Alla Danilkovitch, RN, MS, PhD discusses cellular therapies with specific emphasis on placental membranes. Dr Danilkovitch examines previous and current modalities for preserving viable placental components and discusses appropriate uses for the novel cell preservation.
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Release Date: 03/16/2018 Expiration Date: 12/31/2020
Alla Danilkovitch, RN, MS, PhD
Chief Scientific Officer
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TAPE STARTS – [00:00]
Male Speaker: Our next speaker is Dr. Alla Denilkavich [phonetics] [0:00:04]. I’ve only pronounced this three times introducing this young lady and I’ve botched it every time, so I apologize. She earned a PhD in Cell Biology, MS in Cell Immunology and Microbiology from Moscow State University. She’s an RN, has a degree in Pediatrics from the Moscow Regional School of Nursing. She’s done extensive studies in Wound Care. Actually one of the first approved stem cell drugs, she was involved with that, as well as cartilage repair and coverings for acute and chronic wounds. So you’re in for a treat.
She is extremely bright and will educate us on the things that we, kind of, use in everyday practice and what we’ve been shown that we should be using. But here now, for clarification of what those products are actually supposed to do for our patients, is critically important.
So please welcome, Dr. Denilkavich.
Alla Denilkavich: Thank you very much for such introduction. I’m glad to be here. And I hope that you enjoy the change.
Although I will show some pictures of wounds. I would like to talk about what is going on in the research. But not very far research, not basic research. Research that is tightly connected to practical life. We will be talking about cellular therapies and why cellular therapies are important for wounds and what is going on in the development to kind of speed up development of cellular therapies.
I’m chief scientific officer of [indecipherable] [0:02:05]. I have no other disclosures.
And today’s objectives of my lecture is to give you examples that you understand the key differences between products containing living cells, versus products containing non-living cells. Understand what the challenges companies are facing when they’re developing cellular therapies.
Talk about a new technology that hopefully would help to change face of cellular therapies. And think about and discuss with you where we are going in the future. Cellular therapies – everyone understands how promising cellular therapies are.
In contrast to devices or pharmaceuticals, cellular therapies, the core promise to cure diseases, not just treat symptoms. But if we look on and compare cellular therapies – where we are right now, versus devices, pharmaceuticals or biological drugs, it is very underdeveloped. And there are reasons why, despite on all high promises.
What is so special about living cells? Living cells – they have unique properties that they – like, when we are alive, we respond to different environmental stimuli. So cells, they respond what is going on around. They respond and interact to other cells in the tissue. They respond to soluble growth factors in cytokines. They respond to chemical and physical stress stimuli and in response, they will produce what is needed.
I give you a very simple example, if you look outside, then you see rain, you will take umbrella. This is your dynamic response to change in environment. Very similar to cells, if you place cell, a living cell, in the inflammatory environment, cell will produce anti-inflammatory factors to balance and bring everything back to normal.
So when you will be looking for cellular therapies for wounds – not long time ago, experts in wound care, they get together and they discussed these issues. Whether we need cellular therapies to implement in the wound care and if to do that, why and when it’s appropriate.
So you should look and think about to apply therapy containing living cells when in your office you see a patient at higher risk. Higher risk – it means that this patient will heal from underlying diseases and conditions that you know that this patient will be not good healer.
And if you look on such risk patients on the molecular and cellular level, we know right now that these patients, they have deficiency in cells. Their cells in the area of wound is not functional. They don’t have enough stem cells to support healing. And this is why delivery of therapies with healthy portal cells directly to the wound, would be very beneficial for such patients.
It is very unfortunate when you look what is available right now to you. It’s all on one slide. So you can pull several other examples but this is it. If you ask me to create such list for non-viable therapies, it probably will be 50 slides. So there is a reason why.
Among these therapies right now, you know very well, Apligraf and Dermagraft. So they were bioengineered to more than 15 years ago. You are using maybe sometimes, living human cadaveric skin. How about when new kid on the block is placental membranes. And I would like to talk a little bit more about placental membranes as a new modality for wound care.
There are two placental membranes, amnion and chorion. And they are covering the baby in utera. And people observed long ago that if it’s covering babies, it’s probably good to cover wound. So placental membranes were used in history a long time ago.
But then, we realized that we should test donors for infectious diseases. And we had a gap between the use of placental membranes until number one, we found and developed methods how to test donors. And number two, we found methods how to preserve tissues. Because placental tissues, native placental tissues, has all components and properties that are beneficial for wounds. Placental tissue is anti-microbial, anti-fibrotic, anti-inflammatory, support angiogenesis. It’s exactly what you need for chronic wound especially in compromised high risk patient.
But again, use for first tissues is not practical. You need to have sufficient window to test it for infectious diseases and also you want it to heal with sufficient time that you have product off-shelf and use it as needed. Ideally, the goal of preservation is to retain everything what is beneficial in the first tissue. However, methods were developed and used right now for commercial products. They are different. And some of them may destroy or harm one or several components in the placental tissue.
There are methods that will allow you to have all components like the first tissue. One example, cryopreserved viable placental membrane. So in such membrane, you will retain structural metrics, growth factors and native cells that are in placental tissue. What is very important to understand that placental membranes – very rich source of young, potent stem cells, particularly, mesenchymal stem cells. And right now we know that mesenchymal stem cells are important for wound healing. Normally, they are present in dermis. And there are many MSEs in young children and young adults. However, when we age, the amount of stem cells in our dermis decline. And that’s one of the reasons why all the patients, they’re losing potential to heal.
So this cryopreserved form of membrane retains all essential components that are present in the first tissue. And again, if you do different testing, you can realize and you can find that placental membranes with all components intact will be more potent that placental membrane without one or zero components. There are data showing it in vitro, in animals. And even right now, there are data showing benefits in clinical studies.
So again, if you compare living versus non-living tissue in the Petri dish, you place fresh living or cryopreserved tissue containing all elements including viable cells. Viable cells will be responding to environment. Bar number one showing you place living tissue in a hypoxic chamber and no oxygen. In response to low oxygen such living tissue will produce more vascular endothelial growth factor. If you start thinking how it is relevant to wounds, you want an environment that is hypoxic, meaning that you have very low oxygen.
You don’t have blood vessel because vascularization was compromised during the wounding of the tissue.
So what do you need to be able to close the wound? The step number one, you need to attract endothelial cells and rebuild new blood vessels. So meaning that you need more vascular endothelial growth factor and living tissue can’t do it, can’t provide VEGF that will attract other types of cells and rebuild vascularization.
The second bar showing that cells will respond to higher level of inflammation while producing anti-inflammatory factors. And the last one showing if you expose living tissue to bacteria, bacterial antigens will stimulate production of antimicrobial peptides. And this is the tissue of placental tissue. Placental tissue in utera protect the baby from infection.
So altogether, this is you can get only from living tissue. So that’s the key difference. A non-living tissue may have very beneficial structural metrics and growth factors. However, a non-living tissue has no dynamic response to changing wound environment. And your wound is not static, your wound is dynamic. So today you need VEGF and rebuild blood vessels. Tomorrow you will need to attract fibroblast and build granulation tissue. After tomorrow you will need to induce migration of epithelial cells and close the wound. So that’s the key difference between non-living and living products.
Another example is clinical. So data limited, but I am very pleased to see that every year more and more studies are published. So you see here three studies that are retrospective by nature but analyzing what you are facing in the real practice on a daily basis. So one of the products was bioengineered product. Another one, cryopreserved placental membrane. And the last one was bioengineered fibroblast containing dermal product. So you are very well-known, it’s an uplift graft, derma graft and graphics.
And the effectiveness of these products was compared retrospectively to effectiveness of non-living placental product. And you can see that all three studies uniformly show that on a daily basis, if you look on a higher volume of patients, so the living tissue will give you better outcome than non-living tissues.
Again, if we go back to what is available to you from the viable skin substitutes, question will be how you store them. And this is the key issue with living cells. Living cells, they cannot survive a long time outside of the body, whether it’s isolated cells or it’s a living tissue or its bioengineered product containing living cells. So right now, we have two extremes. Number one, you can store it at room temperature but it will be very limited shelf-life. And another example, you can store long time at very low temperatures.
So by saying that, you can already see advantages and disadvantages of living skin substitutes. A huge advantage that living tissue and constructs will provide you dynamic response to changing wound environment and produce growth factors for a long time as needed. A huge disadvantage is the storage. It’s short storage at room temperature or long storage will require a specialized equipment.
So from the company’s standpoint, it’s very challenging to develop cellular therapies. And one of the challenges is storage in distribution. Again, if you store at room temperature, you have very complex distribution and it’s not convenient for the customer like you because you order the product and then shelf-life is just several days or a maximum of several weeks. So if you wanted to store at long time, it’s another disadvantage that you need specialized equipment like deep freezers or liquid nitrogen. So such products, they ship on dry ice and should maintain specialized low temperature. So meaning that when you receive this product, you are in charge to store it appropriately.
So as I mentioned to you right now or until now, only one method is in use for long storage of cellular therapies is cryopreservation. And here you can see a portrait of the British scientist Chris Polge. He’s actually a founder of cryopreservation method.
In 149 he describes the use of glycerol, that if you add glycerol to living sperm cells and freeze them, then post thawing, you can see that this sperm is still alive and functional. And it’s dramatically changed the face of medicine and veterinary practice. Because of him now we don’t need to run in the field and think how we can breed our animals. So you collect sperm, you can preserve it and you can use it later. The same in the field of medicine. So it created the foundation for in vitro fertilization.
Again, very important. At that time it was huge progress toward development and use of cellular therapies. It’s opened new possibilities. However, it’s huge disadvantage that you need specialized shipment and specialized storage. Meaning that if you practice in a regular office, it might be not be available for you because you cannot provide appropriate storage conditions.
Ideally, what everyone wants, everyone wants to store cellular therapy long time on the shelf, that you just grab it from your regular office shelf without any specialized conditions and specialized equipment. So people started thinking about it a long time ago, and there are examples. Example number one, if you look in the nature, there are small animals that can survive desiccation. Meaning that you raise these animals and then rehydrate them and they come back normal. So also I’ve accumulated a lot of knowledge about the use of cryopreservative agents that are also found at high level in these small animals. And this molecule, they allow these small animals to survive without water for a long time.
So scientists started dehydration of cells and tissues a long time ago. If you check literature, these publications where more than 30 years ago. It showed the proof of concept. However, until now no one was able to create robust product or robust process that allow you to have consistently viable cells of with high viability, functional cells and that you can store it long time. However, all three key components that are already there, that helped us to create a new method, new utilization method, how you can dry tissue or cell but keep cells alive.
Let me show you several examples, experimental data that exist right now. This is a amnion. If you draw immune using a new method and then rehydrate, you will be not able to distinguish tissue from cryopreserved or fresh. And you can see this histologically, it’s also the same. But what is most important, viable cells. You can see here a green staining. Green dots represents living cells, red dots represents dead cells in both layer of immune, epithelial and stromal, you can see high level of viable cells in both lyo-preserved and cryopreserved tissues. Then if you place lyo-preserved tissues in culture medium, you can see that cells not alive for short time. Even after three weeks, there are a lot of living cells in your Petri dish.
How about functionality of such tissue? You can see here that a similar to the cryo-preserved tissue and I can tell you similar to fresh tissue, lyo-preserved living tissue maintain ability to respond to changing environment. So you can see on one graph, inhibition of production of inflammatory factor TNF alpha, or tumor necrosis factor alpha, produced by activated immune cells. On another graph, you can see that this lyo-preserved tissue, similar to cryo-preserved tissue, secretes more vascular endothelial growth factor if you place this tissue in a hypoxic chamber.
The same in the mouse’s wounds. You can see that if you create wounds in a diabetic mice and create chronic wounds, then you’ll apply cryo-preserved or lyo-preserved immune and you can lead to enclosure. In contrast, controlled animals, they didn’t close wounds. So a mice is very happy to receive lyo-preserved tissues.
And again, I would like to emphasize that when you digitize a live tissue and then use the same lyo-preservation process, so your metrics is preserved, your growth factor is preserved.
The difference mid panel and long panel that one tissue has no viable cells, not the tissue that has viable cells, and you can visually see that of the three weeks, tissue… viable tissue close wounds, and non-viable cells, the wounds is still open and very close as a controlled animals.
So, what is very interesting and very encouraging that the data I’m showing to you is applicable not only to immune, it’s applicable to variety of different types of tissues. Whatever we tried wound cartilage, different types of placental tissue, umbilical tissue, chorion, a skin, so every time we apply this new lyo-preservation technique, at the end of the reconstitution, we can see the tissue retain viable cells.
So in summary, if you compare, lyo-preservation is not new. You know, a lot of products and a lot of tissue-based products, they are lyophilized. However, all that you know until now is non-living cells because traditional lyophilization will kill living cells. So it will not use specialized lyo-preserved solution and you need to use it. And then you need to tweak your conditions so that you do lyo-preservation in a way that you are not creating water crystals in the tissue or cells that is detrimental for cells. So at the end, the normal lyo-preservation technique will allow you to retain not only structural metrics and growth factors but what is very important living cells.
So in conclusion, what I’ve talked right now, it’s encouraging that we have a method that it looks like this method will allow us in the future to store living cells and tissues at a room temperature. And that eliminates a lot of hurdles, not only for companies but also for customers. As a customer, you should be very excited, because very soon, you will have living products sitting on your shelf. And I hope that not very far in the future, we will be going to the grocery store and buy stem cell bandage sitting on the shelf and applies to our wounds or when we need to use it.
I hope that this method will start to be used universally by cell biologists and by companies who are developing different remedies, cellular therapists for a very difficult to treat diseases, for genetic diseases, for other diseases and we’ll hope that it will help us to speed up and we will progress toward better medicine. Thank you very much.
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