why do it? 
Particular cells can be isolated and cloned 
	-isolation of mutant cells 
	-investigate cell growth 
	-produce hybrid cells that have applications in biotechnology 
Produce important pharmaceuticals 
	-vaccines 
	-hormones 
Stem cell cultivation 
Fertilized egg 
Blastocyst 
Cultured undifferentiated 
stem cells 
Differentiation 
Blood cells 
Muscle 
Neural cells 
Embryo 
Isolated 
pluripotent 
stem cell 
Origin of Human Embryonic Stem Cells 
Isolation of Human Embryonic Stem Cells 
First done by Dr. 
James Thompson 
and colleagues at 
the University of 
Wisconsin (1998). 
Application of stem cells 
1. Stem cells used for pharmaceutical safety 
2. Cell therapy 
Embryo transfer