Welcome back to the lecture series in animal
cell culture or cell culture technologies.
So, in the first week, we introduced the subject
and then we went on exploring the different
aspect of the biology of the cultured cells.
So, if we recap, when we talked about culturing
the cells, we talked about you know take a
part of the tissue make a single suspension
and then you culture it on a dish.
So, first thing which has to be considered;
what is the demand of the cell of these cells
for oxygen.
So, we talked about some kind of a condition
creating a condition where proper oxygen to
carbon dioxide balance is being maintained,
then the dish where you are growing the cell
should mimic the extracellular matrix of the
cell in your condition that is the animal
and we talked about the role of extracellular
matrix how these cementing materials plays
a critical role in determining the fate of
the cell.
Post that of course, while talking about the
adhering bring of the cell, we talked about
classification one has to understand is it
adhering cell, we are culturing or we are
culturing a non adhering cell then followed
by this, we talked about the cell cycle progression,
are we dealing with cells with limited time
division or we are dealing with fully differentiated
cell and we introduce some of the terminologies;
cell cycle leading to cell division followed
by differentiation and at times differentiation
where the cell loses its as per the definition
permanently loses its function which it is
supposed to do post differentiation, then
we talked about de-adaptation where the cell
which suppose to produce a specific function
de-adapts or does not perform at under a specific
conditions, but that could be rectified see
in other words de adaptation is a reversible
phenomena.
So, these are some of the points and finally,
I insisted you to understand that how many
passages a cell lines; suppose you are using
a cell line which has been immortalized or
it can grow n number of cycles, but then to
a fundamental question come; what is a practical
number, really a cell can be use over possible
you know division; what we are ritualizing.
So, one really has to know that because otherwise
through every passage a cell goes through
a life cycle and the cell loses some of its
telomere and eventually lot of its function
kind of gets compromised, but then it all
depends how you want to treat the cell and
for what purpose you are growing the cell.
Are you using it for a specific sensor application
or you are using it for understanding certain
event happening at the cellular level or you
are using it for some kind of hybridoma technique
like you know antibody production or you are
using it for production of some other x, y,
z chemical.
So, that particular aspect will determine
how much carefully have to be with cell type
you are dealing with.
So, let us resume the second week by kind
of summarizing the aspect what we have already
dealt and couple of other aspect which we
have not talked about to start off with.
So, we are into 
week 2, lecture 1; W 2, L 1.
So, talking about biology of the 
cultured cells, so, the point we have already
dealt is oxygen and CO2 environment, whatever
we are maintaining, we talked about extra
cellular matrix and we talked about adhering
and non-adhering cells, then we talked about
cell cycle progression.
So, we will be having mitosis phase G 1 phase
synthesis phase G 2 phase and talked about
cell division then we talked about differentiation
then dedifferentiation and de-adaptation;
this is what we have already covered.
This part is there with us, there are few
more points in order to completely appreciate
the biology of the cultured cell; what we
will be dealing with and one of the very critical
important things what we will be dealing with
is the PH under what PH a cell is an adapted
to.
So, generally we know our body PH is at 7,
but while you are growing the cell, there
are or let us put it other way; while the
cells are growing in your body, we have this
blood which is slowing through you have these
extra cellular matrix proteins; extra cellular
matrix solution which is continuously washing
out that spot ensuring it to make a PH 7.
So, when we talk about cell culture just for
your imagination sake, think of it, here we
have a dish and, in that dish, we have the
cells which are growing this half moon shaped;
these are the cells and here you have the
medium in which they are growing.
Now how one can handle the PH of this medium
and how do we know what kind of PHB cells
will prefer why I am telling you this.
See for example, we have to culture the cells
of the stomach which digest the food at a
very acidic PH of course, from our knowledge
of physiology, we know that that this acid
production by the cells parietal cells chief
cells which have present in the just for those
taking this example in the stomach which is
which functions at a very acidic PH.
So, it means less than 7.
So, the cells which are involved in this is
parietal cells chief cells, all these different
cells; these cells can withstand at will a
very low PH for a transient period of time
because of course, in the stomach it is not
that it is a place which is filled with acid
these cells are specifically produces the
acid by reacting the hydrogen iron and the
chloride iron.
But what is important for us to realize that
it is not every cell and they utilize this
acid to break the food and soon after that
this acid is kind of you know again re-absorbed
or kind of you know it is destroyed.
So, for transient period of time if I have
a PH scale out in the stomach, if you think
of it, something like PH scale here, so, if
I say these are this is 7 and this is plus
7 onward; these are my below 7, see for example,
less than 7.
So, what we will observe is and this is the
of course, the acidic side.
So, from the base line value during digestion,
thus this environment will go like this for
a finite period of time, this thing happens.
So, then what we interpret from this is the
cells in the stomach can withstand a very
low PH for a transient period of time, but
in order to regain that kind of activity we
have to create such a situation.
So, if we are culturing these kinds of cells,
we should know that what is the level they
can withstand.
Similarly, there are cells if you bring them
down to that kind of PH level, they will die,
they will not be able to withstand that kind
of high proton concentration, right.
So, maintaining the PH of a system is extremely
critical and knowing the window to which a
cell can withstand it because when we are
growing things, we are not removing all are
the Debris which the cells are producing that
kinds of create a situation where the PH may
automatically fail there is always a possibility
and there is always a danger in the cell culture
that your cultured dish goes on an acidic
frame.
So, this is something which one has to be
very careful this is one aspect.
Second aspect in that line is that we have
talked about the PH, now to add up what all
we have talked about.
So, here we have now talked about the PH,
the next what will be talking about is see
for example, here the cells are growing in
a dish adhering cell assuming that these are
all adhering cells and these adhering cells
are dividing, this is the medium where they
are growing.
Now on at this condition these cells which
are dividing will need energy and it is only
source of energy is in medium because none
of these cells are synthesizing their own
food material they are depending on the medium
to supply them with energy.
Now, 2 things; we are introducing one has
to know what kind of medium, we are going
to use because though the basic salt solution
remain more or less same, but at different
spots of the body different parts of the body,
there are several other added molecules for
specific cell types, these are cell type is
specific you might wonder, how that is happening.
So, such thing happens, see for example, if
you consider your body just that old analogy;
what I gave you for example, these are colonies
of cells in your body some are red some are
green 
some are blue like this and blood vessel is
traveling like this which is bringing the
necessary nutrients out here, but still every
cell type has a specific requirement.
So, many of the factors which see for example,
these colony of blue cells will be needing,
they are surrounding brothers and sisters
secrete those and through blood vessels or
through direct contact, these are being you
know transmitted to this cells or blood brings
specific say hormones or growth factor whose
receptors are very unique on specific cell
type.
So, in a way again coming back to that fundamental
concept that cells in a very dynamic environment
extremely dynamic extremely changing, whereas,
whenever we do a cultured we essentially follow
very static model at least as of now the most
of the models which are being followed across
the world are very static model.
So, each cell type possibly needs if we are
growing a pure culture of any specific cell
type, say for example, I grow these blue cell
as separately or the red cell as separately
or the green cell as separately, I mean it
to provide those specific molecules which
are cell type is specific, they mostly come
in the form of hormones and grow factors the
complexity arises if I have to grow, say for
example, 2 different cell types together under
that situation see green needs a different
kind if growth factor and red needs a different
kind of growth factor.
So, how I ensure that the growth factor received
by green which I am providing from outside
out here does not interact with the red, there
is only one possibility either red does not
have a receptor for it then you are very lucky
or red will have receptor for it.
And that adds up to the complexity in a real
life what happens there are blood vessels
which are crawling through and they ensure
that these binds there, but outside the system
that privilege is lost.
So, those adds up the complex situation of
growing cell in a very controlled environment
further, we talked about when we talked about;
we talked about the energy molecule.
So, every cell, if you look at it mostly they
are readily source energy source is glucose.
So, every medium what we develop will have
glucose as one of the major sources of energy
and more over carbon, but that comes with
some set of problems which will be coming
later we use glutamine and other sources there
are reason and rhyme.
So, with this I will close in on to this part
I will add that last tail piece what all we
have covered.
So, we talked about oxygen and carbon dioxide
we talked about extra cellular matrix, we
talked about the cell cycle dedifferentiation
de adaptation and of course, we talked about
the cell lines and in between we have talked
about little bit very little bit about primary
culture 
and now we talked about then we talked about
the PH acetic or basic PH where the most of
the cell groups at 7; PH 7 and then we talked
about medium growth factors hormones and the
need for energy molecules or source of carbons
source of carbon.
So, these are the very basic fundamental;
what one has to keep in mind while one is
planning to do cell culture and if you have
the basic knowledge physiology, then these
things should come very handy fine you know;
what all my body needs; there is the same
condition or close to the same condition,
I will have to provide; now if you remember,
when we talked in one of the very early classes
that whenever I am growing the cells.
So, see for example, this is my knowledge
of in vivo physiology or physiology which
is happening inside the cell and here I am
with in vitro physiology, what we talked about
is in vitro physiology in the form of oxygen
CO2 tension extra cellular matrix PH cell
cycle medium culture medium of course, and
this you have energy source 
growth factors and hormones.
Now, what is critical for it know is after
growing the cells in all these conditions
which is we are our goal is to you know come
as close as possible almost; similar almost
congruent then what we have to do the cells
which are growing here their properties have
to be evaluated.
Say for example, the cell produce certain
level of activity an ensign say for example,
it has an anti oxidative ensign capacity and
it produce a P unit of it, now I have to compare
it with in in vivo condition; how this cell
is functioning and what is the quantification
parameter of this particular one property
or many properties as a matter of fact and
if under in vivo condition see for example,
if I say activity in the y axis.
So, if the activity of a particular thing
is somewhere here seen for example, how far
I am in in vitro condition; am I exceeding
it am I below it is I seem at it.
So, depending on where am I; I have to understand
the biology of this cell which is under growing
in any in vitro setup or I am not even performing
that function I am here 0.
So, this sight the one I am circling with
red are the one which are the in vitro physiology
of the one imaginary aspect the P unit; what
I am trying to telling.
So, there comes the real challenge how much
you have understood the biology of the cell
of the cultured cell and how much we have
appreciated the in vivo physiology of that
particular tissue from where you have derived
the cell sample in order to create an artificial
condition artificial or synthetic 
growing condition, fine.
So, understanding of the basic physiology
and correlating it with in vitro physiology
is very important for a successful execution
of an experiment validity of the data in real
life and understanding the short falls and
short comings what you are under growing while
you know performing this task.
So, with this; this is our first class and
the second week we will move on to the layout
and designs.
So, next our objective will be move on to
with this basic start of I will move on to
layout and design of a cell culture facility.
So, I will close in here please go through
the points and think over it ponder upon it
and develop your own philosophy about the
subject.
Thank you, thanks for your patience listening.
