The document discusses various characteristics of cells in culture. It describes how primary cultures are established directly from animal tissue, while cell lines come from established cultures. Primary cultures have cells taken directly from tissue and placed in growth medium. The document also discusses how to isolate a single cell type, factors that allow differentiation, and how normal cells differ from transformed cells that can grow indefinitely.

1. Characteristics of Cells
in Culture
Shubham A. Chinchulkar
M.Tech (Pharm.)
National Institute of Pharmaceutical education and
Research (NIPER)
shubhamchinchulkar007@gmail.com
1

2. Characteristics of Cells in Culture
 The cell culture as to whether cells are obtained directly from animal tissue or from
a culture collection
 Isolation process is far more demanding and troublesome compared to establishing
a culture from a cell sample that could be obtained from a culture collection
 Mostly the labs are prefer to use cell lines as they are well characterized in terms of
growth, origin and genetic traits
Animal tissueAnimal Organ Cell culture
2

3. A primary culture:
 The cells taken directly from animal tissue are added to growth medium
 The embryonic tissue are use to establish the primary cultures cells are more easily
dispersed and have a superior growth potential
 Tissue structure is highly ordered and consisting many type of cells
 To select a single cell type from this ordered structure we establish the primary
culture
Kidney Tissue Growth Medium
Cells to be
added into the
growth
medium
3

4.  Harrison and Carrel - original methods developed for tissue culture
 Maintenance of tissue fragments (or explant) on a solid surface and supplied with
suitable nutrients
 If individual cells are separated out before culture then this will be of greater use
 This can be achieved by following steps –
Fragmenting
tissue with
forceps and
scissors
Treatment with
a proteolytic
enzyme such as
trypsin or
collagenase
Disaggregates
the tissue into
individual cells
Isolated by
low-speed
centrifugation
4

5.  The contact between cells and with the degradative enzymes should be minimized
otherwise membrane damage may occur and cells can be bathed in trypsin for 10–
20 minutes
 The long exposure leads to the breakdown of the protein components of cell
membranes
 Collagenase degrades collagen and is less harmful to cell membranes
 The major difficulties and reasons for failure at this stage is that the cell population
becomes contaminated with bacteria or fungi
 And to avoid contamination we should maintain the aseptic condition and
instruments should be sterile and working surfaces should be swabbed with 70%
alcohol
 Subculturing or passaging – cells stop growing in primary culture then some of
cells inoculate into fresh medium
5

6.  Secondary culture – established after the first passage of the primary culture
 Cell line – cell population that can continue growing through many subcultures
 There are greatest chance of genetic alteration occurs in the first few passages
following the primary culture as cells adapt to a new chemical environment
 The chick embryo fibroblasts may grow for around 30 passages before becoming
senescent
Cell types:
 The cells are derived from tissues
 Have characteristic shapes that can be observed and recognized easily through a
light microscope
6

7. 7

8. Select a particular cell type:
 It is important to isolate a single cell type from the culture population
 There are several ways this can be achieved –
1. Allow the cells to grow
2. Control the composition of the growth medium
3. Separate cells by using gradient centrifugation
Normal Cell: Characteristics
1. Diploid chromosome number 2. Anchorage dependence
3. A finite lifespan
4. Nonmalignant
8

9. Anchorage-dependence:
 Anchorage-dependence - requirement of cells for a solid substratum for attachment
before growth can occur
 Petri dishes, T-flasks, or Roux bottles – provides the substratum in lab - specially
treated
 glass or plastic
 The combination of electrostatic attraction and van der Waal’s forces involved in
cell and surface interaction
 The divalent cations (usually Ca2+) and basic proteins forming a layer between the
solid substratum and the cell surface plays role in cell adhesion
 There should be a 2.5 nm-thick layer on the substratum prior to cell attachment
9

10. Serum-derived glycoproteins (e.g.fibronectin) =
Conditioning factors =
Cell surface glycoprotein =
The adhesion of anchorage-dependent cells to a solid substratum
Conditioning factors released by cells into the
medium help in forming a bond between cell
surface glycoproteins and the substratum
Cell
Surface
(Negative charge
is provided)
Cells get adhered
on the surface
Density of the
electrostatic charge on
the solid substratum
 Tissue culture-grade plasticware consists of sulfonated polystyrene with a surface charge
of 2–5 negatively charged groups per nm2
10

11. The culture of differentiated cells:
 Differentiations – cells slowly change their characteristics to become specialized
cells with particular characteristics/role
 This process occurs in vivo during embryo development or during wound healing
and leads to the formation of cells with specialized function (differentiated) such as
neurons or muscle cells
 The undifferentiated precursors of this process are called stem cells
 Most stem cells or embryonic cells grow well in culture
 Once cell get differentiated then it loses its capability to grow and this is reflected
by poor growth in culture
 Differentiation is important because specialized cells are used up, damaged or die
all the time during your life
 Specialized cells cannot divide and make copies of themselves, but they need to be
replaced for your body to carry on working
11

12. Adaptive response of cells to the
culture media
When some cells derived from a tissue
are placed in culture there can be an
apparent loss in differentiated
properties in the surviving cell
population
Selective outgrowth of
undifferentiated cell types
Tumor cells
UndifferentiatedGood growth characteristics
12

13. Neuroblastomas
Differentiated tumor cells Fast-growing tumor cells
Used to study
response effects with
nerve growth factor
Differentiated tumor cells retain
the phenotypic characteristics of
normal differentiated cells but are
also able to grow in culture
 Although growth of most differentiated cells is poor
13

14. Neuroblastomas
14

15. Factors may allow
some differentiated
properties of normal
cells to be
maintained in culture
Hormones and growth factors
Keratinocytes, hepatocytes and nerve cells
Chemical agents
dimethyl sulfoxide (DMSO)
Cell interactions
Interaction with the growth surface
Asymmetrical distribution of ion
currents (particularly Ca2+)
Arrest of growth when a cell population has covered an
available growth surface
Contact between cells may allow the formation of gap junctions and allow
metabolites to synchronize the expression of differentiation
15

16. Embryonic stem cells:
 These cells These cells are capable of apparently unlimited growth but have the
capacity, given the appropriate stimuli, to differentiate into any other cell type
Blastocyst
Inner mass cell
Pluripotent
16

17. Important properties:
Pluripotent - capacity for differentiation into the cells of the three major tissue types
(endoderm, mesoderm and ectoderm)
17

18. They can be propagated indefinitely in a non-differentiated state
Directed differentiation
Pluripotent stem cells
Edited Population
Ectoderm
Mesoderm
Endoderm
Cardiomyocytes
High activity level of telomerase - immortality in human cell lines-
Generally, embryonic stem cells are allowed to clump then they form an embryoid body in
which the cells begin to differentiate spontaneously
But in this case the addition of specific growth factors may direct the cells down a specific
pathway of change
Useful for studying developmental changes and also for use in cell therapy
Gene editing
or addition of
specific
growth factor
18

19. Adult stem cells:
 These are undifferentiated cells found among differentiated cells in a tissue or organ
 These cells differentiate along a more limited pathway than embryonic stem cells to
produce cells associated with the tissue
 The cells serve to replace cells or repair tissue damage
 Transdifferentiation or plasticity - Under certain conditions these cells may be
induced into cell types other than those associated with the tissue from which they
were derived
 This can be achieved by manipulation of cell culture conditions
19

20. Hematopoietic pathway
Extensive range of mature cell types
 The hematopoietic stem cells are important
for the continuous replacement of the cells
found in the blood system
 Stem cells, early progenitor cells,
progenitor cells and mature cells are four
stages through which cell differentiation
occurs
20

21. Two distinct progenitor lineages
Lymphoid Lineage Myeloid lineage
T-lymphocytes, B-lymphocytes and natural
killer
Erythroid cells that can differentiate into
monocytes, macrophages, neutrophils,
eosinophils, basophils, megakaryocytes and
erythrocytes
Glycoprotein
and
Erythropoietin,
Low oxygen levels
(hypoxia)
Formation of erythrocytes
Kidney
21

22. Transformed cells:
Expression of foreign genes in bacteria
OR
Change of animal cells from normal to infinite growth capacity
 Established or continuous cell line - cells acquire a capacity for infinite growth
 Transformation - cells to lose their sensitivity to the stimuli associated with growth
control
Normal cells
Lose their
anchorage-
dependenceTransformation
Transformed cells
&
Chromosome
fragmentation
22

23. Aneuploidy - slight alteration from the normal
diploid state
The transformed cells have a high capacity for
growth in relatively simple growth medium and
without the need for growth factors
Not all transformed cells are malignant,
characteristic defined by the ability to form tumors
in animals
However, all tumor-derived cells grow
continuously in culture
Examples include HeLa cells, which are derived
from a cervical cancer and Namalwa cells, which
derive from a human lymphoma
23

24. HeLa cells
These cells are relatively easy to grow
Robust and show good growth characteristics
Short doubling time Low requirement for growth
factors
 Cells can be transformed or ‘immortalized’ by a variety of techniques
24

25. Normal cells Immortalized cells
Mutagens, Viruses or
Oncogenes
That induces the formation of
tumorigenic cells
 Infection by retroviruses is a particularly effective method of immortalizing cells
 Retroviruses express activated oncogenes (e.g. myc and ras) which cause cell transformation
 The retroviruses are also useful for incorporating recombinant DNA into animal cells
25

26. 26