Animal Cell Culture

Presentation
 TOPIC : Introduction to Establishment of Primary
Culture and Disaggregation of Cells

INDEX
 Primary Culture
 Isolation of Explant
 Disaggregation of Tissue
 Mechanical Disaggregation
 Enzymatic Disaggregation
 EDTA treatment
 Culture media
 Physiological Conditions
 Subculturing
Primary Culture :

 It refers to the stage of the culture after the cells are

isolated from the tissue and proliferated under
appropriate conditions until they occupy all of the
available substrate i.e., reach confluence
 There are four stages :
1. Acquisition of the sample
2. Isolation of the tissue
3. Dissection and disaggregation
4. Culture after seeding into
the culture vessel
Steps Involved in Establishment of Primary Culture :
1. Isolation of Explant.
2. Disaggregation of Tissue
3. Culture Media
4. Physiological Conditions
5. Subculturing
 Isolation of Explant :

 Ethical issues must be taken care of during explant

collection.
 Protocols must be followed to avoid contamination.
 Explants must be free of contamination.
 Handling should be under aseptic conditions
 Human explants must be collected only after the
consent from donor and relatives.
Disaggregation of Tissue
Cell cultures are generally started from disaggregated
explants, tissues can be disaggregated by following
methods:
1. Mechanical Disaggregation :
 The tissue is carefully chopped or sliced in small pieces
pressing the dissected tissue through a series of sieves.
 Alternatively forcing the tissue through a syringe
 Cells grow from tissue pieces , which are then
subcultured.
 Gives cell suspension more quickly
 Cause mechanical damage
 Soft tissues respond well to this technique for example
embryonic liver , spleen ,etc.
 Time taken is very less.
2. Enzymatic Disaggregation :

 Intercellular matrix and basement membranes contain

glycoproteins like fibronectin which are protease
sensitive , so enzymes can be used to disaggregate cells
from tissues.
 Commonly used enzymes are Trypsin , collagenase ,
heparinase , hyluronidases , pronases , dispases, etc.
 Yields higher number of cells.
 Embryonic tissue disperses more readily and gives a
higher yield of proliferating cells than newborn or adult
tissues.
 Crude trypsin is by far most common enzyme used as
it is tolerated quite well by many cells and is effective for
many tissues.
Warm Trypsinization Method :
 In this method exposure of cells to active trypsin at 37° C
is minimized to preserve maximum viability, hence
dissociated cells are collected every half an hour.
 Trypsin is removed by centrifugation and neutralized with
serum in medium.
 Useful for disaggregation of large amount of tissue in a
relatively short time.
 Doesn’t work as well with adult tissue containing lots of
fibrous connective tissues.
 Mechanical agitation can be damaging to some more
sensitive cell types such as epithilium
 Time required is upto 4 hrs.
 Number of viable cells is less.
 Cold Trypsinization Method :

 It is a simple method to minimize the damage to cells

during the exposure to Trypsin
 Tissue is soaked in Trypsin at 4° C for 6 – 18 hrs to allow
penetration of the enzyme with little Tryptic activity.
 Followed by disaggregation at 37°C for 20 -30 minutes.
 This method gives a higher yield of viable cells.
 It is more convenient as no stirring is required and
incubation at 4°C may be done overnight.
 Generally used in case of soft tissues like embryonic cells.
3. EDTA Treatment :

 Cell to cell adhesion is mediated by interacting

glycopeptides or commonly called Cell Adhesion
Molecules ( CAMs ).
 Some of these adhesion molecules are calcium
dependent and hence sensitive to chelating agents like
EDTA.
 Tissues like epithilium require Ca2+ for their integrity
and hence can be easily disaggregated by treatment with
EDTA prepared in Balanced Salt Solution.
Steps in Establishment of Primary Culture :
 Culture Media :
 Media used for culture of tissues must be able to support
their survival and growth.
 Choice of media depends on the type of cells to be
cultured.
 Normal cells and primary cultures from healthy tissues
require defined quantities of proteins , growth factors and
hormones.
 Transformed cells can synthesize their own growth
factors.
 The various types of media are broadly classified as
Natural media and Artificial media.
Physiological Conditions :
 Osmolarity
 Temperature
 pH
 Gas phase
 Foaming
 Venting and Viscosity

Subculturing :
 Process of transfer of cells from spent out media to
fresh media
 When primary culture is first subcultured it gives rise o
secondary culture.
Thank you