Advanced Aminal Cell Culture

2013 2nd Semester

Department of Animal Science

Chungbuk National University

1st Lecture

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’

Syllabus

Date TopicsSeptember 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 INovember 7, 2013 Stem Cell IINovember 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..)

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.

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

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…

- Cost effective than animal experiments

- Avoid ethical problem in animal/human experiments

- Production of Protein/Antibody/Virus/Vaccine using cultured animal cell culture

History• 1885 : medullary plate of an embryonic

chickens (Wilhelm Roux)• 1907 : Grow Frog nerve fiber in using

hanging drop culture

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.

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

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)

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)

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

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

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

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

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)

Recent National (US) Bestseller story about Henrietta Lacks

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 : 824 copies of chromosome 123 copies of chromosome 6,8,7

Copy Number variations of HeLa chromosome (Nature, 2013)

Transformed Cells Can Grow Indefinitely in Culture

Type of Cell by Morphology

Fibroblast like

Lymphoblast-like

Epitherial-like

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)

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)

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

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

Buffering in Cell Culture

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

Journal Presentations

• We will have journal presentations every weeks (Two persons per week)

• Detail will be announced by E-mail