Primary Cell culture and cell line | Cell culture basics

hello everyone in this video we'll talk about cell culture cell culture refers to removal of a cell from an animal or plant and subsequently culturing it in a favorable artificial growth situation for example you can see here a lung cell has been taken out of the organ situation and kept in a culture so the lung epithelial cell can be cultured and in the next example you can see the meristem or a portion of the leaf is cultured in an artificial media to grow a intact plant so for both plant and for both animal cells they

can be cultured in vitro or outside of the organ and that is known as cell culture so this video is all about cell culture we would learn about basic terminologies which are super important to understand cell culture first we would learn about primary cell culture so primary cell culture starts with the dissociation of cell from a parental animal tissue it could be also plant tissue and enzymatic digestion or mechanical digestion is performed to get rid of the tissue organization and kind of like free the cells from these tissue-like settings so repeated up and down with

trypsin or several enzymes such as acute etc can get rid of these tissue-like organization ultimately it would become cell suspensions now these cell suspensions are plated onto specific media which would support the growth of these cells and that becomes our primary tissue culture that means from the organ you isolate the cell and plate it on a culture setting and that gives you the primary cell culture now these primary cell culture has different growth kinetics first of all there would be a lag phase where there would be no significant growth followed by a log phase exponential

growth phase where the cells would grow in an exponential fashion and lastly as the cells grow the media gets limiting and also there is not enough surface area for growth so all of these factor leads to a plateau phase so whenever a cell culture or whenever a cultured cell reach these plateau phase you would see this is termed as confluent cells confluency is commonly used as a measure of cell numbers and i mean cell surface area versus the surface area of the culture dish just to get an idea that how much crowded these cells are

if it is too much confluent then it won't get enough amount of media or necessary nutrients for its growth so when they are super confluent then you need to subculture them and first time you subculture from these cell types then they would be known as a cell line so cell line is just a subclone which is derived after the first subculture so from this culture you see these cells are now confluent it's time to separate them into two different hubs and we term it as subculture so after subculturing it would be termed as a cell

line now cell line which is derived from a primary culture have limited life span and as they are passage many cells which are having highest capacity of growth they predominate the culture so this results in a high degree of genotypic and phenotypic similarity or uniformity in these cell culture or in this cultured cell population so let us quickly compare the properties of primary cells versus cell lines so obviously i would say the primary cell has a very high biological relevance compared to a cell line i'll tell you why in a moment but if you talk

about the life span both could have sort of like the primary cell could have like a limited to finite life span where cell lines are generally infinite or unlimited now there are many other points where they differ but these primary cell culture recapitulates the tissue like organization way more than the cell lines i'll tell you why but before that let's try to understand this process in a lab setting in order to understand cell culture we have to virtually explore a cell culture lab so here is a cell culture lab you can see the essential components

it should be very clean and it should be tidy so in this cell culture lab on the right hand corner you can see there is a biosafety cabinet all the actions would take place here before looking at the bioseptic cabinet let us look at other components so you can see just left side of the biosfg cabinet there are two hoods there are two incubators which are important for keeping the cells and they would provide the proper environment for the cell to grow other than that there would be centrifuges microscope cell counter etc so there are

certain norms that you have to follow when you are working on a cell culture lab such as wearing a lab coat all the time a covered lab coat obviously and if you are culturing something which is infectious or any other ah serious material then you should have a additional layer of protection you have you should have your proper ppe kit in order to culture the cells now guys what you need to understand is what is inside the hood these hoods are generally what is depicted here is a classical bsl 2 hood or biological safety level

2 hood however biological safety level 3 and safety level 4 hoods are very different looking and other videos we'll talk about that but not in this video so you can see there are many uh things which are associated with cell culture should be placed in the hood but it should be first sprayed with alcohol and then it should be kept inside the hood such that it is contamination free now while working in the hood it is always permissible that you wear lab coat and having proper gloves and other protective equipments this would prevent contamination and

also save you for from any kind of bio hazards so let's try to understand how we can prepare a hippocampal primary cell culture just to get an example so in order to culture the hippocampal neurons we need to isolate brain from mouse generally in lab mouse are available right so you can take mouse embryo out and take out the embryonic brain in a ice cold pbs solution now after dissecting these brain in an ice cold solution you would dissect out the hippocampus and collect it in a particular tube containing trypsin acutase or pronase so all

of these enzymes are able to dissociate these content of this tissue so all the cell types which are present in that tissue would be dissociated now so obviously you put it in the incubator for some time such that the enzyme can work and after that you pipe it up and down vigorously such that it creates a cell suspension now after that it is necessary to count the cells to get an estimate how much cell you need to plate in a culture roughly about a million or 1.5 million cells are a good material to start with

and it also depends upon flux volume volume or kind of a container where we are culturing the cell i just talked about a 10 centimeter plate now after that you resuspend these uh cells in the media the cell suspension you you make a cell suspension in the media and then what you have to do you have to centrifuge them and wash them because the trip scene which is already there in that media would be harmful so you need to get rid of these trypsin so supernatant is discard whereas the pellet of cells is retained after

the trypsin is gone then again the cells are washed with pbs and ultimately resuspended with the culture media now this culture media which we add we pipe it vigorously in this step to mix our cells to break the clump of cells and make them a suspension and finally we plate them in a 10 centimeter plate or whatever plate that we have in supply and this is how we establish a primary culture now all this thing is just in a animated fashion right so let's look at this process in bit more details in a realistic setup

so ultimately you would put it in an incubator for their growth and monitor them almost every day and change the media every alternate day and lastly you have to check it in the microscope right to check their growth in this case you can see these neurons are taking their shape and forming connections in this culture so let's look at the original process so here you can see a mouse embryo and then you can dissect out the brain like this and from a from this brain you know where the hippocampus is situated so you can micro

dissect this hippocampus and culture them and in a culture setting in a very early stage you see hardly they have new rights and projections and connection in between then whereas at the late stage you can see quite a lot of connection has been formed inside them and if you track their progression over days you would appreciate how in the culture setting they are growing and forming connections with each other and making a functional neural network so obviously this is a mature neuronal network which is depicted in this image and curtsy of course google images now

let us talk about two different type of cell culture paradigm one is known as adherent cell culture another is suspension cell culture so adherent cell culture is basically what the cells get adhered with the plate material generally the plate need to be coated with some kind of extracellular matrix proteins such as lamin fibronectin sometimes such that these cells get a substratum on which they can grow whereas many cells don't need this substrate of our growth imagine a blood cell obviously in in the in vivo setting they are floating here and there so obviously in a

cultured setting they can also float so obviously it's easy to handle a suspension cell culture than uh adherent cell culture and it's easier to sort of like subculture them and a lot of difference has been noted here now let me tell you why these culture systems are not so good these culture systems has some advantage but a lot of disadvantage imagine you have a circuit organization in vivo in a certain fashion so certain amount of glial cell is connecting a certain amount of neuron in a specific stoichiometric ratio that ratio that organization cellular connection these

might be totally jumbled up when you are culturing because you are first breaking the way of connection formation and then you are allowing it to be forming spontaneously so exactly the rules or the micro environment in which these connections form would be very different that means though culture settings can allow the cells to be grown in vitro but they have a lot of limitation as a researcher it is really important to understand the limitations such that we can design our own experiments right so obviously we have certain advantages such as like the primary cell cultures

they are more closer towards the in vivo settings than a cell line because cell lines sometimes there are chromosomal aberrations there are growth related problems so really we have to understand what kind of issue we want to chase in this cell in this cell culture paradigm based on that we would use our cell lines either a primary one or a cell line right so i hope that was uh comprehensive enough but many of the people stopped using this dissociation culture method these days the primary culture method instead of that they started using organotypic culture so

it's impossible to culture the brain in total but if we slice the brain then these thin slices can be cultured because nutrient can reach inside but whereas in an intact brain it is very difficult for this nutrient to flow deep inside the brain and that is why there would be necrosis but in this settings a large surface area is in contact with the culture media so all these neurons would get some amount of culture media to thrive and that is why organotypic culture is a very good culture system these days and lot of researchers are

using these organotypic culture in a different video we'll talk about organotypic culture cell culture media etc so stay tuned and watch my channel if you like this video give it a quick thumbs up don't forget to like share and subscribe and do follow me in patreon you can also be my patreon just paying only four dollars per month and guys i'm also present in an academy so you can access all of my courses in an academy using my code ap 10 you would get a 10 discount so hurry up and thanks for listening thank you

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