Types of animal cell culture; characterization & Their preservation.

Types of animal cell culture; characterization & their preservation

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Types of Animal Cell Culture; Characterization &
Preservation of Animal Cells

Presentation report by
IPSITA SAHOO
Registration No – 200705180160

DEPARTMENT OF ZOOLOGY

Under the supervision of
Mr. Bibhudutta Mishra
Assistant Professor, School of Applied Sciences
Centurion University of Technology and Management, Bhubaneswar, Odisha


SCHOOL OF APPLIED SCIENCES (SOAS)
Centurion University of Technology and Management, Bhubaneswar, Odisha

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CONTENTS
Introduction to animal cell culture
Why do we need Cell culture?
Overview of Animal cell culture
Types of cell culture
Primary cell culture
Different techniques used for primary culture
Secondary Cell Culture
Cell lines
Characterization And Preservation Of Animal Cell
Cryopreservation
Significance of Cryopreservation
Reference

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Introduction to animal cell culture
Cell culture is the technique where cells are allowed to grow
under controlled conditions, usually outside of their natural
environment.
Likewise, animal cell culture is a technique in which the cells
are removed and are allowed to grow in a favorable artificial
environment. The removal of tissue can either take place from
tissue directly or from disaggregation by enzymatic or
mechanical means before culture, or they may be originated
from a cell line or cell strain that has been established earlier.
Cell culture was first successfully undertaken by Ross Harrison in 1907. Roux in 1885
for the first time maintained embryonic chick cells in a cell culture.

Historical events in the development of cell culture
1. 1878: Claude Bernard proposed that physiological systems of an organism can be
maintained in a living system after the death of an organism.

2. 1897: Loeb demonstrated the survival of cells isolated from blood and connective
tissue in serum and plasma.

3. 1907: Harrison cultivated frog nerve cells in a lymph clot held by the 'hanging
drop' method and observed the growth of nerve fibers in vitro for several weeks.
He was considered by some as the father of cell culture.

4. 1916: Rous and Jones introduced proteolytic enzyme trypsin for the subculture of
adherent cells.

5. 1940s: The use of the antibiotics penicillin and streptomycin in culture medium
decreased the problem of contamination in cell culture.

6. 1952: George Gey established a continuous cell line from a human cervical
carcinoma known as HeLa (Helen Lane) cells.

7. 1955: Eagle studied the nutrient requirements of selected cells in culture and
established the first widely used chemically defined medium.

8. 1961: Hayflick and Moorhead isolated human fibroblasts and showed that they
have a finite lifespan in culture.

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Why do we need Cell culture?
Research
• To overcome problems in studying cellular behaviour such as: confounding
effects of the surrounding tissues, variations that might arise in animals under
experimental stress.
• Reduce animal use
Commercial or large-scale production
• Production of cell material: vaccine, antibody, hormone


Overview of Animal cell culture

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Types of cell culture


Primary cell culture
• Separated by enzymatic or mechanical method
• Separate cell directly from the parent tissue ( kidney, liver, heart etc.)
• Maintaining the growth of the cell in a culture medium using glass or plastic
container.
• Liquid or gel (culture media) designed to maintain the growth of the cell. It varies
from different type of cell.
Primary cell culture is further classified based on the type of cell:
Adherent cell Suspension cell
 Cells attach to the surface of the
culture flask.
 Forms a monolayer.
 They have to be detached from the
surface before they get sub
cultured.
 Growth limited to surface area
 Cell does not get attached to the
surface of culture flask.
 They are free floating.
 Cells in blood stream.
 Growth is limited to
concentration of cells.


Advantages: Usually retain many of the differentiated characteristics of the cell in vivo.
Disadvantages: 1) Initially heterogeneous but later become dominated by fibroblasts.
2) The preparation of primary cultures is labor intensive.
3) It can be maintained in vitro only for a limited period of time.
Cell culture
Primary cell
culture
Adherent
cell culture
Suspension
cell culture
Secondary
cell culture
Cell lines
Finite cell
line
Continuous
cell line

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Different techniques used for primary culture


SECONDARY CELL CULTURE
When primary cell is sub cultured then it
is called secondary cell.
Sub culture
o Transfer of cell from one
culture vessel to another
 Method
o Remove growth medium
o Detach adherent cell
(enzymatic method)
 Why required
o To provide fresh nutrients
o To provide more space

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Cell lines
 Cell lines are cultures of animal cells that can be propagated repeatedly and
sometimes indefinitely.
 They arise from primary cell cultures.
 Cell lines is of two types; finite cell lines and continuous cell lines.

Characterization of Animal Cells
 Cell line characterization is performed for Master Cell Banks (MCB), Working
Cell Banks (WCB), and End-of-Production Cells (EPC) of in vitro cell age used
for production.
 According to current regulations, the following aspects must be considered
a) The origin and history of the cell line cellular
morphology and growth characteristics
b) Cellular identity
c) Purity of the cell lines
d) Tumorigenicity and oncogenicity
e) Stability

 The objective of the characterization is to
confirm the identity, purity, and suitability of the
cell substrate for manufacturing use.

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Preservation of animal cell by Cryopreservation
Cryopreservation is the method of keeping the live cells, tissues and other biological
samples in a deep freeze at subzero temperatures for the storage or preservation. The
sample is commonly kept at −196°C.
At such low temperatures, all the biological activities of the cells stop and the cell dies.
Cryopreservation helps the cells to survive freezing and thawing.
The ice formation inside the cells can break the cell membrane. This can be prevented
by regulating the freezing rate and carefully choosing the freezing medium.
Cryopreservation Process
In this process, biological materials including cells, oocytes, spermatozoa, tissues,
ovarian tissues, pre-implantation embryos, organs, etc. are kept in extremely cold
temperatures without affecting the cell’s viability.
Dry Ice and liquid nitrogen are generally used in this method.
Cryopreservation Steps
The complete procedure steps involved in preserving the obtained biological samples
are as follows:
1. Harvesting or Selection of material– Few important criteria should be followed
while selecting the biological materials such as – volume, density, pH,
morphology, and without any damage.
2. Addition of cryo-protectant – Cryoprotective agents such as glycerol, FBS,
salts, sugars, glycols are added to the samples as it reduces the freezing point of
the medium and also allow slower cooling rate, which reduces the risk of
crystallization.
3. Freezing – Different methods of freezing are applied in this method of
cryopreservation to protect cells from damage and cell death by their exposure to
the warm solutions of cryoprotective agents.
4. Storage in liquid nitrogen– The cryopreserved samples are stored in extreme
cold or -80°C in a freezer for at least 5 to 24 hours before transferring it to the
storage vessels.
5. Thawing- The process of warming the biological samples in order to control the
rate of cooling and prevent the cell damage caused by the crystallization.

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Reference
 Butler, M. (2004). Animal cell culture and technology. Taylor & Francis.
 Yao, T., & Asayama, Y. (2017). Animal‐cell culture media: History,
characteristics, and current issues. Reproductive medicine and biology, 16(2), 99-
117.
 Stulberg, C. S. (2019). Preservation and characterization of animal cell strains.
In Culture Collections (pp. 163-170). University of Toronto Press.
 Pegg, D. E. (2007). Principles of cryopreservation. Cryopreservation and freeze-
drying protocols, 39-57.
 Dhali, A., Kolte, A. P., Mishra, A., Roy, S. C., & Bhatta, R. (2019).
Cryopreservation of Oocytes and Embryos: Current Status and
Opportunities. Infertility, Assisted Reproductive Technologies and Hormone
Assays, 1-16

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Acknowledgements
• My subject teacher: Mr. Bibhudutta Mishra
• Dr. Yashaswi Nayak, HoD and Dean SoAS
• All the Faculty members of Department of Zoology, School of Applied sciences,
CUTM
• Family and friends