Section: CME Category: Wound Care

Entering a New World: Ambient Storage of Cellular Therapies

Alla Danilkovitch, MS, PhD, RN

Alla Danilkovitch, RN, MS, PhD discusses the latest technology for ambient temperature storage of viable cellular products. Dr Danilkovitch examines the science behind successful treatment of wounds with viable tissue as opposed to dehydrated tissue, and the improved impact the new method will have on the availability and cost of wound treatment.

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Goals and Objectives
  1. Understand the novel lyopreservation technology for ambient storage of cellular products
  2. Discuss the use of this technology for development of cell therapies for wounds
  3. Analyze the composition and properties of Lyopreserved placental membranes for wounds
  4. Understand the impact of cell lyopreservation on the use of cellular therapies by wound care providers
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  • CPME (Credits: 0.5)

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    Release Date: 03/16/2018 Expiration Date: 12/31/2020

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    Alla Danilkovitch has disclosed that she is an employee of Osiris Therapeutics, Inc.

  • Lecture Transcript
  • TAPE START – [00:00]

    Male Speaker: I would like to introduce Dr. Alla Danilkovitch. Dr. Danilkovitch has a background in PhD, is a nursing as well. She comes to us from Osiris. Osiris is – she held responsibilities to help develop the pipeline products and lead research and development in medical affairs and clinical operations. She's been a researcher for more than 25 years and has been involved in academia in the United States as well as the government. Her areas of scientific expertise include cellular and molecular biology, immunology, cell bio-chemistry, signal transduction, asset development, validation, stem cell biology, cell regenerative medicine and product development.

    She is a member of several professional societies and is a current reviewer of peer-reviewed journals throughout regenerative medicine. Dr. Danilkovitch conducted research at the Cancer Institute of the National Institutes of Health as well as Max Planck Institute of Biochemistry in Munich, Germany, and as well as a number of other facilities. She earned her PhD in Cell Biology and a Master's of Science in Cellular Immunology and Microbiology from Moscow State University. She also retains an RN degree in Pediatrics from Moscow as well.

    Dr. Danilkovitch will be speaking today, Entering a New World: The Ambient Storage of Cellular Therapies. This is obviously very current with all the regenerative approaches that we have. So it's with great pleasure I would like to welcome Dr. Danilkovitch. [Clapping]

    Dr. Alla Danilkovitch: Thank you very much. I always very excited to be part of educational conference and you can see by the title that I wanted to talk about new technology, what is coming to your office tomorrow. The technology is not here today, but it is coming. It's like, you know, announcement about iPhone 10, everyone is talking about it and everyone knows that it is coming. I wanted you to be prepared to meet products in a new configuration that you can store in the future, cellular products at room temperature. So I'm Chief Scientific Officer of the company. And I am full time employee, no other disclosures to make.

    And today, our key objective is to overview a newly developed technology that allows us to bring Cellular Therapies to be stored at room temperature. And we can discuss where the field is going and how quickly and what kind of products will be available for you in the nearest future.

    So, but before we will go to new technology let me go to the basics. So, when you will start using advanced skin substitutes, you usually start with the basic treatment of patients. You evaluate patient and wound and then you apply good wound care. However, a lot of patients they will not respond to your good wound care. And that's usually patients with different comorbidities and aging. So, one patient is not responding to good wound care you will reach your toolbox with the advanced skin substitutes. And here on the slide, you can see variety of different products available currently to you. And if you break down you can see that they may have different compositions, ranking from [indecipherable] [00:03:36] and plus growth factors. And you go to the right side you can see that cellular products are available for you. Then you may ask question, when and why should I use Cellular Therapies for wound treatment?

    Couple years ago a group of experts, and you can recognize names on this slide. They got together and they discuss this particular topic: When to use cellular therapies for chronic wound treatment. And you can see that the key conclusion was that you use this product and you may go ahead and start using this product together with good wound care faster than your standard four weeks. And this group of patients will include aged patients with a lot of comorbidities and you can see a list of such comorbidities that bring patient to at higher risk for non-closure. Why you want to use cellular therapies in such higher risk patients? Because all cells in these patients they are non-functional. They have empire functionality and particularly their stem cell is not functional. These cells, they senescent in the area of wound, they cannot migrate, proliferate. They cannot secrete appropriate levels of growth factors…


    …and extracellular matrix that will lead to wound closure. And as I mentioned that it's also particularly important that they practically don't have stem cells. And here we are coming to the stem cells. So, why stem cells are so important? Because we carry stem cells to the last day in our life. We can more or less regenerate our organs and tissues. So stem cells, they are source of specialized tissue-specific cells that actually participate in the repair of their damaged tissue. I will give you an example. For example, fibroblasts that are sitting in your dermis, they are terminally differentiated mesenchymal stem cells. Your epithelial layers are coming from epithelial stem cells. Your blood vessels are coming from a little stem cells that become mature blood vessel cells, and so on and so on.

    But unfortunately what happening over time that we are losing stem cells. And here on the graph, you can see example what is going on with the number of mesenchymal stem cells. One type of stem cells that right now we are recognizing, very important for healing of all organs and tissues. This type of stem cells in the beginning we were thinking that they can regenerate bone. These cells can become osteoblast and can help to rebuild bone. However, at the present time we know that emesis, they are sitting in each tissue in the organ and they orchestrated organ and tissue repair for every organ, including skin.

    So you can see here that what is going on in bone marrow that with age we were losing a number of mesenchymal stem cells. But not only number also functionality is going down. And this is a very good review, I highly recommend to read it. So this review summarize what was known data accumulated until 2015. And the conclusion of the review was that actually using autologous stem cells not beneficial for many cases in aged and diseased population. So, and this is just what’s going on with the aging, but also with the diseases. Here’s example from the study in mice. If you take normal mice and diabetic mice and isolate stem cells, you can find that significant decrease in the number of stem cells will be in diabetic animals in comparison to normal animals.

    And on another graph, you can see that these cells in the diabetic animals, they do not produce sufficient level of growth factors required to support normal wound healing. So you may ask what to do in this situation? If I see in all the patients with diabetes, with cardiac diseases, with Charcot foot, with all other comorbidities knocking in my door, what can I offer to such patient? Yes, you can offer treatment with Cellular Therapies and particularly stem cells.

    Here’s again example from the literature in mice. If you compare diabetic mice to normal mice, you can see horizontal line representing the rate of wound closure in normal animal. Column number one is control. This is how healing wound happening in diabetic animals. You can see that they are closing their wounds, but they're significantly below the normal wound closure rate. So if you take fibroblast and apply them directly to such wounds, you can see that nothing happened. This wounds continue to close with the same rate. However, in the last column you can see if you add mesenchymal stem cells, mesenchymal stem cells will dictate mouse fibroblast and mouse epithelial cells, endothelial cells what to do in the wound so that it will speed up significantly the rate of wound closure bringing it to the rate what was observed in normal animals. So this particular study will suggest that we need product containing mesenchymal stem cells especially for compromised patients with higher risk for non-closure.

    Why Cellular Therapies are so important and what is unique about cells? What is the difference between cellular versus acellular or devitalized product? Cells like us, they're living creatures who respond to local microenvironment. If you place cell in the area with high level of inflammatory cytokines, cells will respond and produce more anti-inflammatory cytokine. So, cells, they are responding to everything what is around and they are trying to counterbalance it to bring homeostasis in the tissue and then the organ back to normal. You can see examples.


    Can you recognize what right now is available for you, products skin substitutes that contain living cells. There are several different groups. You can break down like you're very well aware about two bio-engineers can substitutes. One is bilayer containing fibroblast and epithelial cells; another one is dermal skin substitute containing fibroblast. Right now, a new kid on the block is placental tissues. Placental tissues were very well known a long time ago and now they are coming back and people who are trying it recognize therapeutic value of placental tissue.

    So if you compare cellular to acellular, unfortunately right now not a lot of data available. However, you can find in the literature three studies. All three studies were comparing the living construct or a living tissue to dehydrated tissue containing non-viable cells. And you can see commonality in the results that in all three studies, cellular construct or a tissue over perform dehydrated. Most studies needed, however, this is the indication that it is a different class of product. And when you are thinking about treatment of patients, you always should consider it from this perspective. If your patient is compromised in higher risk probably you need to start immediately with the cellular therapy.

    So as I mentioned to you, placental right now is becoming available and there are a lot of products, 50 plus products and everyone every day more and more products come into the market. So placental tissues are very well known that anti-inflammatory, anti-fibrotic support angiogenesis and antimicrobial. So placental tissue has all properties that are required for wound healing. If you look at the composition you can see three main components in the placental tissues. So one is extracellular matrix and variety of different extracellular matrix proteins are present in the placental tissue. Also variety of naturally present a growth factors in cytokine, and what is very important is naturally present living cells, so including mesenchymal stem cells. You will have everything if you are dealing with fresh tissue. However, you can understand that it's not practical. You cannot store it for long time and what is very important that you cannot test your donors and your tissue for safety because the window for storage of fresh tissues is very short. So scientists are smart. They develop different methods how to preserve the tissues including placental tissue. And you can see that everyone starts with the fresh placental tissue containing three components of the tissue. However, depending on what method you will apply, you may end with one or two, altered or non-altered, more altered or less altered, destroyed or preserved tissue component. And there are several products that will be cryopreserved that right now available for you that will retain all components of placental tissues. And this is what is ultimate goal for preservation.

    So you all know that usually a living tissue autograph is considered gold standard. What is bone autograph or skin autograph? So it means that it is very beneficial to have methods that allow you to preserve all components in the tissue that will be come your product that you can use for wound management.

    So here is another example, if you have all three components including viable cells, this living placental tissue responsive to local microenvironment. This is dynamic response that, and specific attribute to only living tissue. So if you place living tissue in a hypoxic chamber so you can see again horizontal line representing that you will have some vascular endothelial growth factor in the tissue. However, hypoxia will drive up regulation of the VGF production by living tissue. If you try to repeat this experiment with tissue containing no cells, you will not see such a response to hypoxia. The middle bar show you another example of what is going on in the presence of inflammatory cytokines. In this particular case, tumor necrosis factor alpha. So again, tumor necrosis factor alpha trigger up regulation of inhibitor of TNF.

    And last column represents response to bacteria. If you place living tissue in the presence of bacteria, living tissue will start producing antimicrobial factors.


    So keep in mind that this is the key difference between cellular versus acellular product. So cellular products are very good, very powerful, but it's very expensive to make. I can tell you that our company spent a lot of money to make it and then you need to ship it in a special shippers with dry ice. And it's not available for every physician because you need to have special ultra-low storage like deep freezer or liquid nitrogen. Ideally, you will have Cellular Therapies stored on the shelf. Right now, there are no such option. You can store living products for short time, like for example, your very well-known Apligraf or it’s cryopreserved. If you cryopreserves you can store a long time. However, only recently a new technology was developed and it looks like it will allow us to have such therapists stored at long time at room temperature.

    So scientists were working on it long time and here is the summary of probably 30 years of work from thousands different scientists from different areas. So this is connecting your dots. What we know right now that why it is feasible? Number one examples from nature. There are small animals or bacteria or yeast, they have mechanism that will protect them from desiccation. So they can survive in the dry condition for long time and then when water is available, they are coming back alive. So we also accumulated a lot of data about water substances inside of such animals and bacterial cells that will help you to cryopreserve or dehydrate cells. And you can see a long list of different methods what scientists were trying and different types of cells they were trying to dehydrate and demonstrate the feasibility of this approach.

    So, one called methods it looks like a Lyophilization is very feasible. So what is lyophilization and why it is a good method to use for dehydration of living cells or tissue? So, lyophilization is known for long time and your pharmaceutical drugs, they are mostly lyophilized. When you lyophilize, you preserve structure of your protein or your molecule and you can store it at room temperature on the shelf for long time.

    So lyophilization has three phases. So phase one is freezing. So in other word for lyophilization is freeze-drying. So freezing is phase one. When you bring water in the sample into solid state. And then you go to phase two, which is primary drying. You're taking water from the sample but you transit it from the solid state directly to the gaseous state. So you are escaping the watery or liquid phase again. So, and that allows to find the conditions so to lyophilize cells or tissues because if you look on the step one, if you do it in the presence of special substances, special chemicals that are cryo-protective and lyo-protective. So your phase one, freezing, is the cryopreservation that is much more advanced and people know how to do it in routine viable cells. And then you can, two conditions for phase two-primary drying and secondary drying. And thankful that we have right now good lyophilizers with the programming allows you to change a lot of steps in lyophilization. And such program was found to lyophilize living tissue.

    Next several slides, I will show you just example of how lyophilized immune look like. So you can dehydrate your immune using novel method, but you will retain in contrast to current lyophilization method. All structural in cellular component intact. So here you can see histology that the histological, the structure of the tissue in lyo-preserved immune is the same as in fresh or cryopreserve. When you rehydrate, the tissue look exactly fresh or cryopreserved. But what is more important, you can see here green images that the number of viable cells is the same kind, lyo-preserved versus cryopreserved sampled. Moreover, the cells will remain viable not only immediately after rehydration but if you place lyo-preserved piece of tissue in the cultured medium after three weeks you can see that these cells start crawling out of the tissue, started proliferate.


    And you can see viable cells three weeks after. So viable cells is good. It looks like these cells not only viable but routine functionality. And we did some functional tests. Here you can see still that living lyo-preserved tissue similar to cryopreserve tissue is responsive to local microenvironment. If you place tissue in the presence of high inflammatory cytokines, this viable tissue will release factors and down regulate secretion of inflammatory cytokines by activated immune cells.

    Another example again, if you place lyo-preserved tissue in a hypoxic chamber you can see up regulation of EGF triggered by low level of oxygen. If we go in, in vivo in mice, and this is a special model that allows you really to differentiate between different products. This is not only wounding in diabetic animals, but if you treat such created wounds with inhibitors of anti-oxidative enzymes, you convert this wound from acute into chronic. And if this wound will remain untreated you can see on the – unfortunately you cannot see but believe me that this wounds over time and you can see day 35 becoming larger instead of closing. So when we use viable lyo-preserved or cryopreserved membranes, it was a required multiple application in the same way like we observed in our clinical trials to cause such wounds. So, but the behavior of lyo-preserved tissue was exactly the same as cryopreserved tissue. When we look on what is going on in the wound, you can see here collected samples from the wound. And in the controlled animals, you can see enlarged gene expression on the right. All genes expressing inflammatory cytokines they are up regulated, they are red colored. After application of viable lyo-preserved tissue, you can see that red becoming green, meaning that viable lyo-preserved tissue down regulated expression of inflammatory cytokines are converting this wound environment from highly inflammatory to anti-inflammatory. And this is what is required to transit wound from inflammatory phase into regenerative phase of wound healing.

    So it's not only the gene expression, but the same happen at the protein expression, down regulation of inflammatory cytokines and up regulation of anti-inflammatory. And also very important to down regulated oxidative stress. The presence of higher level of oxygen radicals are actually damaging cells in the area of wound. That's why the cells are dying and becoming senescence. You attract more cells from the healthy periphery of wound, but again, oxygen radicals will kill the cells. So it is important to down regulate oxidative stress. And what we observed that in the presence of viable lyo-preserved issue, the activity of antioxidant enzymes is going up, that again is very helpful to drive wound to the wound closure.

    So at the end, I can summarize that I presented you that there is a method that allows us to have in the nearest future Cellular Therapies or cellular tissues that can be shipped and stored at room temperature. And we believe that it will completely change the face of Cellular Therapies in the future. And what is very important that, more physicians will be able to use cellular treatments that are required especially for high-risk patients or compromised patients not only for wound treatment but there are other diseases and conditions that require Cellular Therapies.

    Thank you very much for your attention. [Clapping]

    TAPE ENDS – [24:09]