Cryopreservation of Cellular Therapies: Is There a Better Way?
Alla Danilkovitch, PhD discusses cellular products and their use in wound healing. Dr Danilkovitch further examines challenges facing their extraction, their successful storage and the best methods discovered to date.
Alla Danilkovitch has disclosed that she is an employee of Osiris Therapeutics, Inc.
Male Speaker: Our next speaker is an RN, MS, PhD, Alla Danilkovitch, who is very much involved in research, in education. She joined Osiris in 2003, responsible for the development of product pipeline and leads research and development medical affairs and clinical operations. She’s been a researcher for over 25 years and experienced academia in US government and industry combined. Her bio can go on and on and on. So rather than do that, I’m going to ask her to come up and share her thoughts with us on the topic of cryopreservation of cellular therapies, is there a better way? So please welcome Dr. Danilkovitch.
Alla Danilkovitch: Thank you very much. If you look on the title, you’re probably wondering what cryopreservation of cellular therapies and why I should care whether there is a better way. This lecture about the new technology, about new technology and new products that will come to your clinic in the nearest future and that’s why I would like to share with you what is in the development right now and what is coming in your future. And I hope that you will get excited and will be welcoming and waiting for new products coming based on new technology.
My disclosure, that I’m full time employee of Osiris Therapeutics but here I’m as a scientist and I will talk exclusively educationally and cover new development in the research. So what I want to overview that I want you to understand what is available right now regarding cellular therapies for wound care and what are differences of cellular therapies from other therapies, what are advantages and disadvantages, talk about new lyophilization technology that allows you to store living cells and tissue at ambient temperature and where we are going with such products in the future in the wound care space.
As you know all, that wound statistic is scary. So number of wounds is not going down. It’s going up and it put a lot of burden on our society on the people who suffer of chronic wounds and people who pay for chronic wounds. So more and more people developing diabetes and more and more people suffer from chronic wounds.
So if you look from the molecular and cellular perspectives, what is going on in chronic wounds? So you can this is from Dr. [Freiburg] [0:03:18] latest review, you can see how many different factors at the molecular and cellular level going around in the chronic wound and I would cite you can see how many factors you need to have on your product to be able to overcome problems with chronic wounds.
So this is what you want from your wound care, advance wound care modalities. You wanted this wound care modality to care good quality, different types of cells found in skin. You want in this wound modality to have antimicrobial property. You wanted to hear for inhibitors of proteases. You wanted to hear factors that will correct inflammatory environment and stimulate angiogenesis to fight hypoxia. So every product you want to touch ideally will have all these factors. Is it possible? Yes, it is possible.
So here’s experiment in animals where diabetic received living cells. In this particular example, mesenchymal stem cells and mesenchymal stem cells are able to correct abnormal wound environment and promote angiogenesis and stimulate wound closure. So on the graph on the left, you can see how long cells are persisting in the wound because there is a thought that living cells, they will not last in the wound forever which is absolutely true. However, this should term persistence in the wound is sufficient to dramatically change wound environment from non-healing to healing.
Looking on all these data presented on this slide, you may make several conclusions. Conclusion number one, that cells are important and can contribute to wound closure. Conclusion number two, that the cell type is important because you can see on the graph – so that here the graph with emesis stimulate wound closure while fibroblast wasn’t – they were not able to stimulate wound closure.
So you also can say that because cells, they do not persist for a while and the number of cells puts – you put in the wound that will be dropping over time that the main mechanism is paracrine. Paracrine meaning that cells secret growth factors and cytokines that will correct wound environment. And you might ask here a question. So why I need living cells when growth factors can do that job? Yes and no. Yes, growth factors are beneficial when you’re adding them to the wound and no because whatever you add to the wound there is no dynamic response.
So this slide shows you what is unique about living cells? The unique property of living cells in any therapy contained living cells would be dynamic response to changing wound environment. So if you have living cells to the wound containing bacteria, living cells will produce antimicrobial factors. If you add the cells to the wound environment containing – with hypoxia, and that such cells will produce angiogenic factors to attract other cells and promote our new blood vessel formation and so on and so on.
You can see other cells will influence what cells produce, metrics proteins, growth factors and cytokines in surroundings, physical factors and different environment and external stimuli also will affect what cells will produce. So that’s why if you are talking about products contained in all living cells, they are beneficial to the wound. However, you never will get dynamic response from your wound product to the wound environment. That’s the key difference between cellular versus non-living cells product.
These look like very crowded table. This is example of wound care modalities containing living cells and you can see and very well recognize two of these, this is our bioengineered skin substitutes known as Apligraf and Dermagraft. So – but also you know that there are products like living cryopreserved skin, that also contain living cells. And there is a new ketone on the block. It’s placental membranes and placental tissue. This ketone is old but new at the same time all because placental membranes were used long time ago. This report of using placental membranes was done by Dr. David at John Hopkins in 1910. It’s longer than 100 years ago. However then people stopped using placental membranes until they figure out how to process tissue and preserve that it can be fully tested and store on the shelf that you can use it.
So let’s talk a little bit about placental membranes. There are two of them, amnion and chorion. Why placental membranes are so beneficial for wounds? Because they have properties that are required to treat chronic wound. However, you will hear for all these properties if you will be able to preserve all components in the placental membranes and there are three of them by categories, extra cellular metrics, growth factors and living cells. So if you preserve everything usually the standard processing method to preserve living cells is cryopreservation. You can optimize your method in the way that you will preserve both structural elements in the membrane as well as viable cells. You can see here green dots represent viable cells and red dots represent dead cells. So it means that you can do cryopreservation of placental membranes that you will not see the difference in the structure in cell viability between cryopreserved and fresh tissue.
So if you look on the living placental tissue, you definitely will see responsiveness to micro environment.
Here’s the basal level, what you can find regarding the vascular endothelial growth factor in the tissue, prostaglandin E2 or antimicrobial defensin. So, if you put such living tissue in hypoxia, you can observe increase in the production of VEGF. If you have inflammatory cytokines for example tumor necrosis factor alpha, you can see increase in production of the prostaglandin E2and this factor is antagonist of TNF alpha. If you had bacteria, you can increase over the basal level production of antimicrobial peptides.
So, that’s your difference between living tissue and nonliving tissue that if you compare it with nonliving tissue, whatever basal level you have, you will not see any responsiveness and increase in production of any factors.
So, here’s another very important for wound treatment example that placental tissues covering baby provide a barrier and protection from microbes. So, that’s why placental cells in the tissue, they are capable to produce antimicrobial peptide and inhibit growth of bacteria. Here, you can see inhibition of bacterial growth in the presence of living placental tissue and all this bacteria, they were isolated from chronic wounds and you can see your regular suspects, so this one, very well known, practically present in all chronic wound.
So, it’s not only in vitro experiments that you can detect this antimicrobial activity in the Petri dish. What is very interesting that there is a correlation with clinical data. Here’s the results of clinical trial with the living placental tissue control multi-center, your level one evidence and using in placental tissue, you can see that in the presence of viable placental membrane, that was used as an adjunct to the standard of care, you can significantly increase wound closure, but not only wound closure. What is interesting that you can reduce significantly the volume of wound related infections. Why? We’ll just look that a placental tissue can inhibit growth of bacteria that always will be present in the wound.
So, level one evidence is good, but also, what we need is comparative studies. And on the next slide, you can see results of the one center with respective analysis when living tissue was compared to nonliving tissue. And what you can see, the difference, that again living tissue was able to close more wounds than nonviable tissue, nonliving. And what is also very interesting that actually nonviable tissue very good to close smaller wounds. However, when we’re talking about larger wounds, this is where you can differentiate between nonviable and viable tissues.
So, if you start thinking let’s say that you have 20-year-old with wound, do you need to use any products? You put a regular Band-Aid and you can see that this wound will be successfully close. If I move into the older patient population with some comorbidities and let’s say that this is 40 years old with just benign diabetes and having wound, a two-centimeter square wound. You probably can use nonviable products or just collagen matrix and be very successful to closing this wound. However, if we are talking about very difficult to treat wound persisting for several years in patients with higher level of different comorbidities, so, it’s probably better to use viable tissue because you will have more chances to close such wound.
Ideally, you do not wait until this patient will fail all your treatments. You already can differentiate and you know based on the patient characteristics and wound characteristics, patients that are at higher risk, you don’t need to wait four weeks when they will fail standard of care. You don’t need to try other simpler products. So, you probably better start with the cellular products and be more successful faster than keep forever this wound to keep open and open to complications, infections and amputations. So, in summary, what are benefits to use cellular products?
Because cells are source of growth and factors in matrix proteins, so, they can provide sustainable, useful growth factors over time until these viable cells persist in the wound. They provide dynamic response to local wound environment because in this particular moment, you may need a vascular endothelial growth factor. But after two hours, you may need factor that will attract fibroblast. And next day, you may need factor that will and use migration of keratinocytes.
So, cells can sense and change the profile of factors that they will secrete. So, this is a big advantages what you will get with cellular products. However, there are disadvantages. If you isolate cells, they cannot live outside of the body long time and then you can see two different things happening with cellular products. One, you can store them refrigerated or at room temperature, but you will have very short shelf-life. What are you going to do? You can cryopreserve. This is what scientists figure out for long-term storage for cells and living tissues. However, if you have living cells or tissues, you will require deep freezers or liquid nitrogen for storage. You require special shippers with dry ice for shipment and you will spend your time in preparation of such products. So, altogether, it’s a huge disadvantage. So, these products cannot be utilized by every physician in every office because if you don’t have a proper storage conditions, you are out of using cellular products especially cryopreserve.
So, what are we going to do? Ideally, you wanted to have cellular product that you can store at room temperature. Can we do that? We believe that we can. So, number one, just to emphasize that again this is all your living skin substitutes. This is storage conditions and you can see clearly you can store these. If it’s refrigerated at room temperature or years, if it’s cryopreserved just to emphasize advantages and disadvantages.
So, there is a hope that we can dry tissues, dry cells and in the future probably organs or entire human being and keep everything alive. When you rehydrate the cells that were stored dry room temperature will come back living again and not only living but functional. So, this was a foundation for drying living tissues. So, a lot of scientists, a lot of groups worked on it more than a decade to form this foundation. Number one, what we know from mother nature that there are animals that can live in a very dry condition, for example, water bear or tardigrade. Tardigrade can survive in the space under radiation, can survive in a dry condition without water for months and years. So there are examples like bacteria, they can create a cocoon outside of them and survive in very difficult conditions. So it is possible.
What also we know that such animals and from our experience with the cryopreservation that there are substances that can help you to protect cells during cryopreservation or drying and all these substances, they have common chemistry and they can be used for both lyo and cryopreservation.
And here, you can see multiple examples when people were trying different methods how to dry or how to preserve living cells and store them at room temperature. And in this column, you can see type of cells people are trying to do. So, the problem was that very often, the results are not reproducible and very difficult was to upscale these results or you can dehydrate successfully, but the shelf life is still very short.
So, we selected a method based on lyophilization. If you start thinking about lyophilization, you know very well this is old method. A lot of pharmaceutical drugs, they are lyophilized because in the dry state, you can store them for a long time. Food industry uses a lot of lyophilization. So, you know that you can buy a lyophilized food. You can take it with you in the mountain and then you can reconstitute it and you can eat this food.
So, it’s a good way to preserve drugs and food. And if you look carefully on the stages in lyo-preservation, so, there are three stages. Stage number one, you freeze. That’s why this method is also known as a freeze dry. You freeze substance what you wanted to lyophilize. And then, when your water is solid, you do a next stage. You evaporate water and you do it by transient water from solid to gas without liquid phase. So, this is primary drying and then you do secondary drying to remove residual water.
So, why lyophilization was so good to try? Because, we have a lot of experience, there are machines, lyophilizers, different grades of lyophilizers, very fancy lyophilizers that can help you to tweak programs. And we already practically, step one freezing, we already know how to do it. This is your cryopreservation. You can use solution containing special cryopreservative that also is good to use as a lyopreservative and then we had only to figure out this step two and step three to play around with conditions how to take water from the living tissue and not damage cells. And it looks like we were able to figure it out.
So, here, example of placental membrane amnion, so, this is how it will look after lyophilization, very similar like you can see other dehydrated placental membranes. But if you reconstitute it, you cannot distinguish lyopreserved from cryopreserved and here, not shown, but fresh placental tissue will look the same. If you look histologically on the structure of placental membrane, you can see that no difference between living tissue, fresh tissue, lyopreserved and cryopreserved. But what is very important is cell viability.
You can see here comparison between cryo and lyo. So, the number of viable cells post thaw and post rehydration is the same on both layers, epithelial layer and stromal layer. And what is also very important, if you put piece of lyopreserve tissue in culture, after three weeks, you still can see a lot of living cells and culture. They will start coming from the tissue. They will start to proliferate. You can see here with trypan blue, some dots blue. This is dead cells, but majority of cells are still alive.
So, now, next question, we see that structure is the same, retained as in first tissue and in cryopreserve tissue. Cell viability is retained. How about cell functionality? This is cell functionality in vitro and you can see that both cryopreserved, viable cryopreserved amnion and viable lyopreserved amnion, they can inhibit secretion of inflammatory factor TNF alpha by immune cells. So, it means that the responsiveness in the same way between the two different types of preserved amnion. If you put the lyopreserve and cryopreserve amnion in the hypoxic conditions, again, you can see response that its upregulated expression of vascular endothelial growth factors.
How about animals? If you go to animal model, so, what you can see, you create wound in animal and by injecting inhibitors of antioxidants, you can convert this wound into chronic wound. If you apply just Tegaderm, this wound will be not closing. It becomes larger that the mice can even die. So, however, when you will start applying viable lyopreserve or cryopreserve placental membrane, you can see that after five weeks, you can completely close the wound. What is very interesting that in such chronic mouse model, you will require to apply product several times. But what is also good that there is no difference between cryopreserve and lyopreserve. So, practically, we are talking about the same placental membrane but two different configuration, one traditionally cryopreserve that require minus 80 storage and another one lyopreserve that now we can see it on your shelf.
So, what could also influence on the wound microenvironment? And here, you can see gene expression. In the control wounds, this cluster, a red cluster represents a lot high level of inflammatory cytokines. If you increase it, you can see here this is example of your tumor necrosis factor alpha present in all chronic wounds. If you start treatment with viable lyopreserved amnion, you can see dramatic shift from inflammatory environment to anti-inflammatory, so, everything becoming green.
So, if you look on the protein level, it’s the same story. TNF alpha, so, it’s upregulated in the chronic wound and downregulated when you use viable lyopreserved amnion. What’s also happening that it’s not only downregulation of inflammatory cytokines, but you can also see upregulation of anti-inflammatory cytokines as interleukin 10. So, the viable cells and placental membrane are changing wound environment in all possible ways. So, that’s again the benefit of having viable cells. They, as you, respond to environment, what is around us, they know what to do what wound will require in this particular moment.
And this graph was showing you also upregulation of enzymes that are very protective for other cells in the wound. They are antioxidant and they will take oxygen radicals and block their damaging activity in the wound.
So, in summary, what I just described, you need to remember the main difference between cellular versus no cell product is in dynamic response to local wound environment. However, it’s not easy to store cellular products. And now, a new method developed that will allow you to store living products on your shelf. So, it will eliminate need for shipment on dry ice. It will eliminate need to store it at minus 80 and it will eliminate need and save you time during the preparation and application of the product.
Now, if you start thinking where we are going, I hope that very soon, the amniotic membrane based on this new technology, living lyopreserve tissue will come to your hospital, to your office, to your clinic.
So, what next step? Next step is to understand and we believe that performance clinically will be the same between cryopreserve and lyopreserve tissue. If you look way in the future and I hope it’s not very far away future, I hope that one day that we all will be going to the grocery store to the pharmacy and grab from the shelves Band-Aid, but the active ingredient in this Band-Aid will be living lyopreserve stem cells. And I hope that I can see it when I am still alive and that’s very encouraging that such treatment already will come in the nearest future. Thank you very much.