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Episode 78 – Liveyon Pure Cast – NO LIVE CELLS? Interview with Dr. Rafael Gonzalez and Margaret Coutts
Dr. Alan Gaveck: Welcome back everybody to Liveyon Pure podcast, Dr. Alan Gaveck Director of Medical Education and Clinical Application for Liveyon and of course this is John Kosolcharoen, CEO and founder of Liveyon.
John Kosolcharoen: Yeah, great to be here today.
Dr. Alan Gaveck: You know John we hear a lot out in the field and we read in the press, they’re some naysayers that are saying umbilical cord blood doesn’t have live cells.
John Kosolcharoen: Live cells.
Dr. Alan Gaveck: Right? Well, they can ask us … You know the sales guys can say, hey no we know there’s live cells because we talk to these people and we know it’s out there, but what we figured, for you, out there what we should do, is really introduce you to the people who work in the field who really, really know this stuff. With us today is Dr. Rafael Gonzalez, very well known Ph.D. researcher in the stem cells field, former education with a4m. And also the Chief Scientific Officer at Liveyon labs, Dr. Margaret Coutts. They’re both at Liveyon labs right now, welcome.
Dr. Margaret Coutts: Hi.
John Kosolcharoen: Thanks for [crosstalk] coming in and joining us. I appreciate that today. I know, Rafael, you have a lot on your plate so I appreciate you being able to come over and jump on this podcast with us, I think a lot of people really wanna hear from you.
Dr. Rafael G.: Great, you have a beautiful lab here and it’s always great seeing Margaret. Actually Margaret and I crossed paths at UCI …
Dr. Margaret Coutts: That’s right.
Dr. Rafael G.: We were both actually at … Well I was at [inaudible], I think I was there before you.
Dr. Margaret Coutts: You were there before …
Dr. Rafael G.: I was there about 12, 13 years ago I was there at University California Irvine. I worked at the Reeve-Irvine Research Center and then I left and she actually went, you were working there after and with the same group. That’s pretty neat. And then later on it was weird how we crossed paths again, of course in the stem cell sector, she’s lucky actually to be here and work with Liveyon and I’m actually here in this beautiful location, actually the lab is beautiful, everything’s really nice here.
John Kosolcharoen: And we’re happy to have both of you here, so thank you. We wanted you guys to come on today because there’s just been a lot of talk in the industry that products derived from umbilical cord blood doesn’t have any live cells, and specifically, there’s been a lot of talk that the Liveyon Pure Product, which we just launched this week, doesn’t have any live cells either. I wanted to actually bring on a cellular biologist and specialist in the stem cell field as well as the Chief Scientific Officer that made the Pure Product, to talk a little bit about that.
Margaret, what would you say to anybody out there that said, the product doesn’t have any live cells.
Dr. Margaret Coutts: I would be really surprised because we thaw vials routinely to check that they’re healthy and viable after they’re thawed and on the average 80.9% of the cells are viable.
John Kosolcharoen: Wow, that’s a high percentage.
Dr. Margaret Coutts: When we worked out the procedure to isolate the product, having a high viability after cryopreservation was very important. I really remember that percent well because 80% viability is a high benchmark so I was very happy when we met that.
John Kosolcharoen: So you actually test the product multiple times in the lab looking for viability during the whole processing of the cord, right?
Dr. Margaret Coutts: Yes. Before we start processing, during the process, and at the end once the cells are in the vial, in the cryopreservation agent, but before they go into the freezer.
John Kosolcharoen: So this process takes a couple of hours to process the cord.
Dr. Margaret Coutts: That’s correct.
John Kosolcharoen: What percentage of cells do you lose from that raw cord that you get when it comes in, cord blood that you get when you comes in, to your finished product. What’s your average loss there?
Dr. Margaret Coutts: It comes in the door, on the average, with 89% viability. After the process is done it’s 88.9 so it’s essentially unchanged and then after freezing and thawing it’s 80.9% on the average.
John Kosolcharoen: Wow. That’s pretty amazing.
Dr. Alan Gaveck: That is pretty amazing.
Dr. Rafael G.: Which it is, I agree. I’ve done cell biology, worked with cells, umbilical cord blood, bone marrow, exedra, tissue for many years and that viability, I applaud you that’s actually a really good job.
Dr. Alan Gaveck: Rafael you’ve been in the industry a long time and you’ve had the opportunity as an independent to test all of the products on the market, all the way from amniotic tissue, amniotic fluid, products derived from the umbilical cord tissue itself, which is the [inaudible] jelly or the connective tissue, and cord blood. And you work very, very heavily with cord blood and cord tissues in general, what would be your response to somebody that said that we don’t have live cells or that you don’t get live cells from cord blood?
Dr. Rafael G.: It’s ridiculous. You really have to mess it up to not get live cells with these type of
products. You would be debunking a complete multi-billion dollar industry with the umbilical cord blood industry. I’ve worked with these samples, I’ve taken these samples and thawed them from various different vendors. We’re discussing cord blood here, not cord tissue, or not the other cells. Tissue types are different, they’re really, really hard. And I can see why that’s out there and there’s been some confusion with that. This is cord blood, it’s a fluidous product it’s fluid, and it’s derived from fluid, and you
obtain these cells and it’s … You’re not basically destroying, for instance, a cord tissue,
you’re destroying a piece of rope and you gotta homogenize this or enzymatically digest this to produce it and then all this DNA is actually released and it causes a ton of cell death. And then you gotta freeze and thaw it, it’s typical …
Dr. Alan Gaveck: And it’s really not that clean of product. I know I was in the lab the other day and Margaret was saying that she looked at some products under the slide that weren’t sourced from cord blood, sourced from cord tissue and other things, not that many live cells. A lot of people say there’s not live cells. A lot of the testing we’ve done on other products, really that’s true, there are a lot of products out there that don’t have live cells, but with what you just described there also a lot of debris. Margaret had said … She thought the slide was dirty, right? We got a product and thawed it out, and she thought that, wow she thought at first it was a dirty slide.
Dr. Rafael G.: It’s all debris. Yeah, it’s all debris.
Dr. Margaret Coutts: Yeah I think people are clumping together a number of different products. I mean as Rafael was saying, some of these products are ground up amniotic lining or they’re ground up umbilical cord tissue, it’s not really meant to have any live cells on it, in it. It’s not cryopreserved properly. It’s dried and ground up tissue. We’re not that product, we’re something completely different.
Dr. Alan Gaveck: What about the naysayers that say, hey these are just a couple of Ph.D.’s and this is just their opinion, is there anything that backs what you’re saying?
Dr. Rafael G.: Oh absolutely. I mean there’s a lot of publications out there in umbilical cord blood looking specifically at different systems for [inaudible] viability. Some of the companies that have these different centrifuge systems and some of the old bag system, Paul I remember had a bag system. There’s a bunch of different companies in the umbilical cord industry, blood industry, that specifically does this because remember they have to. They’re selling a service and they’ve been selling this type of service to freeze these products for future use, for these individuals and the concept is, there’s stem cells in there, there’s cells in there that can help in the future.
Dr. Alan Gaveck: Yeah that’s a multi, multi-billion dollar industry.
Dr. Rafael G.: Yes.
Dr. Alan Gaveck: Why don’t you walk us through the cryopreservation process. What happens? How do you take a live cell bag or a bag full of live cells and cryogenically preserve it and then thaw it out and still get cells? How do you do that?
Dr. Margaret Coutts: Well I think your big enemy, freezing and thawing is water crystals form. If you don’t add some sort of cryoprotectant, as we all know when water freezes and becomes ice, it expands. You would rupture the cell membrane and kill the cells if you didn’t displace some of the water inside the cell with the cryopreservant.
Dr. Alan Gaveck: Kind of like when you put a beer bottle in the refrigerator … Or in the freezer and freeze it. It freezes, it doesn’t have any place to go it just breaks, but if the top’s off of it you might get a little spill, but the bottle doesn’t break, kind of like that?
Dr. Rafael G.: Well it’s somewhat like that but not exactly. Just consider the cell’s full of water, the water has to be displaced out otherwise, like Margaret said, what’s called vitrification process
occurs where crystals from. Those crystals will eventually expand [inaudible] this cell, the cell will blow. This cryopreservation agent displaces the water so the membranes are a little leaky because it has to go in, penetrate through, the water has to be displaced out and then when you thaw it, the complete opposite. The interesting thing is it’s the way when you freeze something, so you don’t mess up the membrane, you do it very, very slowly. It’s a slow process, basically one degree celsius, per minute. You bring it down to negative 80 and then eventually if you wanna go onto liquid nitrogen you go onto liquid nitrogen, but then when you thaw it, the concept is, the faster the better because you wanna avoid that specific crystallization area. That time between, what is it, negative 40 and negative 20.
Dr. Margaret Coutts: Yeah.
Dr. Rafael G.: Around there, that these crystals conform. You thaw it out as quickly as possible so this cryoperservant can go out and water can come back in and then cells feel good, they’re happy again.
Dr. Alan Gaveck: So happy cells … You know, when you’re talking about happy cells and live cells, and cells that, you know, were maybe beat up or broken, or they go through this process, we’ve heard a lot of talk about, well maybe you can thaw cells. Maybe you can freeze them, maybe you can thaw them, they’re still alive, but how many of them are dead or have gone through the Armageddon of the cryo process. Is there a way to measure that? And with the Pure series, what type of cell quality comes off the cryopreservation?
Dr. Rafael G.: I think Margaret did something intelligent with your last … I saw the documentation on it and something intelligent was just let it sit for an hour at room temperature and then test the viability of it and see what transpires. She can tell you more of what of the data she got from that when she did it, on your guys’ …
Dr. Alan Gaveck: I saw the data, but I go back there and there’s like books and stacks of characterizations and papers, and numbers, so please let me know what all that stuff meant.
Dr. Margaret Coutts: The short story was after we thawed the cells, you know, people say the DMSO poisons them, they’re really sick, they’re gonna die, and though we do recommend strongly that the product be thawed and used immediately, we just thought okay what would happen if we just left it out? Only a few percent died, we checked at, 15 minutes, 30 minutes, 60 minutes.
Dr. Alan Gaveck: Really? Wow.
Dr. Margaret Coutts: The vast majority of those cells were alive, but I think more important proof is, you know, you can look at the cells under a microscope, they look fine, you can do that [crosstalk] staining and you can grow them in culture. You see a variety of different cell types when you look at those cells once you’ve put them in a flask and in an incubator. And depending what media you use you can culture out different types of cells.
Dr. Alan Gaveck: [crosstalk] You were talking about live cells and dead cells and I know … You know, some savvy stem cells doctors they have their own stem cell counters, there’s different ways you can do it. We hear terms like they use Trypan Blue and look under the microscope, but is there any way, the way that you guys count cells, that there’s some type of subjectivity that it says 80% on your computers, but is that subject to change at all? Or you have both a way to count both live and dead cells?
Dr. Margaret Coutts: Well it’s really important to have a cell counting process that has absolutely no subjectivity whatsoever. We stain the cells, you know, one a green dye that is fluorescent green in lives cells and a red fluorescent dye that stains red cells. If the cell is sick and dying the
staining stains it red, so our count is conservative. It goes into a chamber slide and is loaded into, basically, a microscope that has a counting … An imaging and counting
capability. It looks at four different microscopic fields, counts the cells, averages the four fields, and produces a number. It’s not …
Dr. Alan Gaveck: So when you’re saying there’s 80.9% viability this is … On average and some of them I saw are a lot higher, some of them are 89%. This is a true live count, dead count, post-thaw, in a machine that doesn’t have any subjectivity.
John Kosolcharoen: And to be clear you do it on every single batch.
Dr. Rafael G.: And then to top it off too, John, this is a cell counter that count nucleated cells. Some people do what’s called the Trypan Blue and use a hemocytometer, and you can use some machines too to do it, and that basically counts almost every single cell in there, meaning red blood cells too, and nucleatic cells, white blood cells that are actually in there. That, of course, will give you a much higher cell count and a much higher viability. This is a cell counter that incorporates, one of the stains incorporates, into the nucleus to count a nucleated cell.
It tells you that is it a nucleated cell, it’s not gonna count the red blood cells that are
there, if there’s any red blood cells there, it’s not gonna count that. It only counts the
nucleated cells, so that tells you that you do have a viable cell for sure.
John Kosolcharoen: When you’re talking about live viable cells, how are you thawing that? I mean, in the lab of course you can do things and thaw out cells differently that would give you those higher viabilities, but when you’re talking about doing this at every lot, do you do it the same way a physician would do it in the clinic? Or how do you thaw them for this testing?
Dr. Margaret Coutts: We thaw the cells exactly the same way, we recommend the doctors …
John Kosolcharoen: How is that?
Dr. Margaret Coutts: You just basically hold them in a gloved hand and just let the heat of your hand thaw the cells. It takes, you know, four or five minutes or so. We extract the cells from the vials the same way the physician would. We don’t have any special equipment or fancy way to do it. We try to get the same count that the physicians would get.
John Kosolcharoen: So you do the … Go ahead Rafael.
Dr. Rafael G.: And John, I’m sorry … Like John asked earlier, you’ve tested these products. I have taken these products post thaw and have cultured them, and like Margaret says, there’s a bunch of different cell types there and it does grow. The ones that can adhere, adhere, and they
propagate. They do grow.
John Kosolcharoen: And that brings up a good point because if there are no live cells, if you try to culture it nothing will grow. Right?
Dr. Rafael G.: Nothing would adhere either.
Dr. Margaret Coutts: Yeah.
John Kosolcharoen: Nothing would adhere, nothing would grow.
Dr. Margaret Coutts: I would be really amazing if I could take dead cells and make them [inaudible].
John Kosolcharoen: But Margaret that’s kind of the second point, that what you’re doing at the lab right now, yes you see viability, you do the testing, you count the cells, we’re saying there’s viability. Can you tell us what you’re doing now at the lab with growing these cells, that you are, and why you’re doing that?
Dr. Margaret Coutts: Well, the thing about growing cells in culture the media or the food that you’re feeding the cells … You basically select the type of cells that will grow in a selective media. You have to use a different media depending on what type of cell type you wanna grow because it’s not as complete and perfect as being in the body.
John Kosolcharoen: Right.
Dr. Margaret Coutts: Right now we’re very excited about culturing out [inaudible] stem cells using a selective media for those and we’ll get counts and we’ll try differentiating them into different lineages,
cartilage, bone, fat, we can do tendon. And doing a flow analysis to really be certain that these fit the complete definition of MSC.
John Kosolcharoen: So you’re working right now on specific media to culture out MSC’s, we’ve identified all the markers that are required on the cells, now it’s that last step of showing it adhering to plastic, but what are some of the other cell types in there? I mean it’s … It can’t just be
these regenerative medicine products aren’t just all centered around pure MSC’s, there’s gotta be other things that are in the product that’s helpful for healing and really, maybe even
primarily, the cell types that help heal your body.
Dr. Rafael G.: I mean I personally know there’s only, in every one of these products, there’s very few of, you know, stem cells and although that doesn’t really matter because, you know, immune cells are really, really important in healing, the most important cell actually in healing is your immune cell, in your body that floats around on a daily basis has to deal with everything that you’re… You know, from mutations, from DNA mutations from being exposed to a virus and bacteria, exedra, exedra, it has to react. It reacts basically by secreting all these different factors, protein, growth factors that deal with this on a daily basis, that’s truly a regenerative cell in and of itself. And the [epithelial] cells which are in large abundance in there and it makes it … When you start culturing these cells you’ll see, I mean there’s a ton of them. She showed me a flask of what she was culturing just recently and it was pretty neat, because like I say, oh wow look how neat this is, we can see a clear endothelial lining that’s actually forming and it’s an
endothelial barrier that’s actually forming within the culture system. So it’s pretty neat. It’s just a matter of picking the right culture media because cells compete for specific food and some of them do well in one type of food verses the other one does in another type of food, but there’s an abundance of other cell types in there that have capabilities to form new blood vasculature to suppress the inflammatory response, and these are not only the stem cells that do this, but all these other cell types are in your body.
John Kosolcharoen: So that’s kind of the point. The heterogeneous mix of all these cells working together, it’s nothing by itself, right?
Dr. Rafael G.: Yes.
Dr. Margaret Coutts: Yeah, I mean you look in the flasks and you see the different shapes, the different sizes of cells, and you think, wow there’s a whole little ecosystem in here.
Dr. Rafael G.: Yeah.
John Kosolcharoen: Are we gonna be able to see some pictures? I know that we … I just bought that beautiful, beautiful microscope so we can really take a look at some of the cell types and take pictures. We gonna use that finally?
Dr. Margaret Coutts: Yes, yeah. I [crosstalk] …
John Kosolcharoen: You know one other question I’m [inaudible] with all those cells and different medias, and growing and excreting all these growth factors … I mean obviously there’s tons of live cells, how does that compare to a PRP that doesn’t have live cells. When you’re talking about doing a PRP injection into a knee verses a live cell product in the knee, where these cells are
excreting all these different growth factors, what’s the difference? What’s the big difference there?
Dr. Margaret Coutts: You wanna take that one Rafael?
Dr. Rafael G.: Yeah, I think I have a suggestion or a hypothesis on this and actually we’ve done some studies in vitro to look at this too … Is, cells require another cell, basically to survive, and they
work off of each other and feed off each other. When you put cells in a harsh environment, they secrete all these factors and it’s sort of like exosome based business that you’re hearing a lot about and what cells are secreting … And the harsher the environment, the more that cells … Your body goes into a starvation mode, into a survival mode, that it needs to survive and then the cells actually cling closer and closer together. You can see this actually in cell culture when you put them in, for instance, in a situation where you starve them off, you don’t give them any proteins. In this case normally when we do it in cultures fetal bovine serum or [inaudible] serum that we put them in and they starve off, and they wanna … They try so hard to survive, and this is what makes them secrete a bunch of stuff out, a bunch of these growth factors [inaudible] beneficial.They react and they live, and they work for a longer period of time because they have to. Because they have to find a way to survive verses when you use PRP you basically put this stuff in and you have these growth factors, and you have these proteins that have half-life’s, they have a purpose and they do work. They do some work, but then once they do work one time they’re done. They cause this massive [inaudible] inflammatory response and then they’re done doing their job. And then your body has to do the natural healing. Cells are gonna be in there as long as they can survive, they’re gonna fight for survival inside of you. And then they’ll just die off.
Dr. Margaret Coutts: And PRP, it’s valued for the platelets, which are not nucleated so …
Dr. Rafael G.: Correct.
Dr. Margaret Coutts: Sounds like they’re stressed they secrete what they have and then they’re done.
Dr. Rafael G.: Yes.
Dr. Alan Gaveck: Well I could ask you questions all night, but we want to be respectful of your time. Wanna thank Dr. Margaret Coutts, Chief Scientific Officer at Liveyon, it’s been such a pleasure to really watch you and your work. And of course Dr. Rafael Gonzalez very well respected in the stem cell field, thank you very much. Thank you for your time.
John Kosolcharoen: Yeah, thank you for coming on, appreciate it.
Dr. Rafael G.: Thank you both.
Dr. Margaret Coutts: Thank you.
Dr. Rafael G.: Have a great evening, thank you.
Dr. Alan Gaveck: Alright everybody I hope you enjoyed that as much as we did. Love to have your input and we’ll keep talking about it. And get us some good questions and we’ll see if we can get these guys back.
John Kosolcharoen: Alright, sounds great.
Dr. Alan Gaveck: See ya next time.
John Kosolcharoen: Thanks.