충북대학교 축산학과 대학원 동물세포배양특론 강의 1

1. Advanced Aminal Cell Culture
2013 2nd Semester
Department of Animal Science
Chungbuk National University
1st Lecture

2. Basic Information about Courses
Lecturer : Suk Namgoong
Room 443, S21-5
suknamgoong@chungbuk.ac.kr
HP: 010-4103-2415
Biweekly : 1st, 3nd, 5th Thursday 9:00-12:00
Grade : Midterm (20%) + Attendance(10%) +
Seminar (20 %) + Final (50%)
Test will be done as ‘Take Home Exam’

3. Syllabus
Date Topics
September 5, 2013 Introduction : What is Cell Culture?
September 12, 2013 Cell Culture As Model System For Research
September 26, 2013 Cell Culture For Antibody / Protein Production
October 10, 2013 Protein Production/Purification
October 24, 2013 Stem Cell I
November 7, 2013 Stem Cell II
November 21, 2013 TG/KO Animals
December 5, 2013 Genome Engineering/NGS
December 12, 2013 Final Exam
Each Person will Present a Assigned Paper (To be announced..)

4. What is Cell Culture?
• The process by which prokaryotic,
eukaryotic or plant cells are grown under
controlled conditions
• But in practice it refers to the culturing of
cells derived from animal cells/tissuel.

5. Why we need to culture animal cells?
- Cell is the Basic Unit for the Life
- Understanding functions and roles of various cell
are crucial approach for modern biology

6. With isolated cell culture, we can study/investigate
desired cell using various techniques without
interference of other cells/tissues/organ
* Microscopic
* Biochemical
* Molecular Biology
* Toxicology…

7. - Cost effective than animal experiments
- Avoid ethical problem in animal/human
experiments
- Production of
Protein/Antibody/Virus/Vaccine using
cultured animal cell culture

8. History
• 1885 : medullary plate of an embryonic
chickens (Wilhelm Roux)
• 1907 : Grow Frog nerve fiber in using
hanging drop culture

9. History of Cell Culture (Continued..)
• 1912 : Alexis Carrel culture chicken heart using chick
embryo extract
• 1916 : Trypsinization and subculture of explants
• 1923 : Development of first cell culture flask
• 1925 : Subculture of fibroblastic cell lines
• 1940s : Discovery of Antibiotics -: The use of the
antibiotics penicillin and streptomycin in culture
medium decreased the problem of contamination in
cell culture.

10. 1952 : Cloned Tadpoles (Briggs and King)
1954 : Discovery of Contact Inhibition (Abercrombie)
1955: nutrient requirements of selected cells in culture
and established the first widely used
chemically defined medium.(Eagle)
DMEM : Dulbecco’s Modified Eagle’s
Medium

11. 1961: isolated human fibroblasts (WI-38) and
showed that they have a finite lifespan in culture.
1965: first serum-free medium which was able to
support the growth of some cells. (Ham)
1975: First hybridoma capable of secreting a
monoclonal antibody (Milstein)
1978: development of
serum-free media from
cocktails of
hormones and growth
factors. (Sato)

12. 1981 : Mouse Embryonic Stem Cell
1982: Human insulin : the first recombinant protein to be
Licensed as a therapeutic agent.
1985: Human growth hormone produced from recombinant
bacteria
1989 : Knockout mouse using Mouse ES Cell (Capecchi,
Evans, Smithies)

13. 1996 : The First mammal cloned from adult cells (Dolly, the
sheep)
1998 : Human Embryonic Stem Cell
(Thomson)
2006-2007 : First Induced Pluripotent
Stem Cell (iPSC : Yamanaka and
others)
2013 : First Human stem Cell
generated from SCNT

14. Primary Cell Culture
Cells taken directly from living tissue (e.g. biopsy
material) and established for growth in vitro
Undergone very few population doublings
Proteolytic enzymes (trypsin and Collagenase)
are commonly used to break the protein

15. Primary Cell Culture
Cons
• They are not well characterized,
• Have limited life span,
• Slow in proliferation
Pros
• more representative of the main functional
component of the tissue

16. Established Cell Line
- After the first subculture, primary culture may be called secondary cultures
- Thereafter, if continued passage is possible, a cell line.
- Established or immortalised cell line : ability to prolierate indefinetely by
•Random Mutation
•Artificial Modification : expression of telomerase, insertion of cancer antigen
Cell Line Organism Origin Tissue
HeLa Human Cervical Cancer
293-T Human Embryonic Kidney
A-549 Human Lung carcinoma
ALC Murine Bone Marrow
CHO Hamster Ovary
HB54 Hybridoma Hybridoma

17. HeLa
• The oldest and most commonly used
human cell line
• 1951 : Derived from cervical cancer cell
taken from Henrietta Lacks (1920-1951)
Henrietta Lacks (circa 1945)

18. Recent National (US) Bestseller story about Henrietta Lacks

19. HeLa is famous for…
Immotallised Cell Line : Due to mutation, it can
evades normal cellular senescence and can keep
undergo division
Used to test the first polio vaccine / virus Culture
More than 60,000 scientific articles has been
published using HeLa
Extensively Used for the Cancer Studies
It has abnormal chromosome number : 82
4 copies of chromosome 12
3 copies of chromosome 6,8,7

20. Copy Number variations of HeLa chromosome (Nature, 2013)

21. Transformed Cells Can Grow Indefinitely in Culture

22. Type of Cell by Morphology
Fibroblast like

23. Lymphoblast-like

24. Epitherial-like

25. Type of Cells
• Anchorage-independent
- can propagate in the suspension cultre
- Blood Cells
- Cancer Cells
- Hybridoma
• Anchorage-dependent
- propagate as a monolayer attached to the cell culture
vessel
- Will cease profliferating once they become confluent
(completely cover the surface of cell culture vessel)

26. Used for bulk protein production, batch harvesting, and
many research applications
Spinner Flask
Suspension Culture
Appropriate for cells adapted to suspension culture
and a few other cell lines
that are nonadhesive (e.g., hematopoietic)

27. Easier to passage, but requires daily cell counts
and viability determination to follow growth
patterns;
culture can be diluted to stimulate growth
Can be maintained in culture vessels that are not
tissue-culture treated, but requires agitation
(i.e., shaking or stirring) for adequate
gas exchange

28. Adherent Cell Culture
- Adherent cell require surface to attach to grow
Appropriate for most of cell types (including primary cultures)
- The majority of the cells derived from vertebrates are
anchorage-dependent
- have to be cultured on a suitable substrate that is
specifically treated to allow cell adhesion and spreading
- Growth is limited by surface area
- Cells are dissociated enzymatically or mechanically from surface

29. Buffering in Cell Culture

30. Utilization of Cell Culture
• Model System for Basic Science
• In Vitro Cell Toxicity / Screening
• Animal Cell Culture for Protein Productions
• Tissue/embryo Engineering
• Cloning/KO/TG animals

31. Journal Presentations
• We will have journal presentations every
weeks (Two persons per week)
• Detail will be announced by E-mail