Episode 2 | Mini Organs | Back to the Future | Virtual Lab
[Music] hi guys Welcome Back to the Future my name is kasturi and I'm a science Communicator at the center for predictive human model systems or as we like to call it cphms in today's episode we'll be taking you to not one but two Labs both of which work on creating human relevant 3D cell culture models [Music] thank you so let's talk about spheroids and organoids these are examples of complex 3D cell culture models which are made up of one or multiple cell types organoids are Miniature self-assembling and simplified 3D versions of organs produced in vitro from stem cells self-organization is when cells interact with neighboring cells and arrange themselves to form higher order structures organoids are used to understand structural and functional complexities of organs as well as map disease progression and enable drug discovery spheroids on the other hand are simple clusters of either single or multiple cell types you could use tumor cells or hepatic cells or any kind of cells to create the spheroid you want these cells stick together and form aggregated 3D structures which are not capable of self-assembly or regeneration today we'll be taking you to Dr tulide's lab to learn about breast cancer spheroids and then hop over to Dr indomati mariyapan's lab to understand retinal organoids hi I'm Abhishek Delhi and I am a PhD student working in tumor biology lab led by Dr thuli day we are trying to develop an input route to a breast tumor model using multicellular tumor spheroids so today I am going to show you how to fabricate and characterize this multicellular spheroid and I'm also going to talk to you about how we have used this multicellular spheroids to understand breast tumor Invasion and metastasis foreign [Music] ERS in our body the undergo genetic changes which makes them divide uncontrollably so these uncontrollably dividing cells they will form a column which we called as tumor so few tumors they remain in place and others they can spread a few cells from a tumor they can extend themselves and enter into surrounding tissue which is known as invasion and other cells they can also enter circulation move to distinct organ in our body and can form a secondary tumor the process is known as metastasis our lab growth spheroids are pathophysiologically similar to The in Vivo tumors this means the look and behave like the tumor that we find in cancer patients in our lab we work with mcf7 which is a breast cancer cell line these cells require a surface to attach and grow in a two-dimensional culture cells will attach to the bottom of the culture flask but in an agarose coated three-dimensional culture play these cells are prevented from binding to the plastic surface in this case cells will prefer to stick to each other once they are centrifuged and brought together the first step to culturing spheroid Aggregates is to prepare an agarose solution agarose is boiled in PBS cooled and then used to coat the bottom of the well plate once the agarose solidifies it is ready to use cells are detached from the culture flask using trypsin after which they are centrifuged at 1200 G for about 2-3 minutes and counted using a hemocytometer these are what the cells look like when they're floating in the media after detachment by counting these cells we know the exact volume and the exact cell number that we'll be seeding into the culture plate [Music] once we add the specific volume of cell suspension into each well the plates are spun in the centrifuge at 2000 RPM for about 3 minutes this facilitates the cells to settle close together and form aggregates the plates are incubated under standard cell culture conditions at 37 degrees Celsius 95 humidity and 5 CO2 [Music] thank you [Music] first we will collect spheroids from an agar coated plate and transfer them into a 1.5 ml ependorf tube [Music] staining cells involves many different steps to prevent the cells from falling apart we preserve them using a fixative agent called PSA or periformaldehyde in order to allow antibodies to enter the interior of the cell we use a mild detergent like Triton X100 to create small pores in the otherwise impermeable plasma membrane so this process is called permeabilization in between all these steps we will wash the cells with phosphate buffers Saline this helps remove leftover reagents from the previous steps blocking is essential to prevent the non-specific binding of antibodies in the cell and to ensure that only the target protein is being stained for this process we are using bovine serum albumin finally we're going to stay in our sense the first stain we're using is the fungal protein called phylloidine which binds to actin actin is a cytoskeletal protein found in the cell this protein is bound to rhodamine which emits the red fluorescence the second stain is the blue DNA binding die called dappy [Music] in pseudo 3D migration assays steroids are transferred to a conventional cell culture plastic surface where migratory cells exit the spheroid in a movement similar to the way ants will move towards a piece of food here cells are allowed to migrate without any extracellular barrier unlike a real tumor to mimic the barrier that cells penetrate during cell Invasion we perform an extracellular Matrix Invasion assay where spheroids are embedded in a protein-rich matrix metastatic cancer cells will make holes in this barrier through which they migrate whereas non-metastatic cancer cells will simply remain in the steroidal structure and this essay helps us distinguish between the two types of cells for now you have seen that how the tumor migration can be studied using different protocols and how you can prepare tumors uh in the lab by using differently available cell lines and mixing them together from the point of view that from a budding scientist I can say that this is a field of enormous importance you can design this kind of tumor and the industries are these days working on it in very extensive way they are finding out different ways of testing drugs testing inhibitor molecules against such Platforms in the research also people are now interested in 3D culture more than the conventional 2D culture because they represent your biology and they represent the pathophysiology in a more better way or they mimic our biological system in in a more honest way we can say so um I hope that this kind of model system and this kind of experimental setup will interest you and in future when you are also interested in doing some kind of a research or develop some anti-cancer drug develop some any other kind of drugs for different other diseases you will think 3D models or specifically spheroid as a viable platform [Music] foreign [Music] [Music] Prasad Eye Institute and at the stem cell research laboratory here we look at understanding retinal development and disease and we also use stem cell based model system for developing regenerative therapies to address some of the problems of retinol and corneal disorders today our team will be demonstrating how we generate these organoids in the dish and how they look like how we identify and isolate them how we use them to understand disease biology in the lab thank you the retina is a thin light sensitive tissue which is found inside the eye any damage to the retina causes Vision impairment in most of the cases the damage caused cannot be reversed therefore it is important to study and understand these kind of diseases miniature retinal tissues or organoids can be derived from Human induced fluoripotent stem cells or ipscs these cells when differentiated can form many structures of the parts of the eye like the retina cornea and the lens the retinal organoids comprises of the major retinal cells like the retinal ganglion cells interneurons and the photoreceptors foreign [Music] let's find out how to culture these retinal organoids which can be used as a model to study various eye diseases a small 2 by 2 mm punch biopsy of the skin is taken from consenting volunteers by a trained plastic surgeon under local anesthesia the tissue is chopped into pieces and cultured using a fibroblast growth supporting medium fibroblast cells are then reprogrammed using yamanaka factors which transform them into pluripotent cells [Music] oh [Music] [Music] this is what a confluent fibroblast culture looks like these cells have the capacity to differentiate into several different cell lineages these cells are also known as induced pluripotent stem cells the ipsa-like colonies are selected and expanded in culture using a standard growth medium for the expansion of stem cells the cells are grown until they are about 70 to 80 confluent stable ipsc clones are passaged at least 10 times and then characterized through stemless Market expression these stemless markers allow for identification of stem cells in different cell populations [Music] once the cells reach confluency the growth medium is changed into a differentiation medium which will induce the ipses to differentiate into eye Fields over a period of three to four weeks [Music] at three to four weeks well-formed eye Fields can be seen with a central island of 3D neural retinal structures these are surrounded by rpe or retinal pigmented epithelium progenitor sellout growths the rpe cells can be isolated and cultured separately for basic research offer transplantation studies so basically these are itac derived ices so what happens when we differentiate the ipaces into the eye cells David's self-organized themselves into a 3D cup like structure so when they are growing in the adherence condition so they will be like a donut shape surrounded with uh type of cells like RP oscs like Oculus of the sector Norms so when we scoot them and put it in the suspension so they will become better so on that time we call them as a retinal Arguments for it [Music] in order to harvest the neuroretinal islands within the eye Fields a glass pasture pipette is used this thin glass pipette is heated over a Bunsen burner and molded using heat to create a smooth and shaped capillary hook this glass capillary is used to gently nudge and lift out the neuroretinal cups from the eye field clusters the smooth angle of the hook prevents the eye feels from any damage these eye filled clusters are then maintained as a 3D culture in non-adherent culture dishes first we stain our organoids with a dye called eosin which will help make these small organoids visible to our eyes while sectioning the organoid undergoes a series of washing steps in alcohol which helps to dehydrate it after the washing step we pour hot paraffin on these organoids to embed them in the wax when we embed our organoids in paraffin the goal is to create thin slices that we will stain with specific antibodies to identify different cells [Music] once the paraffin solidifies we'll cut very thin sections of about 5 microns in width using a machine called a microtone [Music] [Music] thank you [Music] once our sections are sliced and mounted on slides we'll remove the paraffin and rehydrate the samples using xylene alcohol and water [Music] next comes the antigen retrieval step the slides will be dipped in a sodium citrate buffer and heat it for 15 to 20 minutes to remove any leftover paraffin which could mask the antigens if the wax is not removed completely our sections will show higher background noise and affect the specific staining of the cells [Music] another way to prepare these sections is by freezing the organoids using a cryo medium called optimal cutting temperature compound or OCT [Music] once Frozen the oct compound embedded tissue is cut into sections of about 6 Micron thickness these sections are mounted on a glass slide and go forward for staining [Music] because we skip the use of paraffin and the antigen retrieval steps in the crime sectioning there is a lower chance of antigen masking or degradation therefore we can expect reduced background noise and improved antigen specific signals [Music] today we're using three antibodies to stain these organoids dapping to identify all the cell nuclei pac-6 to identify early eye field cells and chx-10 to identify early neuroretinal progenitor cells chx10 marks the lineage committed cells that form the neural retina foreign [Music] which is used to place on top of the 40x objective lens which is also an oil immersion lens oh and how does the oil help so the refractive index of the oil is similar to that of the glass slide which we are going to place on top of the objective lens also the oil helps in better accumulation of Light which in turn forms a sharp image foreign [Music] so here we are looking at the bright field images of retinal organoids obtained from a control line versus the patient specific line so if we look at them morphologically we cannot find any difference but at cellular level we can definitely find some differences here what you see is a day 40 organoid from a normal line if you see over here you do not see any CRX positive cell but it arises at day 55. but when you compare these images with that of a retinal organoid obtained from a patient suffering with retinoblastoma you will find that CRX positive cells are present at day 40 and it increases even more at day 55 so this tells us that in retinoblastoma the differentiation of photoreceptor precursors occurs quite early as compared to the normal line [Music] in order to understand the molecular differences between the normal line versus the patient specific line which is the mutant line we have performed Western blotting [Music] first the resolving gel solution is prepared and loaded into the plates which are assembled between the spacers [Music] foreign [Music] sets the stacking gel solution is prepared and added on top of the resolving gel a plastic comb is inserted into this gel creating the wheels into which we will add our samples this gel is allowed to set for about half an hour [Music] once the gel has been set the normal and mutant patient cell lysates are loaded onto the stacking gin along with a molecular weight ladder [Music] the electrodes are connected to the appropriate ends and a suitable voltage is set the samples will run until the end of the gel to ensure protein separation [Music] these proteins are now present in the gel in a ladder-like fashion this gel is transferred onto a nitrocellulose membrane the proteins will transfer from the gel to this nitrocellulose membrane through which it will be cut into sections depending on the location of the protein of Interest [Music] the genus blocked to prevent non-specific binding and incubated overnight with a primary antibody followed by a secondary antibody for one hour finally the membrane is placed in a cassette and a chemiluminism solution is added to it foreign [Music] the expression of the protein can be captured on an X-ray film this will reveal the presence or absence of the protein [Music] the X-ray result is captured using a gel dock machine and once captured the image is saved and stored for further analysis this is how the result of Western blot looks like so here in the first Lane you can see ladder it has got many bands which are of different molecular weights and it helps us to estimate the weight of our protein in the second lane a band is visible this band is from the normal control line and this expression of this band confirms that the protein is expressed in the third lane is the protein sample from a mutant line but there is no band seen over here and this tells us that the protein is not expressed and therefore by this method we can find out whether the protein is expressed or not now we know that these organoids can be used to understand the developmental differences at cellular and molecular level the retinal organoids that we have generated are used to study the differences in retinal development in disease conditions versus the normal conditions the retinal pigmented epithelium or RP cells be generated can be used for basic research or subratinal transplantation to prevent retinal degeneration and restore Vision in patients with AMD or stargard's disease these new retinal organoids can also serve as tissue source for basic and transplantation applications patient-specific RT cells and retinal organoids can also be used for testing of newer drugs in vitro this would help in reducing the need for a large number of animals for evaluating new drugs in pre-clinical studies these are also human relevant models and may help to reduce the risk of drug failures at Advanced stages of clinical trials patient-derived organoids can also be used for devising personalized therapy by pre-screening of drugs or their combinations to eliminate the non-responding toxic drugs this can help in planning better therapeutic strategies and improved clinical outcomes there are some limitations in using these model systems in vitro organoid models are often immature and closely resemble the fetal stage organs tissue found natively in our body comprises of both tissue specific and other supporting cell types like the blood vessels and immune cells most of the self-assembling organoids derived from stem cells do not possess all the cell types that truly represent the whole organ that is why it is important to employ these model systems based on their maturity and biochemical validations that are feasible in vitro [Music] [Music] thank you [Music] today we exploit the world of miniature organs saw how they are created and learned all about how they are used in research these structures have given us many insights into diseases like autism dementia and have even been used to grow primitive eyes this goes to show that the impact of these mini organs is not so many and they hold enormous potential to understand health and disease that brings us to the end of today's Virtual Lab we hope your neurons are firing and look forward to all your interesting questions in the next B12 circle make sure to check out this episode's notebook and to find your assignment questions tune in next time and come with us to a new lab where we will learn how computational and mathematical modeling is used to predict drug Behavior see you soon [Music] thank you
2023-04-19 19:11
