warning the following video contains images of the dissection of a chicken embryo with the purpose of instructing biotechnology students on how to create primary culture viewer discretion is advised welcome to the lab i'm drew collup in today's lab we're going to be disaggregating our tissues you can see here this is a kidney that we dissected in a previous video here we have a liver and that black spot is the gallbladder the purpose of disaggregation is to break apart the organs into individual cells here are the chicken thighs with the feet still attached we'll be taking the meaty part of the thighs here is the heart now watch carefully did you see that because the sinoatrial node is found in the heart that's the pacemaker the heart can beat on its own outside the body and that's what you just saw keep an eye on this when you're doing your dissection here we have the stomach with a bit of intestine hanging off the side here is the intestine itself you can see it's very thin looks kind of like spaghetti here we have pieces of the brain the brain does not come out as a solid organ and finally we have the eyes we remove the viscous fluid inside we'll just be culturing the black part for the remainder of this video i'll be disaggregating the heart to start with we'll sterilize our forceps and our scalpel blade and we will cut the heart up into very small pieces did you see that again it just beat once again these are sterile and i will cut this heart up into very very small pieces the purpose of disaggregation again is to break this solid organ up into individual cells with the technique we use in the lab we must have them as single cells growing on the flask this is for diffusion of waste as well as nutrients and oxygen and carbon dioxide into the media i apologize for it being out of focus it appeared to focus in on my hand not on the tissue itself we'll use three methods of disaggregation in this video the first is explant but we'll take these cut up chunks place them into a dry fresh dish and adhere the tissue to the dish the second method is cold trypsin we'll keep our trypsin in the fridge until we need it we'll put these small chunks into a conical tube and we'll let the cold trypsin infuse get into the tissue overnight finally we'll do mechanical disaggregation we will literally squish the tissue through a sieve to mechanically break apart the cells you can see now i have cut up the tissue and here is a fresh dish i'm going to take about half of the tissue chunks and transfer them over to the dish we used pbs previously to prevent the tissue from sticking to the plastic that is the opposite of what i want to happen here as a result i'm trying to dab off as much of the pbs as i can on the side of the other dish to try and get the tissue as dry as possible please spread them out we need space for the cells to migrate out from the explant as you can see the tissue does get sticky once you remove the pbs from it you don't need too many chunks fewer high quality explants is better than the entire surface covered with a goo of cells on the day of your disaggregation please start with this because we must incubate this at 37 degrees for half an hour this way the tissue will stick any tissue that does not stick will not be useful in our explant disaggregation we will now move on to our second technique of desegregation using a 15 mil conical tube we will transfer the other half of the heart into this tube once again this should all happen in a sterile environment with sterile forceps don't worry about getting all the tissue down to the bottom this first technique is going to cool the tissue down initially we'll place all the tissue into the 15 mil conical tube cap it up and put it in the fridge at 4 degrees for 15 minutes to cool down the tissue please remember your tryption should also be at 4 degrees this is a cold trypsin technique not a room temperature trips and technique while we're waiting for our explant and our cold trypsin to incubate i will demonstrate the mechanical disaggregation method this method is best used on soft tissues such as the liver or the brain i will remove this gallbladder place it off to the side we'll come back to that later and using sterile forceps and a scalpel blade i will cut up the liver into small chunks i will get a 50 ml conical tube 5 0 50 mils i need to obtain a 5 mil sterile syringe and i would like to get a 100 micrometer nylon mesh this is our cell strainer this can fit in the top of our 50 ml conical tube let's open it up look for the tear point there it is right there tear it open we can pull this out with our sterile forceps and place it into the lid of our 50 ml conical tube try not to do that place it inside and then place the cap on top this will not screw down it will just rest on the top preventing any contaminants from being blown into our tube we will cut up our tissue with sterile forceps and a scalpel blade remember when switching between tissues make sure you sterilize this is extremely important so we do not get cross contamination mixing say our heart cells in with our liver cells we will transfer the entire cut up organ into the cell strainer there they are before i move on i do want to show you the gallbladder it looks black it's not black it's green in nature bile is made by the liver and stored in the gallbladder it's used to help absorb fats into the intestine let's continue on with our mechanical desegregation now we're going to use the plunger of this 5 mil syringe to press the tissue through the cell strainer this is sterile and i will use the rubber plunger to press it through the strainer you won't see the tissue go through but the cells will be going through some cells will be damaged in this process which is why it's best to use soft organs skeletal muscle does not work well with mechanical disaggregation we are pushing the whole liver through this cell strainer don't worry about getting the entire liver through we just need enough cells that we can culture now that we've applied pressure and broken apart the cells we will now place some media into our flask we're going to be using our mem our minimal essential media that has been finalized we'll use a serological pipet to pipet about five mils of media through the strainer in doing so this will wash any of the cells that have been broken apart through the strainer and will end up in the bottom of the 50 mil conical tube sometimes there's a bit of vacuum pressure that forms so you have to lift up the side of the cell strainer depends on how much tissue is covering the cell strainer on the inside so if it it's not flowing through just lift it up a little bit that'll break the vacuum seal and your cells will run through we can now throw that in biohazard and here we have a nice slurry of liver cells the protocol is now complete we will now seed directly from the 50 ml conical tube into a few of our t25 flasks the concentration of cells in that five mils of media is very high as a result you don't want to add too much or else you'll create a biofilm and these cells will just cover the incomplete surface of your t25 flask and that will not grow very effectively i'm not exactly sure what the true concentration should be of cells added into the t25 class as a result i will seed several flasks creating a dynamic range i will add half a mil to one flask one mil to a second flask and then two mils to a third flask a t25 flask requires five mils total so i will use mem to top up to that i'm looking for the ideal concentration because i'm unsure what that is i created three different flasks with three different concentrations looking for the goldilocks flask if it's too high i can use the low concentration one if my concentration was too low i can use the high concentration one liver cells often look different than what perhaps you've been used to with our elongated cells liver cells often grow as more rounded cells they do not elongate as much as other cells you've probably worked with in tissue culture i will grow these up overnight come back the next day and determine which one would be the best flask to stay with if it's too high i'll throw them away if it's too low i'll throw that away as well i'll probably take one that is the best concentration our goldilocks flask we'll call it and we will try and culture that there's plenty of cells in there we'll put those to bed at 37 degrees now we'll continue on with our cold trypsin method we have cooled down the tissue in our 15 mil conical tube for 15 minutes at 4 degrees i will now add in cold trypsin here's our trypsin it's dissolved in a solution of edta and pbs we'll use a pasture pipet to add in the trypsin now please note we do not need to fill this entire tube with trypsin that would be a waste of our resources all we need to do is add in enough trypsin that the tissue is covered in it also you would like to make sure you don't drop your cap but also make sure that the tissue is all found in the bottom of the flask you can see this is stuck on the side i can spray it with some trypsin it did go down but if there is a problem you can press it down you can see the chunks of trypsin in the bottom and again i'm trying to demonstrate the technique of pushing down the chunks with our glass pasteur pipet you can see the small chunks of heart tissue found in our trypsin we're going to place this at 4 degrees until our next lab during this time the trypsin will not be active it only activates at 37 degrees during that time the trypsin will infuse go into the tissue we will then incubate in a 37 degree water bath to activate it here is our explant plate that was incubating at 37 degrees for half an hour when labeling it please label it along the edge so we do not block the view from the microscope the tissue looks like it's stuck pretty well we will now add in two and a half mils of our mem media do this very gently any of the tissues that come unstuck from the dish will be useless two and a half mils and you can see i will gently drip it in the purpose of the explant method is to put a small chunk of tissue on a dish it will adhere and then the cells on the outside will divide they'll have no place to go but outwards so it will appear like the cells are migrating away from the x plant it appears one of my chunks has come unadhered and that chunk will be garbage i could remove that right now if i wanted to it appears i have four good explants they stuck initially and they survived the addition of our mem staying stuck to our flask we'll now incubate that at 37 degrees until our next lab here is the explant plate from one of my students in the class this is about a day or two after the explant was created we will place this on our inverted microscope it's connected to a camera which is connected to a monitor in the lab and we can view this at low magnification initially we'll bring this into focus and we'll take a look at the explants and the cells once again these are heart cells and you can see we had success here now the explant should look darker why because it's not a single layer individual cells should look clear and healthy cells should look elongated here's an example of a large explant and you can see the heart cells have migrated away from that explant what we'll do is we'll sterilize forceps we'll remove that explant chunk leaving behind the single monolayer of heart cells we'll then continue to culture that and we will have our single layer of cells zoom in at medium magnification you can see them a bit better the explant and the cells you can start to see nuclei here and finally add high magnification now only time will tell if this will become a continuous cell line these cells are senescent and may not survive after a couple days if the cells do not transform they will all die that's the nature of primary culture it's not as easy as it looks [Music] if you enjoyed this video give it a like and consider subscribing you can click on this link to check out our experiment comparing an ostrich egg to a chicken egg i hope you enjoy it thanks for watching until next time [Music] you