Cryopreservation of Cellular Therapies: Is There a Better Way

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Residency Education / Wound Care
Cryopreservation of Cellular Therapies: Is There a Better Way?

Alla Danilkovitch, RN, MS, PhD
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.
CME (Credits: 1)
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Alla Danilkovitch, RN, MS, PhD

Chief Scientific Officer
Osiris Therapeutics
Columbia, MD
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Lecture Transcript
biomedical
TAPE STARTS device manufacturers, or other corporations whose products or services are related to the subject
– [00:00]
matter of the presentation topic. The intent of this policy is not to prevent a speaker with a potential conflict of
interest from making a presentation. It is merely intended that any potential conflict should be identified openly
so
Malethat the listeners
Speaker: mayspeaker
Our next form their own
is an judgments
RN, MS, PhD,about the presentation
Alla Danilkovitch, whowith themuch
is very full disclosure
involved of the facts. in
in research,
education.
--- She joined Osiris in 2003, responsible for the development of product pipeline and leads research
and development medical affairs
has nothing to and clinical operations. She’s been a researcher for over 25 years and
disclose.
Alla Danilkovitch
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.

TAPE STARTS – [00:00]
Male Speaker: Sure.
Male Speaker: OurThank

Alla Danilkovitch: next speaker
you veryismuch.
an RN, MS,look
If you PhD,onAlla
theDanilkovitch, who is very
title, you’re probably much involved
wondering what in research, in
education. She joined
cryopreservation Osiristherapies
of cellular in 2003, responsible for thecare
and why I should development of product
whether there pipeline
is a better way. and
Thisleads research
lecture about
and development
the new technology,medical
aboutaffairs and clinicaland
new technology operations. She’sthat
new products been a researcher
will come to yourfor clinic
over 25 years
in the and future
nearest
experienced academia in US government and industry combined. Her bio can go on and on and on. So rather
and that’s why I would like to share with you what is in the development right now and what is coming in your
than doAnd
future. that,I hope
I’m going to ask
that you willher
gettoexcited
come upandand
willshare her thoughts
be welcoming and with us on
waiting forthe
newtopic of cryopreservation
products coming based
of cellular therapies,
on new technology. is there a better way? So please welcome Dr. Danilkovitch.
Alla Danilkovitch:
My disclosure, Thank
that you.
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
Male
you toSpeaker: Sure.
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
Alla Danilkovitch:
lyophilization Thank you
technology thatvery much.
allows you Iftoyou look
store on the
living title,
cells andyou’re
tissueprobably wondering
at ambient whatand where we
temperature
cryopreservation of cellular
are going with such products therapies and why
in the future I should
in the woundcare
carewhether
space. 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
As you knowwhyall,
I would like tostatistic
that wound share with you what
is scary. is in theofdevelopment
So number right
wounds is not nowdown.
going and what is coming
It’s going in your
up and it put
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
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
a lot of burden
antimicrobial on our society
property. on thetopeople
You wanted hear for who suffer of
inhibitors of chronic wounds
proteases. and people
You wanted whofactors
to hear pay forthat
chronic
will
wounds. So more and more people developing diabetes and more and more people suffer from chronic
correct inflammatory environment and stimulate angiogenesis to fight hypoxia. So every product you want to
wounds.
touch ideally will have all these factors. Is it possible? Yes, it is possible.
So
So if you look
here’s from theinmolecular
experiment and cellular
animals where perspectives,
diabetic what
received living is going
cells. In thison in chronic
particular wounds?
example, So you can
mesenchymal
this
stemiscells
fromandDr. [Freiburg]
mesenchymal[0:03:18]
stemlatest review,
cells are ableyou can seeabnormal
to correct how many different
wound factors atand
environment the promote
molecular and
cellular level going
angiogenesis around inwound
and stimulate the chronic wound
closure. So onand
theI would
graph cite youleft,
on the canyouseecan
how many
see howfactors youare
long cells need to
have on your
persisting product
in the wound tobecause
be able to overcome
there problems
is a thought with chronic
that living wounds.
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
So this environment
wound is what you want
fromfrom your wound
non-healing care, advance wound care modalities. You wanted this wound care
to healing.
modality to care good quality, different types of cells found in skin. You want in this wound modality to have
antimicrobial
[05:05] 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
Looking on allwill have
these all these
data factors.
presented on Is it possible?
this Yes, it
slide, you may is possible.
make several conclusions. Conclusion number one,
that cells are important and can contribute to wound closure. Conclusion number two, that the cell type is
So here’s experiment
important because you in can
animals where
see on diabetic
the graph received
– so living
that here thecells.
graphInwith
this emesis
particular example,
stimulate mesenchymal
wound closure
stem cells and mesenchymal stem cells are able to correct abnormal
while fibroblast wasn’t – they were not able to stimulate wound closure. wound environment and promote
angiogenesis and stimulate wound closure. So on the graph on the left, you can see how long cells are
persisting
So you also in can
the wound
say thatbecause
becausethere
cells,isthey
a thought
do notthat living
persist forcells, they
a while willthe
and notnumber
last in the wound
of cells forever
puts – you which
put in
is
theabsolutely
wound thattrue.
willHowever, this over
be dropping should term
time persistence
that in the wound
the main mechanism is sufficientParacrine
is paracrine. to dramatically
meaningchange
that cells
wound environment from non-healing to healing.
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
[05:05]
when you’re adding them to the wound and no because whatever you add to the wound there is no dynamic
response.
Looking on all these data presented on this slide, you may make several conclusions. Conclusion number one,
that cells
So this are shows
slide important
youand
whatcan contribute
is unique aboutto living
woundcells?
closure.
TheConclusion number
unique property two, that
of living cellsthe cell therapy
in any type is
important
contained because you
living cells can see
would on the graph
be dynamic – so that
response here thewound
to changing graph with emesis stimulate
environment. So if youwound closure
have living cells to
while fibroblast wasn’t – they were not able to stimulate wound closure.
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
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
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
and
livingpromote ourthere
cells. And new is
blood vessel
a new formation
ketone and soIt’s
on the block. onplacental
and so on.
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
You can see other cells will influence what cells produce, metrics proteins, growth factors and cytokines in
placental membranes was done by Dr. David at John Hopkins in 1910. It’s longer than 100 years ago. However
surroundings, physical factors and different environment and external stimuli also will affect what cells will
then people stopped using placental membranes until they figure out how to process tissue and preserve that it
produce. So that’s why if you are talking about products contained in all living cells, they are beneficial to the
can be fully tested and store on the shelf that you can use it.
wound. However, you never will get dynamic response from your wound product to the wound environment.
That’s
So let’sthe
talkkey difference
a little between
bit about cellular
placental versus non-living
membranes. There arecells
twoproduct.
of them, amnion and chorion. Why placental
membranes are so beneficial for wounds? Because they have properties that are required to treat chronic
These look like very crowded table. This is example of wound care modalities containing living cells and you
wound. However, you will hear for all these properties if you will be able to preserve all components in the
can see and very well recognize two of these, this is our bioengineered skin substitutes known as Apligraf and
placental membranes and there are three of them by categories, extra cellular metrics, growth factors and living
Dermagraft. Sopreserve
cells. So if you – but also you knowusually
everything that there
the are products
standard like living
processing cryopreserved
method skin,
to preserve thatcells
living also is
contain
living cells. And there
cryopreservation. You is a new
can ketone
optimize on method
your the block. It’s way
in the placental membranes
that you and both
will preserve placental tissue.
structural This ketone
elements in
is old but new at the same time all because placental membranes were used long time ago. This report of using
the membrane as well as viable cells. You can see here green dots represent viable cells and red dots represent
placental membranes was done by Dr. David at John Hopkins in 1910. It’s longer than 100 years ago. However
dead cells. So it means that you can do cryopreservation of placental membranes that you will not see the
then people stopped using placental membranes until they figure out how to process tissue and preserve that it
difference in the structure in cell viability between cryopreserved and fresh tissue.
can be fully tested and store on the shelf that you can use it.
So if you look on the living placental tissue, you definitely will see responsiveness to micro environment.
So let’s talk a little bit about placental membranes. There are two of them, amnion and chorion. Why placental
membranes
[10:07] 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
Here’s themembranes
basal level, and
whatthere are three
you can of them by
find regarding thecategories, extra cellular
vascular endothelial metrics,
growth growth
factor in thefactors
tissue,and living
cells. So if youE2
prostaglandin preserve everything
or antimicrobial usually the
defensin. So, standard
if you putprocessing
such livingmethod
tissue intohypoxia,
preserveyou
living
cancells is
observe
cryopreservation. You can optimize your method in the way that you will preserve both structural elements in
increase in the production of VEGF. If you have inflammatory cytokines for example tumor necrosis factor alpha,
the membrane as well as viable cells. You can see here green dots represent viable cells and red dots represent
you can see increase in production of the prostaglandin E2and this factor is antagonist of TNF alpha. If you had
dead cells. So it means that you can do cryopreservation of placental membranes that you will not see the
bacteria, you can increase over the basal level production of antimicrobial peptides.
difference in the structure in cell viability between cryopreserved and fresh tissue.
So, that’s your difference between living tissue and nonliving tissue that if you compare it with nonliving tissue,
So if you look on the living placental tissue, you definitely will see responsiveness to micro environment.
whatever basal level you have, you will not see any responsiveness and increase in production of any factors.
[10:07]
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,
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
So, here’s another
significantly very important
the volume of wound for wound
related treatment
infections. example
Why? We’ll that placental
just look that atissues covering
placental tissuebaby provide a
can inhibit
barrier and protection from microbes. So, that’s why placental cells in the tissue, they are capable to produce
growth of bacteria that always will be present in the wound.
antimicrobial peptide and inhibit growth of bacteria. Here, you can see inhibition of bacterial growth in the
presence of living
So, level one placental
evidence tissue
is good, butand allwhat
also, this bacteria,
we need they were isolated
is comparative from chronic
studies. And on wounds
the next and
slide,you
youcan
can
see your regular suspects, so this one, very well known, practically present in all chronic wound.
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,
So, it’s not only in vitro experiments that you can detect this antimicrobial activity in the Petri dish. What is very
nonliving. And what is also very interesting that actually nonviable tissue very good to close smaller wounds.
interesting that there is a correlation with clinical data. Here’s the results of clinical trial with the living placental
However, when we’re talking about larger wounds, this is where you can differentiate between nonviable and
tissue control multi-center, your level one evidence and using in placental tissue, you can see that in the
viable tissues.
presence of viable placental membrane, that was used as an adjunct to the standard of care, you can
significantly increase
So, if you start wound
thinking closure,
let’s say but have
that you not only wound closure.
20-year-old What do
with wound, is interesting
you need tothat
useyou can
any reduce You
products?
significantly the volume of wound related infections. Why? We’ll just look that a placental tissue can inhibit
put a regular Band-Aid and you can see that this wound will be successfully close. If I move into the older
growth of bacteria that always will be present in the wound.
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
So, level
matrix andone
beevidence is good,tobut
very successful also, this
closing whatwound.
we need is comparative
However, if we arestudies. And onvery
talking about the difficult
next slide, you can
to treat
see results
wound of the one
persisting center years
for several with respective
in patientsanalysis when
with higher living
level of tissue was
different compared toso,
comorbidities, nonliving tissue.better
it’s probably And
what you can see, the difference, that again living tissue was able to close more wounds than nonviable tissue,
to use viable tissue because you will have more chances to close such wound.
nonliving. And what is also very interesting that actually nonviable tissue very good to close smaller wounds.
However,
Ideally, youwhen we’re
do not waittalking about
until this largerwill
patient wounds, this is
fail all your where youYou
treatments. canalready
differentiate between nonviable
can differentiate and you and
know
viable tissues.
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
So, if you start thinking let’s say that you have 20-year-old with wound, do you need to use any products? You
probably better start with the cellular products and be more successful faster than keep forever this wound to
put a regular Band-Aid and you can see that this wound will be successfully close. If I move into the older
keep open and open to complications, infections and amputations. So, in summary, what are benefits to use
patient population with some comorbidities and let’s say that this is 40 years old with just benign diabetes and
cellular products?
having wound, a two-centimeter square wound. You probably can use nonviable products or just collagen
matrix
[15:00] 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
Because cellstissue because
are source you willand
of growth have more in
factors chances to close such
matrix proteins, wound.
so, they can provide sustainable, useful
growth factors over time until these viable cells persist in the wound. They provide dynamic response to local
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
https://podiatry.com/lecturehall/description/4684/Cryopreservation-of-Cellular-Therapies-Is-There-a-Better-Way
Page 8 of 12
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
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
wound environment
products. because
So, altogether, it’s ainhuge
this particular moment,
disadvantage. you may
So, these need a
products vascular
cannot endothelial
be utilized growth
by every factor. But
physician in
after two hours, you may need factor that will attract fibroblast. And next day, you may need factor that will and
every office because if you don’t have a proper storage conditions, you are out of using cellular products
use migration
especially of keratinocytes.
cryopreserve.
So,
So, cells
what can sense
are we andtochange
going the profile
do? Ideally, of factors
you wanted that they
to have willproduct
cellular secrete.thatSo, you
this can
is a store
big advantages
at room what
you will get with
temperature. Cancellular
we do products. However,
that? We believe there
that are disadvantages.
we can. So, number one, If you
just isolate cells, they
to emphasize thatcannot live is all
again this
outside of the body long time and then you can see two different things happening with cellular products. One,
your living skin substitutes. This is storage conditions and you can see clearly you can store these. If it’s
you can store
refrigerated at them
roomrefrigerated
temperatureororatyears,
room iftemperature, but you
it’s cryopreserved willtohave
just very short
emphasize shelf-life.and
advantages What are you
going to do? You can cryopreserve. This is what scientists figure out for long-term storage for cells and living
disadvantages.
tissues. However, if you have living cells or tissues, you will require deep freezers or liquid nitrogen for storage.
You require
So, there is special shippers
a hope that we canwith dry
dry ice for dry
tissues, shipment andinyou
cells and the will spend
future your time
probably organsin preparation of such
or entire human being
products. So, altogether, it’s a huge disadvantage. So, these products cannot be utilized by every physician in
and keep everything alive. When you rehydrate the cells that were stored dry room temperature will come back
every office and
living again because if you
not only don’t
living buthave a proper
functional. storage
So, conditions,
this was you for
a foundation are drying
out of living
using tissues.
cellular products
So, a lot of
especially
scientists, cryopreserve.
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
So, what areTardigrade
tardigrade. we going can
to do? Ideally,
survive youspace
in the wanted to have
under cellular
radiation, product
can survivethat
in ayou
drycan store atwithout
condition room water for
temperature. Can we do that? We believe that we can. So, number one, just to emphasize that again this is all
months and years. So there are examples like bacteria, they can create a cocoon outside of them and survive in
your living skin
very difficult substitutes.
conditions. So This is storage conditions and you can see clearly you can store these. If it’s
it is possible.
refrigerated at room temperature or years, if it’s cryopreserved just to emphasize advantages and
disadvantages.
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
So, there is a hope that we can dry tissues, dry cells and in the future probably organs or entire human being
have common chemistry and they can be used for both lyo and cryopreservation.
and keep everything alive. When you rehydrate the cells that were stored dry room temperature will come back
living againyou
And here, andcan
notsee
only living but
multiple functional.
examples when So, this was
people werea foundation for drying
trying different methodsliving tissues.
how to drySo, a lotto
or how of
scientists, a lot of groups worked on it more than a decade to form this foundation. Number one, what we know
preserve living cells and store them at room temperature. And in this column, you can see type of cells people
from mother
are trying nature
to do. So,that there arewas
the problem animals
that that
very can livethe
often, in a very dry
results are condition, for example,
not reproducible water
and very bear was
difficult or to
tardigrade. Tardigrade can survive in the space under radiation, can survive in a dry condition without water for
upscale these results or you can dehydrate successfully, but the shelf life is still very short.
months and years. So there are examples like bacteria, they can create a cocoon outside of them and survive in
very difficult
So, we conditions.
selected a method Sobased
it is possible.
on lyophilization. If you start thinking about lyophilization, you know very well
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.
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.
[20:05]
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.
this is old method. A lot of pharmaceutical drugs, they are lyophilized because in the dry state, you can store
them
So, whyfor lyophilization
a long time. Food industry
was so good touses
try?a Because,
lot of lyophilization.
we have aSo, you
lot of know that there
experience, you can
arebuy a lyophilized
machines,
food. You can take it with you in the mountain and then you can reconstitute it and you can eat this food.
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
[20:05]
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
So, it’sand
tissue a good way to preserve
not damage drugs
cells. And and like
it looks food.weAnd if you
were ablelook carefully
to figure on the stages in lyo-preservation, so,
it out.
there are three stages. Stage number one, you freeze. That’s why this method is also known as a freeze dry.
You freezeexample
So, here, substance what youmembrane
of placental wanted to amnion,
lyophilize.
so, And
thisthen, when
is how your
it will water
look afterislyophilization,
solid, you do very
a next stage.
similar like
You evaporate water and you do it by transient water from solid to gas without liquid phase. So, this is primary
you can see other dehydrated placental membranes. But if you reconstitute it, you cannot distinguish
drying and then
lyopreserved you
from do secondaryand
cryopreserved drying tonot
here, remove
shown, residual water.
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,
So, why lyophilization was so good to try? Because, we have a lot of experience, there are machines,
fresh tissue, lyopreserved and cryopreserved. But what is very important is cell viability.
lyophilizers, different grades of lyophilizers, very fancy lyophilizers that can help you to tweak programs. And
we
Youalready
can seepractically, step one
here comparison freezing,cryo
between we and
already
lyo. know
So, thehow to do of
number it. viable
This iscells
yourpost
cryopreservation.
thaw and postYou can
use solutioniscontaining
rehydration the same special
on bothcryopreservative
layers, epithelial that
layeralso
andisstromal
good tolayer.
use asAnda what
lyopreservative
is also veryand then weifhad
important, you
only to figure out this step two and step three to play around with conditions how to take water from the
put piece of lyopreserve tissue in culture, after three weeks, you still can see a lot of living cells and culture. living
tissue and not damage cells. And it looks like we were able to figure it out.
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, here, example of placental membrane amnion, so, this is how it will look after lyophilization, very similar like
you can see
So, now, nextother dehydrated
question, we seeplacental membranes.
that structure But ifretained
is the same, you reconstitute
as in firstit,tissue
you cannot distinguish tissue.
and in cryopreserve
lyopreserved
Cell viability isfrom cryopreserved
retained. How aboutand
cellhere, not shown,This
functionality? butisfresh placental tissue
cell functionality will and
in vitro look you
the same.
can seeIf that
you look
both
histologically on the structure of placental membrane, you can see that no difference between living tissue,
cryopreserved, viable cryopreserved amnion and viable lyopreserved amnion, they can inhibit secretion of
fresh tissue, lyopreserved and cryopreserved. But what is very important is cell viability.
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
You can seeagain,
conditions, here comparison
you can seebetween
responsecryo
thatand lyo. So, the number
its upregulated of viable
expression cells post
of vascular thaw and
endothelial post factors.
growth
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.
How about animals? If you go to animal model, so, what you can see, you create wound in animal and by
They will start coming from the tissue. They will start to proliferate. You can see here with trypan blue, some
injecting inhibitors of antioxidants, you can convert this wound into chronic wound. If you apply just Tegaderm,
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
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.
[25:05]
So, what could also influence on the wound microenvironment? And here, you can see gene expression. In the
this wound
control will be
wounds, notcluster,
this closing. It becomes
a red larger that athe
cluster represents lotmice
high can
leveleven die. So, however,
of inflammatory whenIfyou
cytokines. youwill start
increase
applying viable lyopreserve or cryopreserve placental membrane, you can see that after five weeks, you can
it, you can see here this is example of your tumor necrosis factor alpha present in all chronic wounds. If you
completely close the wound. What is very interesting that in such chronic mouse model, you will require to
start treatment with viable lyopreserved amnion, you can see dramatic shift from inflammatory environment to
apply product several
anti-inflammatory, so, times. But what
everything is alsogreen.
becoming 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
So, iftraditionally
you look oncryopreserve that require
the protein level, it’s the minus 80 storage
same story. and another
TNF alpha, so, it’s one lyopreserve
upregulated that
in the now we
chronic can see
wound and
it on your shelf.
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
[25:05]
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
So, whatknow
us, they couldwhat
also to
influence
do whaton the wound
wound microenvironment?
will require And
in this particular here, you can see gene expression. In the
moment.
control wounds, this cluster, a red cluster represents a lot high level of inflammatory cytokines. If you increase
it,
And you can
this see here
graph this is example
was showing of upregulation
you also your tumor necrosis factor
of enzymes alpha
that present
are very in all chronic
protective wounds.
for other cells inIf the
you
start
wound. treatment with
They are viable lyopreserved
antioxidant amnion,
and they will you can
take oxygen see dramatic
radicals shift
and block from
their inflammatory
damaging environment
activity in the wound. to
anti-inflammatory, so, everything becoming green.
So, in summary, what I just described, you need to remember the main difference between cellular versus no
So, if you look on the protein level, it’s the same story. TNF alpha, so, it’s upregulated in the chronic wound and
cell product is in dynamic response to local wound environment. However, it’s not easy to store cellular
downregulated when you use viable lyopreserved amnion. What’s also happening that it’s not only
products. And now, a new method developed that will allow you to store living products on your shelf. So, it will
downregulation
eliminate need forof shipment
inflammatory cytokines,
on dry buteliminate
ice. It will you canneed
also see upregulation
to store of 80
it at minus anti-inflammatory cytokines
and it will eliminate need as
interleukin
and save you10.time
So, the viable
during thecells and placental
preparation membrane
and application ofare
thechanging
product. 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,
Now,they know
if you what
start to do where
thinking what wound
we arewill require
going, in this
I hope thatparticular
very soon,moment.
the amniotic membrane based on this new
technology, living lyopreserve tissue will come to your hospital, to your office, to your clinic.
And this graph was showing you also upregulation of enzymes that are very protective for other cells in the
wound.
So, whatThey
nextare antioxidant
step? Next stepand they
is to will take oxygen
understand and weradicals
believe and
that block their damaging
performance clinicallyactivity
will be in
thethe wound.
same
between cryopreserve and lyopreserve tissue. If you look way in the future and I hope it’s not very far away
So, in summary, what I just described, you need to remember the main difference between cellular versus no
future, I hope that one day that we all will be going to the grocery store to the pharmacy and grab from the
cell product is in dynamic response to local wound environment. However, it’s not easy to store cellular
shelves Band-Aid, but the active ingredient in this Band-Aid will be living lyopreserve stem cells. And I hope
products. Anditnow,
that I can see whena Inew
am method
still alivedeveloped
and that’s that
verywill allow you that
encouraging to store
suchliving products
treatment on your
already shelf. So,
will come it will
in the
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.
nearest future. Thank you very much.
TAPE ENDS - [28:39]
nearest future. Thank you very much.
TAPE ENDS - [28:39]

Cryopreservation of Cellular Therapies: Is There a Better Way

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PRESENT Podiatry Online CME & Conferences | Cryopreservation of Cellular Therapies: Is There a Better Way?

Residency Education / Wound Care

Cryopreservation of Cellular Therapies: Is There a Better Way?

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1. Review available cellular wound care products

Alla Danilkovitch, RN, MS, PhD

RN, MS, PhD

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nearest future. Thank you very much.

TAPE ENDS - [28:39]

nearest future. Thank you very much.

TAPE ENDS - [28:39]

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