Sie sind auf Seite 1von 33

A BRIEF OVERVIEW 2

1. Introduction 15. Phases of Cell Culture


2. History 16. Secondary Culture
3. Idea of Cell Culture 17. Cell Culture System
4. Equipment's used in Cell Culture 18. Quantitation of Cell Culture
5. Cell Culture Environment 19. Cell Morphometry
6. Types of Cell Media 20. Preparation of Chick embryonic DRG cultures
7. Cell Culture Media 21. Preparation of neuronal cell culture from
8. Supplements to basal media embryonic Rat brain
9. Replacement of Serum 22. Intestinal drug metabolism using Caco-2
10. Disadvantages of Serum-Free Media 23. Conclusion
11. Types of Culture 24. References
12. Types of Cell Culture
13. Techniques for primary culture
14. Preparation by trypsin disaggregation
3
SO WHAT IS CELL CULTURE?
• Cell culture can be defined as the process of cultivation cells and tissues
outside the body of an organisms in an artificial environment, which
stimulates the in-vivo conditions, such as temperature, nutrition and
protection from microorganisms.
• Tissue culture is used as a generic term to include the in-vitro cultivation
of organs, tissues and cells. Originally the term is not limited to animal
cells, but includes the in-vitro cultivation of plant cells. Tissue organ
culture can be subdivided into 3 major categories; organ culture, explant
culture and cell culture.
4
HISTORY
“Tissue Culture is not a new technique”

Cell culture was first successfully undertaken by Ross Harrison


in 1907.

Wilhelm Roux in 1885 for the first time maintained


embryonic chick cells in a cell culture.
GENERAL IDEA OF CELL CULTURE 5
Harvest Cells

Isolate cells with the use of


appropriate enzymes

Apply the isolated cells to an appropriate


growth media in a culture dish

Culture cells by placing the culture


dish in a cell incubator

Verify the cultured cells are


of the cell type of interest Subculture cells to obtain a
pure culture or to bypass some
Cells are ready to be manipulated or problems(such as senescence)
modified for experimented procedures
BASIC EQUIPMENT’S 6

Tissue culture ware Incubator Inverted Microscope


Some More Equipment's 7

Facilities for counting cells

Filter Sterilization

Refrigeration and freezer

Water Still or reverse osmosis apparatus


Variety of equipment's used in animal cell culture 8
Essential equipment Beneficial equipment Useful additional equipment

Incubator Laminar Flow Hood Low- Temperature Freezer


Sterilizer Cell Counter Glassware washing Machine
Microscope Vacuum Pump Closed-Circuit TV
Washing-up Equipment Co2 Incubator Colony Counter
Sterilizing & Drying Oven Preparation & Quality Cell Sizing
Water Purification Control Time-Lapse
Centrifuge Upright Microscope Cinemicrography
Cell Freezing Temperature Recorder Controlled-Rate Cooler
Bulk Culture Centrifugal Elunator
Pipette Aids & Fluorescence- Activated cell
Automatic Pipetting sorter
Cell culture environment (in vitro) 9
Components of Basal Media

Keto acids (oxalacetate and pyruvate)


• Keto acids added to the media as additional energy source
• Maintain maximum cell metabolism
Carbohydrates
• Energy source
• Glucose and galactose
• Low (1g/L) and high (4.5g/L) concentrations of sugars in basal media
Vitamins
• Precursors for numerous co-factors
• B group vitamins necessary for cell growth and proliferation
• Common vitamins found in basal media is riboflavin, thiamine and biotin
Trace Elements
• Zinc, Copper, selenium and tricarboxylic acid intermediates
Cell Media Type 10
Media type Examples

Biological Fluids Plasma, serum, lymph, human placental


cord serum, amniotic fluid
Tissue Extracts Extract of liver, spleen, tumors ,
Natural Media
leucocytes and bone marrow, extract of
bovine embryo and chick embryo
Clots Plasma clots

Balanced salt solutions PBS, DPBS, HBSS, EBSS


Artificial Media
Basal media MEM DMEM

Complex media RPM1-1640, IMDM


CELL CULTURE MEDIA 11

1. Basal media

2. Reduced- serum media

3.Serum-free media
12
Supplements to basal media
• L-glutamine
• Non-essential amino acids(NEAA)
• Growth Factors and Hormones (e.g.:insulin)
• Antibiotics and Antimycotics
• Foetal Calf/Bovine Serum (FCS & FBS)
• Heat Inactivation (56 degree C for 30 mins)-why?
Replacement of Serum 13
• The essential factors in serum have been described and include

1. Adhesion factors such as fibronectin.


2. Peptides such as insulin, PDGF and TGF-8, that regulates growth and
differentiation.
3. Essential nutrients such as mineral, hydrocortisone, estrogen and triodothymine.

• All these constituents regulates membrane transport, differentiation and the


constitution of the cell surface.
• Some of these constituents are included in the formulation of serum –free media,
others are not and may require addition and optimization.
14
Disadvantages of Serum-Free Media
1. Multiplicity of media: Each cell type appear to require a different
recipe, and cultures from maligmant tumors may vary in requirements
from tumor to tumor, even within one class of tumors.
2. Reagent purity: The removal of serum also requires that the degree of
purity of reagents and water, as the removal of serum also removes the
protective, detoxifying action that some serum proteins may have.
3. Cell proliferation: Growth is often slower in serum- free media, and
fewer generations are achieved with finite cell lines.
TYPES OF CULTURE 15

Primary Culture
• Explant culture
• Enzymatic dissociation culture

Secondary culture
• Monolayer culture/anchorage dependent culture
• Suspension culture/non anchorage dependent culture

Primary culture- cells were directly taken from the tissues.


Secondary culture- derived from the primary culture.
Types of Cell culture 16

Primary Cultures
Derived directly from excised tissue
and cultured either as:

• Outgrowth of excised tissue in


culture
• Dissociation into single cells (by
enzymatic digestion or mechanical
dispersion)
Different techniques used for primary culture 17
Selection and isolation
of organ

Disection and removal of


necrotic cells and fat cells

Mechanical disaggregation Enzymatic disaggregation Primary explant techniques

Chopping or slicing of tissue, Collagenase


Sieving,syringing,pipetting etc Other enzymes Explant
Trypsin
Cold (bactarial proteases)
Trypsinization
Warm trypsinzation
Resuspended and seed

PRIMARY CULTURE
subculture

CELL LINE
Preparation of primary culture by trypsin disaggregation 18
(A) Warm trypsinization (B) Cold trypsinization
19
Phases of Cell Culture 20
21
Cell line

Normal Transformed Stem Cell

Taken from a tumor They are Master


Normal cells underwent
tissue and cultured Cells that generate
a genetic change to be
as a single cell type Other differentiated
tumor cells
Cell types
SECONDARY CULTURES 22
• Derived from a primary cell culture and isolated by selection or cloning.

• Becoming a more homogeneous cell population.

• Finite life span in vitro.

• Retain differentiated phenotype.

• Mainly anchorage dependant.

• Exhibit contact inhibition.


 Contact inhibition is a growth mechanism. In most cases when two cells
collide they attempt to move in a different direction to avoid further collision.
Cell Culture Systems 23

• Cells may be roughly divided into two main types:


1-Suspension cell culture
(Anchorage-independent)

• Derived from cells which can be divided and


survive without being attached to a substrate.
• Eg. Cells of haemopoietic lineage
• Can be maintained in culture vessels that are
not tissue-culture treated.
• Requires agitation for adequate gas exchange
• Easier to passage
Cell Culture Systems CONTD. 24
2-Adherent cell culture
(Anchorage-dependent)
• Must adhere to a surface to survive
• Form monolayers
• e.g. cells derived from different tissue
(breast, liver)
• Growth is limited by surface area
• Will cease proliferating once they become
confluent ( completely cover the surface of
cell culture vessel).
• Cells are dissociated enzymatically or mechanically
from surface.
Quantitation of cell culture 25

For properly run experiments, it may be necessary to count the cell numbers before, after
and even during the experiment. Day to day maintenance of cell lines also requires
quantitative assessment of cell growth so that optimum cell densities for sub-culturing
and storing can be determined.

We can divide the methods available for determining cell growth into two sub-groups.
• Direct methods
• Indirect methods
 Direct methods for quantitation of cells in culture
26
• Counting chambers
• Coulter counters

Counting chambers Coulter Counter


 Indirect methods for determining cells in culture
• Trypan blue exclusion
• LDH leakage

Trypan Blue LDH leakage


Cell Morphometry 27
Specialized morphological characteristics of cultured cells can be determined and
analysed by using computerized image analysis e.g., neurite length and soma size
of cultured neurons.
Simplified method for the preparation of chick 28
embryonic dorsal root ganglion (DRG) cultures
Preparation of neuronal cell cultures from the embryonic 29
rat brain
Using Caco-2 cell culture as an in vitro model for the 30
study of intestinal drug transport/metabolism
31
CONCLUSION
In recent years the use of animal cell culture has undergone a major expansion from
Being a purely experimental procedure to become an accepted technological
Component of many aspects of biological research. This presentation summarizes
Theoretical background and basic techniques of culturing animal cells in a format
That is readily accessible to all researchers in the field.
32
REFERENCES
• Benford, D.J and Hubbard, SA.(1987) Preparation and culture of mammalian cells, in Biochemical
• Toxicology: A Practical Approach (Snell, K. and Mullock, B.eds) pp57-82,IRL Press, Oxford.
• Butler, M.(1991) Mammalian Cell Biotechnology: A practical Approach. Oxford University Press, Oxford.
• Conn, P.M.(1990) Methods in Neuroscience Volume 2: Cell Culture. Academic Press, California.
• Darling, D.C and Morgan, S.J.(1994) Animal Cells: Culture and Media. John Wiley & Sons, New York.
• Doyle, A., Griffiths, J.B. and Newell, D.G. (1993) Cell and Tissue Culture.
• Freshney, R.L.(1992) Animal Cell Culture: A Practical Approach. 2nd edition, Oxford University Press, Oxford.
• Pollard, J.W and Walker, J.M.(1990) Methods in Molecular Biology Volume 5.
• Wikipedia
33