Alla Danilkovitch, RN, MS, PhD discusses the value of preserving various tissues via cryopreservation and lyopreservation technology. Dr Danilkovitch outlines the advantages and disadvantages of these methods and cites multiple studies related to the use of preserved tissue, such as amnion, in chronic wound healing.
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Alla Danilkovitch has disclosed that she is an employee of Osiris Therapeutics, Inc.
TAPE STARTS – [00:00]
Moderator: Now, our next speaker I have to say if there’s any one person who I learn the most from on stem cell and tissue regeneration, it’s Alla Danilkovitch who is the Chief Scientific Officer at Osiris Therapeutics in Columbia, Maryland. This is not going to be a sponsored talk, but this is scientific talk on cell preservation. Alla has worked for many, many years on stem cell technologies and bringing such stem cell products to the market. We’ve published a number of papers on stem cells, particularly mesenchymal stem cells. So, with that said, please let’s welcome Alla Danilkovitch to the stage.
Alla Danilkovitch: Thank you very much, a pleasure to be here. And today, I would like to focus my talk on one of the challenges with cellular therapies. It’s cell and tissue preservation that will be directly linked to how we can store such products and how we can distribute it to very practical aspects, we all suffer because a products wiggling in cells cannot survive long time at room temperature. And I would like to also to overview and novel technology that it looks like that using this technology, we should be able to store cellular product on the shelf at ambient temperature for long time.
I’m full-time employee of Osiris Therapeutics, but today I am not representing company. I’m talking outside, not promoting company, talking exclusively about scientific data, recent data what is available in the literature. So, learning objectives of this lecture is number one, to understand benefits of cellular therapies and discuss methods so what is available right now for us to preserve living cells and tissues. I will leverage you, as I mentioned a novel lyopreservation technology for ambient storage of cellular therapies. And we will discuss future steps and what are challenges we are facing in this area.
So, let me start with the simple definition. If we look broadly, what is cellular therapy? Cellular therapy is an application of cells with the intention to correct or cure a medical problem. This is a broad definition and as you know there are different types of cellular therapies that can correct or cure variety of different conditions and diseases. So, cellular therapies represent the fourth technological platform together with pharmaceutical drugs, devices and biologics. Cellular therapies are used for a long time; however, we still don’t have completed infrastructure, what we still need to build to have a full picture and full benefits for our health care.
Let me talk a little bit more about why cellular therapies are quite different from pharmaceutical devices and biological drug. So, unique property of cell is the dynamic response to local microenvironment. Cells can respond to internal and external stimuli. They respond to other cells in surroundings to cytokines growth factors around to extracellular matrix, bacteria, physical factors and so on and so on. And very often I can hear that growth factors can replace cells. Even if cells are the main mechanism of action through secretion of growth factors, you still cannot replace cells by growth factors. Definitely you can use growth factors therapeutically and that might be beneficial but growth factors they don’t have ability to respond dynamically to change in local environment at the site of injury or wound.
So, let’s look, a closer look on, for example, mesenchymal stem cell, we all know right now that this type of cell’s very beneficial to modulate and down-regulate inflammatory response. So, let’s say that patient has outer immune disease and in response to self-antigens T lymphocytes will become activated and start production of for inflammatory cytokines. One of them will be tumor necrosis factor-alpha TNF.
So, if you deliver MSC’s, they will go to the site of inflammation and TNF will react with the cell receptor or MSC and trigger cascade of reactions. So, in response to TNF number one, MSC will produce variety of different factors, one of them prostaglandin E2, that will work directly on T-lymphocytes and block secretion of TNF and proliferation of activated T-cells. On the other cell, again, the same TNF will trigger production of growth factors that require to help damaged by TNF tissue to regenerate. You can see complexity of responses by living cells. You never will get such complex respond from any other types of our products whether it’s pharmaceutical drugs or devices or biologics.
However, as I mentioned that the problem with cellular therapies that cells unfortunately they cannot live long outside of the body. You cannot store them at room temperature for long time and that will complicate distribution and storage. So, what we do to resolve this problem. We develop different methods of our cell and tissue preservation. The idea is that you wanted to develop the method that you can store a long time your cellular therapy. You of course need to retain cell viability and most important cell functionality. So, you also wanted to control cost and have meaningful shelf life that you will be able to store and distribute to deliver your therapy to the patient and not have a lot of vestige of the product.
Let’s look closely what is available right now, what kind of commercial products we have and how you store it. So, one way you may not need to store. This is for example when you do skin autograph. How you do it, you take patient in OR, you take skin from one place and then you implant it in a different place where it is required, for example, for burn patient. In this case, the skin you remove from one place you keep it for minutes or maximal several hours. So, another example, when you can store products for several days at room temperature, for example, everyone knows bioengineered skin substitute Apligraf. So, shelf life for Apligraf 10 days. Or for example, you can store tissue allograph or blood components are refrigerated for days or even weeks. So, using this method of storage, the cost is relatively low. You retain cell viability and functionality; however, the downside that very short shelf life and in many cases you even have to use this therapy at risk because you don’t have enough time to complete test for safety, to complete all microbiological tests to guarantee that you are not transmitting any infectious agents.
So, very popular, probably widespread right now, the way how we preserve tissue and cells is cryopreservation or with vitrification which is an alternative way of cryopreservation. So, using a cryopreservation, you can store cell therapies for very long time. However, the downside of that that you need specialized equipment. You need specialized equipment to be able to have stage graded or whatever is your protocol, control temperature drop during cryopreservation and you need specialized equipment to be able to store such products and distribute it. It’s usually deep freezers or liquid nitrogen. It is more expensive and what is also a peachy that not a lot of physicians can use cryopreserved products because their office is not equipped with specialized equipment.
So, let’s look on the cryopreservation. So, cryopreservation is a process that allows you to store cellular products in a very low temperatures. Successful cryopreservation was laid through discovery of cryopreservative agents. And you can see a portrait of Dr. Porch [phonetics]. He discovered glycerol. The glycerol when added to sperm, you can cryopreserve sperm and then get it back alive and it was a revolution for how we can handle cattle reproduction.
You can imagine how it’s a nightmare logistically when you have to collect and use fresh sperm. So, this discovery was done by accident. But I loved such quote from one of the famous Hungarian scientists who discovered vitamin C, Szent-Györgyi. He was saying that a discovery is an accident that is meeting a prepared mind. So, in this case it was exactly the case because that Dr. Porch was already looking for cryopreservatives to preserve spermatozoa.
So, right now, you’re very well aware that there are several classes of cryopreservatives and we still widely use glycerol. We use DMSO, dimethyl sulfoxide and a lot of sugars also can be used as cryopreservatives. But again the downside of this technology is that it’s costly and requires specialized equipment.
So, lessons from nature that there are animals that can survive extreme loss of water, desiccation. Such examples from the nature telling us that it should be a way to dehydrate mammalian cells and tissues continually in mammalian cells and then rehydrate them and get them back to life. So, when we investigate different organisms that can do that, we discovered several molecules that are present during desiccation. And what is very interesting that some of these molecules they served as a cryopreservative agents, for example, sugars like sucrose, trehalose or aminos. They all can be used for cryopreservation process. There are other molecules that also very helpful even to it during cryopreservation.
And I believe that if we will continue to investigate, how such animals can survive, what is going on during the desiccation, it should help us to find a way how to dehydrate mammalian cells and tissues. So, together with these examples from the nature, there are other data accumulated in the literature that forming foundation and telling you yes, we can do it. So, in addition to examples from nature, we know a lot about cryo and lyoprotectants. And also you can see examples from the literature how many different methods people already tried and reported proof-of-concept success for different types of cells. So, the problem was until now that you can get viable cells, but percent of cell viability after rehydration was low. And it was very difficult to have a consistent result and also another difficulty that the shelf life was not what exactly we wanted. Until now, recently new data were reported that you can dehydrate human tissue and keeps cell alive after rehydration at 100% and you can store this dehydrated product for long time.
So, this novel method is based on lyophilization. What is lyophilization? Lyophilization is a process when you dehydrate or remove water from frozen sample. Lyophilization is widely used of pharmaceutical drugs. Why lyophilization was picked up because at several origins for that. And a reason number one is yes we have a lot of experience with lyophilization. By telling that we have a lot of already equipment available and to do a lyophilization. First step of lyophilization is freezing or cryopreservation. So, you can do practically you already know your step number one, you need to find conditions for step number two and step number three, primary and secondary. And as I mentioned that the same substances that we use for cryopreservation can be used for lyopreservation.
So, let me switch gear and talk a little bit about placental membranes as an example of tissue that already was lyopreserved and demonstrated that this tissue after lyopreservation can be rehydrated and have a viable functional cells. So, placental membranes are known for time for their therapeutic properties. They are anti-inflammatory, are anti-scary, promote and endogenizes and do a lot of other things.
So, placental membranes were used for wounds and burns and the ocular diseases for long time. You cannot use unfortunately fresh tissue. You have to use processed tissue. And different methods of processing can give you products with different composition. So, you can destroy cells and have just extracellular matrix or retain matrix and growth factors, but no viable cells. However, the recent data showing that you need to preserve all the three components in placental tissues to have maximal benefits of the tissue. So, such data accumulated from and these experiments from animal models as well as right now we have even clinical evidence that clinically placental membrane that retains all components of fresh tissue, will perform significantly better than other types of processing used for placental membrane that will alter or destroy one or several components.
So, here is a publication reported last year, comparing good cryopreservation technique that you can retain cellular architecture as well as cell viability. On the top images, you can see a staining showing comparable tissue architecture between cryopreserved and fresh amnion. And on the bottom pictures, you can see staining for live dead cells. Green dots represent living cells and red dots represent dead cells. You can see that after cryopreservation post-thaw, you have exactly the same number of viable cells as in fresh placental immune.
Now, let’s look on the novel lyopreservation technique. And in the reported study just recently in April at the SAWC Wound Healing Society conference. One of the presentations done by a scientist was about comparison between lyopreserved amnion with cryopreserved. You can see visually how lyopreserved amnion look like. And after a rehydration, it’s exactly the same like cryopreserved post-thaw. If you look again on the tissue architecture, there are no differences between fresh tissue lyopreserved or cryopreserved. What is more interesting and we’re interested in whether we can retain living cells after rehydration of a lyopreserved amnion. Here is the result of cell viability. You can see both epithelial and stromal cells. which are MSCs and fibroblasts, remain viable and viability is very high in comparable with the cryopreserved amnion as I showed you on the previous slides that is similar to living tissues.
So, cells are not always viable immediately after rehydration, but if you put this rehydrated lyopreserved amnion in tissue culture and keep it for weeks, you can see that cells will start migrate from the tissue. And after three weeks, majority of the cells will be still alive. So, by using this novel lyopreservation technique, you retain tissue architecture as well as cell viability.
Next question, how about functionality. And here you can see functional response to inflammatory and hypoxic environment in vitro. So, you can take peripheral mononuclear blood cells and stimulate them so that they will become activated and produce a lot of inflammatory cytokines. For example, tumor necrosis factor alpha. So, if you put in the petri Dish, lyopreserved or cryopreserved tissue, you can see that the level of TNF will be going down. So, that these placental membranes will down regulate secretion of inflammatory cytokines by activated immune cells.
On the next graph, you can see if you put lyopreserved or cryopreserved tissue in hypoxic conditions, hypoxia will trigger pathways leading to upregulation of vascular endothelial growth factor. The bottom line, the conclusion from this is that similar to cryopreservation, lyopreservation routine functionality of amniotic tissue.
Now next step, in viva. So, we use in this particular experiment diabetic mice chronic wound. These chronic wounds are not only because they are in diabetic animal, but this wound if you treated once in the beginning of the wounding to block antioxidant, such wounds will become chronic, will become populated with bacteria and actually for closure of such wound, it will require multiple applications of products, not just one application.
And to close this wound, it will require several weeks. You can see here that it required four to five applications of amnion whether it’s lyopreserved or cryopreserved to be able to close such wound. Again, the bottom line, the conclusion of this experiment is that the performance of cryopreserved and lyopreserved amnion is the same in the chronic wound in diabetic animals. So, we use another model, more classical model in diabetic mice to investigate molecular mechanism because we know mechanism of how cryopreserved amnion works. Now, we wanted to see and confirm that lyopreserved amnion doing the same thing. So, here you can see wound closure in comparison to controlled animals who didn’t receive the amnion, lyopreserved amnion accelerate wound closure. When you take tissues, pieces of tissue, so you can see that lyopreserved amnion was able to change inflammatory environment of the wound to anti-inflammatory. You can see a red color, this is the higher level of inflammatory cytokines present in the sample obtained from controlled animals. If you take samples from animals treated with lyopreserved amnion, you can see everything is green. So, what happened at the level so far, inflammatory cytokines including tumor necrosis factor, which is framed are going down. So, not only are TNF was going down.
So, you also can observe in the wound that anti-inflammatory cytokines is going up. In this particular example, interleukin 10 is going up. And again, it is a dynamic response. You can see that one week after use of lyophilized amnion, the level of interleukin 10 is higher, then TNF is going down and IL-10 is going down because it’s no need any more to inhibit inflammatory reaction. So, wound already passing this step and go into the next step for wound healing.
The same we observed with upregulation of antioxidants. So, you know that in wound you have a lot of oxidative stress. And oxidative stress triggering senescence of cells and this will preclude wound from healing. So, if you use amniotic tissue, you place amnion and you can detect increase in cartilage and other enzymes that are important to down-regulate oxidative stress. So, the novel method is not only applicable to one type of tissue. Here you can see several examples, staining for cell viability for human skin, umbilical cord tissue, placental chorion, born or cartilage tissue. Fresh and lyopreserved are everything retained regarding cell viability in such tissues.
So, where are we right now and what would be our next step? We need to establish collaboration between industry and academia because we need to understand molecular mechanisms so that we can control much better what is going on and develop a robust manufacturing processes that will lead to commercialization of such products. Again, we believe that widespread commercial implementation will take long time, why? Number one, because the models of lyophilizers are developed for pharmaceutical drugs and they are not very user-friendly to lyophilize cellular products.
Another thing is stability. We believe that we can match cryopreserved products and it will have long-term stability. However, right now, for example, for cord blood cells, after banking we know you can store it 25 years in liquid nitrogen and then you still retain viability and functionality of such cells. Can we start lyopreserved cord blood? Probably yes, but we need 25 years to confirm that the life shelf will be 25 years. I hope that we can utilize shorter test and approximate with the pretty higher probability that we can say that, yes, such storage will have shelf life 25 or longer. But to know for sure it will take long time.
And of course next step would be to answer. Can we lyophilize solid organs because all examples what I showed you this is non-vascularized type of tissues. The next question, can we do it with more complex vascularized tissues and of course with organs. Let me stop here and take your questions if you have any.
Participant: I missed that and what is the-- I just had a question and wanted her to review again because I am very bad short-term memory. The process involved for lyopreservation, the lyophilize preservation of the amnion and how is it different than the cryopreservation?
Alla Danilkovitch: So, to simplify it, practically you can use special solution and you need to use, doesn’t matter cryopreservation or lyopreservation. So, you take your amnion, you add special solution then you cryopreserve it. So, in a way like you do cryopreservation, so practically you need to avoid ice crystal formation. And then you can store it as cryopreserved and it will be your cryopreserved amnion or you can take it, put in the machine create vacuum and start taking or sublimate water from your sample. So, it’s practically you go further with your cryopreserved sample, you remove water and now you have dehydrated amnion, but because of the steps, a proprietary steps developed on how to remove this water from the sample, after rehydration you have your viable cells.
Participant: As long as we kept frozen--
Alla Danilkovitch: Not really, you kept it frozen only on the step one and you can do it directly in lyophilizer or you can do it without the lyophilizer. But the final lyopreserved product you will store on the shelf at room temperature.
Participant: So, the multiple million dollar question is, what does it cost. You know, I can see enormous applications, you know, many doctors as you say clinicians do not have the ability to defrost cryopreserved specimens in their lab to help patients so there would be much more application. In this country, I can see it being used, in other countries where you don’t have a lot of banks with liquid nitrogen in them but, you know, what is the current cost per unit and how do you think it can be brought down?
Alla Danilkovitch: It’s actually, I don’t know exactly and it’s difficult for me as a scientist. I don’t think about cost as a primary thing, but definitely, absolutely I can tell you that cost is tremendously low in comparison to cryopreservation. So, your cost is during lyophilization but then after that cost for storage and maintenance. What is very important, if you store clinical grade material, you deep freezers or liquid nitrogen, they should be on their alarm system then you should take good care of this pieces of equipment to guarantee that the storage of your products are not compromised.
On the shelf, you don’t need to control actually humidity or control temperature. So, it significantly decrease your cost for storage. You significantly decrease your cost for shipment because right now to ship cryopreserved product, you use validated shippers with dry ice or you will use validated shippers with liquid nitrogen. It’s costly. So, to ship lyopreserved product, you put it in a box and just in a regular envelope. So, the logistics for storage and distribution is so user friendly for manufacturer and it’s very user friendly for physicians.
Participant: Thank you.