| 1 | Thermo Fisher Scientific | How to culture pluripotent stem cells in suspension: Passaging of PSC ... | 72358 | 229 | 8 | 60.8 | positive | 4:20 | Passaging of stem scale PSC suspension cultures. Passaging of the PSC's feroids is recommended when they approach 400 micro-muller in size. To avoid the formation of an acrotic core and loss of pluripotency. When using stem scale PSC suspension medium, this generally occurs after 4 to 5 days of growth. To passage the stem scale PSC suspension cultures, you will need. Stimpro acute cell dissociation reagent completes stem scale medium, Y27632 compound, and a CO2 resistant shaker. When PSC's feroids are readily be passaged, prepare the desired number of suspension culture vessels as described earlier. To passage the PSC's feroids, start by swirling the culture vessel slowly in a circular motion to gather spheroids to the center of the well. Collect the spheroids by pipetting or pouring the spheroid containing medium into a 50-millilater conical tube. Watch the walls of the emptied culture vessels with stem scale medium to collect any spheroids that may have been left behind. Collected spheroids should be centrifuged at 200 times gravity for 4 minutes. After centrifugation, aspirate the spent stem scale PSC suspension medium. Add the recommended volume of pre-warmed stem pro acute cell dissociation reagent. Do not use a P1000 pipet to chriterate the spheroid pellet as this may negatively impact cell viability. Allow the spheroids to dissociate in a 37-degree Celsius water bath for 10-15 minutes. During the 10-15 minutes, periodically mix the spheroids by flicking or gently shaking the tube at intermittent intervals. The cell suspension will become cloudy as more spheroids have dissociated into single cells. After 10-15 minutes of incubation and stem pro acute cell dissociation reagent, triturate the cell suspension 5-7 times using a P1000 micro pipet to further break up the spheroids into single cells or small clusters. Once the spheroids have completely dissociated add 3 milliliters of stem scale medium per 1 milliliter of gupco stem pro acutease. To inactivate the dissociation reagent and mix by gentle inversion, the single cell suspension should then be centrifuged and cells resuspended in fresh stem scale PSC suspension medium. Supplement of a 10-micromolar of Y27632. Similar to initiating the PSC suspension cultures count and seed 100 to 150,000 cells per milliliter of medium. In a new non-tition culture treated vessel, before placing a vessel on the CO2 resistant orbital shaker in the incubator. More instructional videos are available but cover the entire stem scale PSC suspension workflow. To find out more, visit thermofisher.com slash stem scale. | ↗ |
| 2 | Thermo Fisher Scientific | How to induce pluripotent stem cells in to definitive endoderm | 491 | 11 | | 50.1 | | 4:03 | In this video you will see how to generate definitive endoderm cells from PSCs using the GIPCO PSC definitive endoderm induction kit. The GIPCO PSC definitive endoderm induction kit is a complete ready to use media system for efficient induction of chloropotent stem cells to definitive endoderm lineage in two days. You can access the product insert and the quick reference protocol online. A supplemental list of reagents used in this protocol can also be referenced here. First, coat the plate with prepared vitro-necked insulation, faw vitro-necked in at room temperature and dilute with PBS. After dilution, add 1 milliliter to each well of a 6-well plate. Incubate at room temperature for 1 hour. Then you'll need to prepare complete essential 8-medium. Find details for this preparation on the web link listed below. Next, prepare a cell recovery solution by adding 250 microliters of Revitis Cell Supplement to 25 milliliters of essential 8-medium. Let media warm to room temperature. When you're ready to seed cells, aspirate the vitro-necked insulation from the coated plate and add 1 milliliter of cell recovery solution to each of the wells. When PSC culture has reached 70% confluence, cells are ready to be seeded for induction to definitive endoderm. Remove, spent essential 8-medium and rinse each well with PBS. 1 milliliter acutease cell dissociation reagent to the well and incubate for 5 minutes, or until the colonies freely come into suspension. Collect cell clumps in a 15-milliliter tube containing 8 milliliters of essential 8-medium. Wash off well with an additional 1 milliliter of essential 8-medium to collect leftover cells. Centrifuge the cells at 200G for 5 minutes. After centrifification, aspirate the supernatant. Flick the tube to dislodge cells and resuspend cells in 10 milliliters of cell recovery solution prepared previously. Seed PSC's clumps at about 1-10 split ratio into vitro-necked encoded plates, so that 15-30% confluence is achieved by day 1. It is suggested that you perform a range-finding study prior to starting definitive endoderm induction to identify optimal seeding density, which will achieve 15-30% confluence by day 1. Faw-diffinative endoderm induction medium A to room temperature. Shake the bottle several times to ensure even distribution of the components in the medium. If the cells are 15-30% confluence, proceed with induction. Completely aspirate spent essential 8-medium from the wells and add pre-warmed definitive endoderm induction medium A. Faw-diffinative endoderm induction medium B to room temperature. Shake the bottle several times to ensure even distribution of the components. Completely aspirate spent medium from the wells and add pre-warmed definitive endoderm induction medium B. After 24 hours, cells are ready to be assayed for definitive endoderm characteristics or further differentiated to downstream lineages. You might observe some floating cells at this stage. These can be easily removed by aspiration without being detrimental to the cells. | ↗ |
| 3 | Thermo Fisher Scientific | Advances in Stem Cell Research: Cell Reprogramming with Episomal iPSC ... | 4214 | 20 | | 48.6 | | 2:39 | [Music] reprogramming you know we're talking a science here that's only a few years old but the truth is that reprogramming in itself isn't that difficult if you willing to put the effort in just to develop your method we're pretty good at it now and it's a really reproducible method there's really almost no reason to be doing it in it the system that leaves insertional mutagenesis as part of the backround we can't build a standard method if your goal is to make ad do anergic neuron from 10 different individuals if every clone you make has a variable number of the reprogramming factors inserted in variable location with variable silencing um and then you add on top of that all the other variables in this science and you you can't make a reproducible population of dop energic neurons [Music] there's several different ways you can do this footprint free episomal we think works really well um we get footprint free colonies we get the numbers you'd want and they're fully plur potent and when you use episomal reprogramming in combination with a defined feeder-free system like the vittin and like essential 8 you end up with a more stable platform the colonies tend to be the same every time they tend to behave the same CDI prefers the episomal vectors because it's a very straightforward technique it's a single transection it's a nontoxic reagent it's just DNA you don't have to handle viruses that are potentially pathogenic you don't have a workflow that requires you to transfect the cell nine times in a row on nine different days you've got a somatic cell you transfect it you wait 16 to 20 days you pick colonies and it doesn't leave a residue it's pretty well documented at this point that there's examples now of fully sequenced genomes where it does not get integrated and doesn't leave a residue we can measure for its loss over time so it's a beautiful reagent you use it it does its job it goes away after the transection we have pretty good data now showing that you can go directly into vitr and you can go directly into E8 we've done E8 lots of times cells grow they behave can reprogram directly into that defined media [Music] | ↗ |
| 4 | Thermo Fisher Scientific | Dr. Uma Lakshimipathy presents Generation of Transgene-Free Induced Pl... | 3357 | 17 | 2 | 45.0 | neutral | 4:53 | So to start with the most common method for using, for generating IPSE is transduction of the four factors shown here is the Yamanaaka factors. And after a black box even which takes anywhere between three to four weeks you end up with IPSE colonies. So the biggest bottleneck right now one is the efficiency of IPSE formation depending on what kinds of cells you start with the efficiency is really low. And the second thing the second bottleneck is how do you detect these emerging IPSE colonies depending on the expertise of the users and the field of pre-repertance stem cells. People can either pick it really easily or there's always like a issue on what clones you place here bet on. So when it comes to efficient methods there are several methods starting from viral, non-integrating and more small molecule methods such as mRNA, microRNA and small molecules. But one of the methods that we've really worked on is a non-integrating viral method based on send-i-virus. The reason why this method is superior to the other current methods is its efficiency. It is a non-based on send-i-virus which is RNA virus. So again coming back to the different methods that are out there. If you were to this is a graph that I took from a published paper. If you look at the efficiency versus the safety obviously methods such as small molecule, microRNA, RNA and protein they don't leave a footprint of the extremely safe to use in a clinical setting. However the efficiency of generating IPSE right now is pretty low at this point of time. The highest efficiency so far has been obtained with viral methods such as lenty and retro. More recently the cytotune which I'll show you some data actually excels the efficiency that you can actually get with the traditional viral systems and at the same time it's relatively much safer because it's RNA virus and it's non-integrating. Therefore it will not leave a footprint in the genome of the cells or the IPSEs that are created. So this is again a brief introduction. This was a system that was developed by a company in Japan called DNAVAC. The original paper was published. There has been several papers since then starting from generation of IPSEs not only with fiber blast but also blood cells. So I won't go into more details there. What I would like to show you is that using the cytotune system that we actually sell as a product the process of generating IPSE is extremely streamlined. The four factors comes in four tubes which can be transduced overnight onto maps. Most of our protocols right now are for fiber blasts but we are developing methods for other cell types mainly blood lineages. After it's a one time contact you don't have to do repeated transduction. So in that way it's really workflow friendly. After transduction you have to give it around three to four weeks. At the beginning of three weeks is when you start colony formation and at the end of four weeks you have sufficient colonies to pick up and choose. There are actually more colonies than you really want because there are way too many colonies there. What we've done here this method actually shows you that it's integration free. Using PCR we're able to show that there's no absolutely no viral genome left in the clones that were established. This is 10 independent clones that were generated. You can also use an antibody although I would say that the antibody is not very great because you can see the haze in a negative cell type and you actually can tell whether it's negative only when you have a true positive control because the positive staining is so much more robust. These cells are pluripotent both in their marker expression and differentiate into different lineages when randomly differentiated via embryo body formation. These clones were all generated on feeder dependent systems in case are based period but since then we've also been able to generate IPSE clones both and the feeder free conditions using a stem pro sfm media as well as xenofre conditions which is basically a case are xenofre media in the presence of growth factor cocktails on human feeders. | ↗ |
| 5 | Thermo Fisher Scientific | The Evolution of PSC Culture Media -- Generation of new iPSC lines wit... | 714 | 2 | | 43.2 | | 6:30 | I'm going to talk about the uses of E8 or essential aid and vitronectin to derive new IPS cells directly into these conditions. And these are some of the experimental conditions that we have used. The cell types that we used have been neonatal dermal fibroblasts from commercial vendors, adult dermal fibroblasts from commercial vendors, four skin dermal fibroblasts obtained from patient biopsy, and adult dermal fibroblasts also obtained from patient biopsies. For Plasma combination, we purchased those from adjeene. These were plasmids that were deposited by Thompson Lab into adjeene, and they're freely available. They follow the OKSM properties, and you can transform them, and you can have enough plasmids to do transfections for several years. The reprogramming methods that we have tested are the Amaxa II nuclear factor by Lanza. It comes preloaded with protocols. It's kind of the only negative thing about it. Bioregene pulser to electroparator. People may have to work out specific protocols because they do differ based on whether you're using neonatal or adult dermal fibroblasts. And then the neon transfection system, we're currently working on optimizing the efficiency for that system. Essentially, to say that you can use any system to derive your IPS cells and use E8 and V8-connect to culture them and derive your new IPS cell lines. This is our derivation scheme. I know it's a busy chart, but I just wanted to point out a few things. You can count the cells. We use about more than 1 million cells. Use the DNA that I just mentioned. Transfect the cells. You can adjust the ratio of transfected cells depending on whether they're neonatal or adult into six-well plates. Once you reach confluency, you start changing media. And then we do add sodium butyrate as a small molecule. Colonies appear between day 20 and 25. You can pick the colonies. You can culture them on vitro-nextin or metri-jell in E8. Initially, we add rock inhibitor. And then you can continue to propagate them and expand them to derive your own new IPS cells. Why sodium butyrate? The sodium butyrate is one of the small molecules. You can choose any of the small molecules that have been published and they work well. Sodium butyrate is a natural, small, fatty acid molecule. It is an H-dack inhibitor as all of you know. It significantly increases reprogramming efficiency and also increases the ratio of IPS cell colonies to total colonies by reducing the frequency of partially reprogrammed colonies. This is a big concern for the scientific community that a lot of times you will have a large number of colonies. And the ones you pick may be partially reprogrammed. There's not a lot of sophisticated testing yet without spending a lot of time and money to do that. So the favorable treatments nowadays is to use a combination of small molecules or a single small molecule to make sure that the partially reprogrammed colonies do not make it until day 25. And the beauty of this system is if you actually mark the colonies and follow them every day, the partially reprogrammed colonies will disintegrate and disappear right in front of your eyes. So it saves you a lot of time and money and aggravation because you don't end up picking a partially reprogrammed or a completely reprogrammed colonies by using small molecules. These are just some pictures. This is normal, neonatal derma fibroblast ready for transfection. These are transfected cells. It about 20 to 30 confluency. These are transfected cells at day six. And you can start seeing colony morphology appearing right in these areas. The fibroblast distinct appearance of spindle shaped cells is now changing into round looking cells. If you look at them at 10X, you can see that indeed they are different. We get about 60 to 100 colonies per 10 million transfected cells. The beauty of that is they are not partially reprogrammed. So there's a lot of colonies for you to pick from. And then the other thing which I mentioned is if you are using a skin biopsy, you can add EGF and thrombin right from the beginning, derive your fibroblast from the skin biopsy and continue your transfections. These are colonies that appear. This is day 15, day 20, day 17, day 17. Just to give you examples that the colonies are very distinct in morphology and appearance. And it is very easy to pick the colonies and start culturing them to derive IPS cells in very easy ways. These are cells that we picked and started propagating. So at 2.5, this is how they look at day 2, day 3, and day 4. And as you can see, this is a very familiar picture to all of us who do stem cell research. These do look like human blue reporting stem cells. And you can continue, you can characterize them, you can bank them, and you're ready to go. One thing that we do is while we are culturing them, we want to know whether they maintain the characteristics of blue reporting sea. So we actually use the Alkfoss live stain that life technology offers. It's a really quick and easy stain. You don't have to fix your cells, which is a very distinct advantage. The stain disseminates in two hours. So you can quickly check your colonies and move on. You don't have to dedicate plates that are fixed to do any kind of staining. And these are two colonies. They kind of have a funky shape, but you can tell that they are positive for Alkfoss. We have characterized the newly derived IPS cells for viability proliferation, blue report and sea differentiation potential by making EBS as well as carry your type. | ↗ |
| 6 | Thermo Fisher Scientific | Human pluripotent stem cells in understanding genetic cardiovascular d... | 711 | 8 | | 26.6 | | 1:00:59 | No transcript | ↗ |