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Liver-based in vitro modelling — Transcript

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0:00much time if you're
0:01this meeting is being recorded
0:04and i was about to say until you got the
0:07the notice um this meeting is being
0:08recorded so
0:10um i hope you don't mind um and the idea
0:12would be that we can put the meeting up
0:14the webinar up
0:28sorry i'm just gonna
0:30can i can i ask everyone to mute
0:32themselves please
0:34yeah
0:35thanks and and so
0:37so when i said today welcome to today's
0:39uh webinar from of into talks the
0:41specialty section of eurotox it is
0:44recorded so that you can put it on
0:46that you can watch it again
0:48on the eurotox website um well and today
0:51is a very special
0:52day uh
0:54it is my pleasure in fact to introduce
0:56our chair of the specialty section
0:58professor matthew finken
1:00um and he he will deliver today's
1:03webinar on liver-based and vitro models
1:05and i'm pretty sure that's why most of
1:07you are here it's a fascinating subject
1:10where there's a lot to cover
1:12mature finca is a full professor
1:14affiliated with the free university of
1:16brussels in belgium and he's also
1:18visiting professor at the university of
1:20sao paulo in brazil
1:22and he has a background in
1:24pharmaceutical sciences and specifically
1:26in vitro toxicology he's a european
1:29registered toxicology and the past
1:31president of the european society of
1:33toxicology in vitro or esteve who will
1:36have a conference later on this year and
1:39his uh research focus is situated in the
1:41field of mechanistic and in vitro
1:43modelling of liver toxicity specifically
1:46and that's why we asked him to uh to
1:48give this uh to give you this webinar
1:51he's also an editor of a couple of the
1:53toxicology journals and published more
1:55than 200 papers
1:57he's coordinated a number of european
1:59projects among which is the ontox
2:01project that quite a number of us are
2:03involved in which intends to set up
2:05animal-free approaches for predicting
2:07chronic toxicity induced by chemical
2:10compounds specifically also chemicals
2:12that are associated with liver toxicity
2:15and as i've said before
2:17for today's webinar my job will focus on
2:19liver-based and vitro models
2:21to be used for pharmaceutical
2:23pharmacological and toxicological
2:25testing purposes and
2:28the
2:28webinar will be recorded what we ask you
2:31to do is that you
2:33put your questions in the chat and then
2:35we can cover them after
2:37uh his uh around 30 minutes uh
2:4130 minute presentation
2:43and please feel free then to to speak up
2:46and meanwhile enjoy enjoy the lecture
2:49um and then for you mathieu thank you so
2:52much for joining and thank you for
2:53giving us this presentation um and i
2:56will now upload your
2:58um
2:59upload your presentation so that you
3:02can
3:03watch us
3:04okay thank you so much and actually it's
3:07it's me having to thank you because it's
3:10usually me introducing the speakers and
3:12sharing these kind of winners so
3:14it's nice for a change that somebody
3:16else does this so thank you so much
3:18so welcome everybody um indeed today i
3:21will talk about liver based and future
3:23models um we did have a couple of
3:26technical issues i was not able to
3:28upload at least not in presentation mode
3:30the presentation from my own computer
3:32that's why ninka kindly offered to do so
3:35from her computer and i suppose that we
3:37are trying to pull up the presentation
3:39now
3:40yeah so elaine can you make me presenter
3:44thanks
3:55sorry there is a technical issue it
3:57worked perfectly yesterday
3:59elaine could you please
4:02give me the rights ah perfect thank you
4:06okay here we go guys we we have it going
4:11and there we go
4:33yep so now i will ask for
4:37control
4:42okay let's see if this works yes it does
4:46hooray yes it does thank you so much so
4:48i assume that all of you can see my full
4:51screen now at least i can
4:53so please give me a shout or if you
4:56don't see it um
4:58all right so as already announced so
5:01today i will talk about liver based in
5:03vitro mode specifically
5:05bird
5:07okay
5:08wait all right this isn't i should not
5:10touch it
5:11yeah i think that's a good idea thank
5:14you so yeah indeed uh today i will talk
5:16about these liver-based and feature
5:18models that can be used for toxicity
5:20testing pharmaceutical testing but by
5:23extension you can you know you should
5:25consider this in a bit of a broader way
5:27uh the liver based and future models
5:29that i will present to you today in a
5:31nutshell at least while they are
5:33applicable for any kind of biomedical
5:36application if you will
5:37now before doing so i think it is
5:39important that we sketch a little bit
5:42the context of all of this and again i
5:45will focus here today on the use of
5:47these liver-based and virtual models for
5:50toxicity testing i think that you all
5:52know that before any chemical any
5:55chemical compounds can be placed onto
5:57the market like the european market that
6:00its safety towards men and his
6:02environment must be assessed and in
6:04order to do so this kind of a safety
6:06assessment or toxicological dossier must
6:08be made
6:10now this dossier contains i would say
6:12very obvious kind of of data like you
6:14know the physical chemical properties of
6:16the of the chemical um if that is
6:18available also epidemiological data even
6:21clinical data but more and more we see
6:23like this qsar data also popping up in
6:26this kind of dose so quantitative
6:28structure activity relationships
6:31now the reality is that for you know
6:34retrieving all of this
6:36of most of this information i should say
6:38that we still rely to uh i would say a
6:40big extent on animal experimentation so
6:43in vivo experimentation however more and
6:46more there is a tendency to also use
6:48alternative methods for that purpose and
6:50that can be in silico information so
6:53that's computational testing but in
6:55particular in vitro information
6:58and that is fully in line with the 3rs
7:00concept that i'm pretty sure that most
7:03of you are familiar with this so these
7:05three rs have been introduced already in
7:071959 by two uk scientists called russell
7:10and burch and as such calls for
7:11refinement replacement and reduction of
7:14animal experimentation and as you might
7:16know this three rs concepts forms the
7:19basis for very important at least in
7:21this context a very important piece of
7:22european legislation about the
7:24protection of animals used for
7:26scientific purposes
7:28which was introduced already in 1986
7:31but that was
7:32kind of revamped if you will about 12
7:34years ago so that there's the directive
7:372010 uh 63 as you might know
7:41now another again at least in this
7:43context relevant piece of european
7:45legislation is the legislation about
7:48cosmetics
7:49so there is already since many years
7:52very important uh legislation on
7:54cosmetics so the finalized or the final
7:56products but also their ingredients that
7:58was first established in 1976 so then it
8:01was a directive
8:03but you are probably aware of the fact
8:05that it has been very drastically
8:07changed several years ago about 13 years
8:09ago when it was turned into a regulation
8:11which means that it becomes immediately
8:13valid in all of the european member
8:15states
8:16and well as we all know according to
8:18this uh regulation we are no longer
8:21allowed in europe to use animals for the
8:23safety testing of cosmetic products in
8:26every region so that's a testing ban but
8:28there is also something called the
8:30marketing ban
8:31which
8:33let's say prohibits bringing onto the
8:35european market
8:37finalized cosmetic products but even
8:39they're ingredients that have been
8:40tested on animals outside of the
8:42european union
8:45and then a final piece of legislation
8:48which is at least seemingly a bit in
8:50contrast to all of this is the european
8:53regulation called registration
8:55evaluation authorization and restriction
8:57of chemicals may be better known as
8:59reach and as such reach demands the
9:02safety the knowledge dependent uh safety
9:05assessment of several ten thousands of
9:07chemicals that already are on the market
9:09so as you can imagine in order to meet
9:12the requirements that are imposed by
9:14reach well that's several of ten
9:15thousands of animals actually would be
9:17required but at the same time which also
9:20tries to at least introduce and to
9:22propose the use of these three r
9:24alternative methods
9:27so let's switch gears now uh let's talk
9:30about the boring part legislation so
9:32let's talk about the signs again
9:35so we will be talking in this
9:36presentation about liver based in retro
9:39models and for those not familiar with
9:41the deliver so what you see here on this
9:44slide on the left hand side above is
9:46what you would see if you would look
9:48through a microscope and you would have
9:50a look at liver tissue you would see
9:53several ten thousands of this hexagonal
9:55structure which is called the livo
9:57lobule which has a diameter of about one
9:59to two micrometer and in which cells are
10:02organized radially around the branches
10:04of the hepatic vein
10:07and at each of its six corners a livo
10:09lobule meets to other lobules which is
10:12called the portal triad and that is what
10:14is magnified on the left hand side below
10:18and as you can see this portal triad
10:20well it consists of branches of the
10:23hepatic artery the portal vein and the
10:25body
10:26as is the case for all of the tissues
10:28and organs in our
10:30organism while the liver also harbors
10:33several different cell types which is
10:35demonstrated to you on the right hand
10:37side below
10:39so the most important liver cells are
10:41both in function as we will see in a
10:43minute but also in number are these
10:45brown cells or at least brown in the
10:47figure here so these are the parenchymal
10:50cells called the hepatocytes which
10:52really can be considered as let's call
10:54them the workhorses of the liver if you
10:56will because they do take care of most
10:58of the liver specific functions and i
11:00will be coming back with this further on
11:03but nevertheless there are also some
11:04very important non-peripheral liver cell
11:07types present and these are also shown
11:09to you so for example these endothelial
11:11cells these blue cells at least in the
11:14figure of world blue cells that make up
11:16the lining of the blood flow which is
11:18called the sinusoids as you probably
11:20know and by doing so they also create a
11:23space of this
11:25so this is very important because this
11:26is also where the extracellular matrix
11:28component at least most of those will
11:30reside and i will be coming this back to
11:32this uh further in the presentation so
11:34please do keep this in mind
11:37and finally in the sinusoidal lumen
11:39there are also some other very important
11:41um let's say non-parenchymal cell types
11:44present like these kept for cells and
11:46dendritic cells that have an
11:48immunological function and i shouldn't
11:49forget of course about these stellar
11:51cells
11:52that actually reside in the space of
11:55this and they have a very important
11:57physiological function so what do they
11:59do they actually store vitamin a but you
12:01might know that in specific types of
12:03liver disease and toxicity like in
12:06fibrosis they adopt a very different
12:08phenotype and i also will exemplify this
12:10further on they lose their vitamin a
12:12content and they start to produce
12:14excessive amounts of extracellular
12:16matrix components
12:19so well the liver is a vital organ as
12:22you probably know you cannot live
12:24without a liver and indeed the liver
12:26performs a number of very important
12:27vital functions and as already alluded
12:30to the previous slide most of them are
12:32actually performed by the hepatocytes
12:35which as i call them the workforces of
12:37the liver
12:38so just a couple of important functions
12:40performed by the liver is it plays a
12:42central role of course in the metabolism
12:45of lipids carbohydrates um but also in
12:48the synthesis and the secretion of
12:50essential proteins think about blood
12:52coagulation factors albumin for example
12:55has already exemplified the liver also
12:57starts a number of vitamins and
12:59nutrients but from the toxicological and
13:02more specifically the
13:03pharmacotoxicological point of view the
13:06most prominent liver function is of
13:08course the biotransformation of these
13:10foreign chemicals which we collectively
13:12designate as xenobiotics
13:15and as a consequence of this function of
13:17course the liver also
13:19represents a primary target for systemic
13:22toxicity
13:23and for this reason it's not very
13:25surprising that over the last four to
13:28five decades a lot of attention has been
13:31paid and is still being paid to the
13:33development but also the further
13:35optimization of liver based in vitro
13:38models and as a result of all of these
13:40efforts going around in different labs
13:42all over the world there are two major
13:45groups of these liver-based individual
13:47models available today this is what is
13:49shown to you on this slide
13:51so the first group are the liver derived
13:53in vitro models and as the name already
13:55suggests they are directly derived from
13:57the liver and they can range from the
13:58whole isolated diffused liver to liver
14:01slices isolated primary parasites and
14:04their cultures liver derived cell lines
14:06all up to subcellular fractions and as
14:09we will see further in this presentation
14:11they all differ in complexity and hands
14:14and longevity
14:16the second group or the second class of
14:19the liver based infection models that
14:21have been introduced more recently so
14:24that is in the last two decades or so
14:26are the stem cell derived individual
14:28models in which stem cells from
14:30different origin
14:31like embryonic stem cells adult stem
14:33cells femoral stem cells and
14:35reprogrammed somatic cells can be
14:37differentiated in vitro in what we call
14:40hepatocyte-like cells we do not call
14:42them hepatocytes yet
14:45because there's a lot of discussion
14:46around this but i will be coming back to
14:48this in the presentation why we still
14:50prefer to call them hepatocyte like
14:52cells and not full-blown hepatocytes
14:55and actually this defines the contents
14:57of the
14:58remainder of this presentation so i
15:00would like to do
15:02is to give you an overview of each of
15:04these different models so how are they
15:06build up their advantages disadvantages
15:09and as you will see i will pay most of
15:11my attention to this first group
15:12delivered derived in virtual models for
15:14the very simple and pragmatic reason
15:16that these are the models that are still
15:18at least at this point being mostly used
15:22in regulatory settings and
15:23pharmaceutical settings and industrial
15:25settings in general especially compared
15:28to the steps of the future models
15:30so let's get started so we will be
15:32starting with the liver derived in vitro
15:34models and the first model here is
15:36actually not a true in vitro model it's
15:38what we tend to call an x-feeder model
15:41namely the whole isolated fuel sliver
15:44so to set up this model you need to
15:46first start from a freshly isolated
15:48liver that is then embedded in the kind
15:50of device that you can see here on the
15:53slide that's kind of mimics if you will
15:55the blood flow
15:57so the obvious advantage of this kind of
15:59x-vivo model is if you will is that you
16:01preserve the entire structure and the
16:03function as we discussed in one of the
16:05previous slide of the liver which makes
16:07it a very appropriate representation of
16:09the hepatic and fifa situation
16:12among the disadvantages is the fact that
16:14it's only suitable for small animals
16:16like rodents for example and that in
16:19general an isolated liver the whole
16:22isolated fused liver has a short
16:24viability of only a couple of hours but
16:27nevertheless it can be used in
16:29pharmaceutical industry for example for
16:31short-term kinetic studies
16:35the second example so remember that we
16:37are still in the first group delivered
16:38derived in virtual models and the second
16:41example here is a true in vitro model is
16:43what we call the liver slice model
16:46so here again you need to start from a
16:48freshly isolated liver or at least
16:50freshly isolated liver tissue in which
16:52as you can appreciate from the images on
16:55the upper panel
16:57well that you first need to you know
16:59isolate a kind of a fragment with a kind
17:01of a corkscrew if you will
17:03which is then further processed by means
17:05of the slicer using or yielding i should
17:08say slices of about 100 to 250
17:11micrometer in thickness that are then uh
17:13being incubated as you can see from the
17:16panel below in a typical cell culture
17:20dish in the presence of you know any
17:22kind of cell culture medium
17:25so here again is the advantage that you
17:27preserve of course not the entire liver
17:30structure but at least the lobular
17:31structure and function so remember this
17:33hexagonal structure
17:35and as a consequence again that you have
17:38kind of a good representation of the
17:40hepatic in fever situation
17:43among the well let's call them
17:44disadvantages is the fact that the setup
17:47of this kind of liver slice model of
17:49course requires high technical skills as
17:52you probably already understood from the
17:54figures shown on this slide
17:56and again that the viability is pretty
17:58short although somewhat better if not to
18:00say quite better compared to the whole
18:02isolated produce ever so in the early
18:04days of the liver spices you can only
18:06keep them alive for five to six hours
18:09but if you look in literature today
18:10there are already protocols in which you
18:12can keep them for a couple of days
18:15and again uh this liver size model is
18:18quite popular especially in
18:19pharmaceutical industries so for
18:21instance if you want to elucidate the
18:23metabolite pattern of a candida drug so
18:25metabolic profiling or if you want to
18:27shed new light on you know the mechanism
18:30of action of be it a drug or a toxicant
18:36okay so the third example in still the
18:39first group of the liver derived in
18:40vitro models or what we call uh the gold
18:44standard in vitro model so this is you
18:47know what is seen as the most commonly
18:49used in vitro model in the field of
18:51liver based in mutual modelling namely
18:53the use of primary hepatocytes and
18:56preferably human primary hepatocytes
19:00now they are typically isolated from
19:02freshly isolated liver using this two
19:04steps collagenous diffusion technique
19:06using the device which is schematically
19:08shown to you on this slide
19:10by the way the figure that you see on
19:12the uh
19:14left hand side is a figure taken from
19:16our own lab but i still used to do
19:18some bench research uh in the lab
19:21but this is demonstrated here with a rad
19:23liver we don't do this anymore in our
19:25lab we are now working with human
19:27hepatocytes which by the way we don't
19:29isolate ourselves but this is still a
19:31common technique so
19:33to be isolated from let's say rodents
19:36liver and the technique that is
19:39still being used and that has been
19:41introduced in the
19:4270s if i'm not mistaken of the previous
19:44century the two steps collagenous
19:46confusion technique and as the name
19:47suggests
19:48this consists of two steps the first
19:50step being the perfusion of the freshly
19:52isolated liver with the calcium-free
19:54buffer so the idea here is to abolish a
19:56cell cell context you probably still
19:58know from the cell biology course that a
20:00lot of cells sell complex so think about
20:02these caterines and academies while they
20:05fully rely on the presence of calcium so
20:07the ideas if you take away calcium you
20:10basically also will mess up all the cell
20:12cell contacts
20:14the second step is then to further
20:16diffuse the liver with collagenase which
20:17is an enzyme that abolishes cell
20:20extracellular matrix complex
20:22so i think you got the rational by now
20:24of this technique so you want to break
20:26up all of the cellular contexts
20:28so in general at least if you apply this
20:30for example to a red liver well this
20:32will yield between 200 and 300 million
20:35cells of which most are hepatocytes with
20:38a viability of at least 80
20:40and although this is a technique that is
20:42still being you know abundantly used all
20:45over the world the main disadvantage of
20:47this two-step splashiness perfusion
20:49technique is that it elicits a so-called
20:52de-differentiation process that is
20:55pursued upon further cultivation of the
20:57hepatocytes so after the isolation
21:00and which is the very reason for which
21:02conventional cultures of freshly
21:04isolated primary hepatocytes and by
21:06conventional i mean just seated on a
21:08plastic culture dish well the reason
21:11that they cannot survive for more than
21:13two days so to say and this is really
21:15due to this d differentiation
21:17and actually what you see here on this
21:19slide is of course an oversimplified
21:21scheme of how this d differentiation
21:23process looks like at the molecular
21:25level
21:26so in the normal liver of course in vivo
21:29uh well the cells
21:31are in intimate contact with each other
21:34of course but because of this two steps
21:36collagenous perfusion technique you mess
21:38up all the cellular context so you force
21:40the cells to re-enter the cell cycle and
21:43this is associated with the induction of
21:46both an inflammatory response but also a
21:48cell growth response which is as you can
21:50see from this fight
21:52mediated by nf capability which is a
21:55transcription factor as you know and
21:57then a
21:58mitogen activated protein kinase which
22:01is a cell growth mediator i won't go
22:03into the details here but what this
22:05actually does is that it uses a lot of
22:08transcriptional changes
22:10all that uh you know or at the expense
22:12of the functional differentiated status
22:15so this is what we call d
22:16differentiation
22:17and the unavoidable consequence of this
22:20is the onset of spontaneous cell death
22:22mainly by apoptosis and again this all
22:25happens in one or two days after
22:28cell isolation and cultivation and that
22:31is again the reason why you cannot keep
22:33these cultures for longer than two days
22:35of course
22:36in course of time a lot of efforts have
22:39been focused on trying to reduce and
22:41counteract this de-differentiation
22:43process
22:44uh with the idea of setting up primary
22:46hepatocyte cultures that can be used for
22:48longer testing purposes so think about
22:50weeks or even months
22:52and we do have quite some tricks
22:55available if you will to achieve this
22:57and they are mainly if not uniquely
22:59based on trying to mimic again or to
23:01reestablish the natural in vivo uh micro
23:05environment so to fully understand what
23:07we call this
23:08anti-differentiation strategies we need
23:11to have a look again to the natural
23:13micro environment of the hepatocytes so
23:15we already touched on this in one of the
23:18first slides but
23:19i would like to come back to this again
23:22so in the natural environment of the
23:24hepatocytes there are at least three
23:27factors that keep the hepatocytes in
23:29their fully functional and
23:31differentiated status and this is what
23:33exemplified or demonstrated to you on
23:35the right hand side
23:36so first of all as i already mentioned
23:38we have all of these extracellular
23:40matrix components that mainly reside in
23:42the space of this that give the
23:44three-dimensional configuration
23:46to these hepatocytes which in turn is
23:48indispensable for the hepatocytes to
23:50perform their functionality so that's
23:52the first factor
23:54the second factor are you know hormones
23:56metabolites whatever kind of soluble
23:59mediators that are present in the blood
24:01of course that directly affects
24:03positively affect the functionality of
24:05the infrastructure that's the second
24:06factor and then the third factor or
24:08direct contacts between neighboring
24:11hepatocytes or even indirect contacts
24:14between the hepatocytes and any of the
24:15non-parenchymal cells that also have an
24:18effect on hepatocyte functionality
24:21now each of these three factors forms
24:24the basis for one of the three what we
24:26call classical
24:28anti-differentiation strategy so kind of
24:30strategy to re-establish the individual
24:33micro environment
24:35in an attempt to keep the cells
24:37functional for longer periods of time in
24:39culture
24:41so first classical anti-differentiation
24:44strategy is pretty straightforward it's
24:46the addition of what we call
24:47differentiation promoting compounds to
24:49the cell culture medium and these can be
24:51of course physiological factors like
24:53hormones insulin vitamins like vitamin c
24:56but also non-physiological factors think
24:58about phenobarbital which is a drug that
25:00induces biotransformation capacity but
25:03even things like dmazo which is a
25:05solvent but that has at least in low
25:08concentrations quite beneficial effects
25:10on hepatocytes
25:11and of course all of this can be done in
25:14a static culture system what you see on
25:16the left hand side above so the
25:18monolayer classical monolayer culture
25:20and by the way the microscopic picture
25:22that you can see here on this site is a
25:24picture taken from our own lab already
25:26many many years ago so freshly isolated
25:29rather parasite cultured for two days in
25:32a classical monolayer culture but of
25:34course you can make this a more dynamic
25:36system if you will like the fusion
25:38culture system in which you have a
25:40continuous renewal of all of these uh
25:43cell culture media which is pretty
25:45expensive by the way but that of course
25:47much more reflects the individual
25:48situation so that is the first trick if
25:51you will the first anti-differentiation
25:53strategy
25:55the second one is to reestablish uh cell
25:58cell context which of course will fully
26:00messed up during the two steps
26:01collagenous perfusion technique by
26:04seeding the cells the hepatocytes
26:06together with another cell that saw a
26:08co-culture fuel and the second cell type
26:11can have an hepatic origin like red
26:13liver epithelial cells of any or any of
26:16the non-parenchymal cells like hyper
26:18cells or it also can be a non-hepatic
26:21kind of cell like fibroblastic cell
26:24lines or stem cells or whatever
26:26by the way the microscopic picture that
26:28you see on this slide here that is a
26:31picture of uh cultures of freshly
26:34isolated primary parasites taken if i'm
26:36not mistaken at day seven of cultivation
26:38and you see these white cells which are
26:40the hepatocytes and the more black cells
26:43or these red liver epithelial cells you
26:45can already tell you know just by doing
26:47the scope culturing that hepatocytes are
26:49in a much better shape than they would
26:51be if they would be cultivated alone
26:54and here again there's a kind of an
26:56alternative an improved alternative
26:58that's a steroid culture so you can do
27:00all of these co-culture
27:02uh you know techniques in a
27:04three-dimensional configuration so these
27:06forwards that even better reflect the
27:08foresight situation
27:11so that was the second classical
27:13anti-differentiation strategy the third
27:15one is the re-establishment of cell
27:17extracellular matrix context which can
27:20be achieved by seeding the cells the
27:22freshly isolated hepatocytes on a single
27:24layer of extracellular matrix components
27:27or between two layers of extracellular
27:29matrix components which produces what we
27:32call the sandwich culture system
27:34so the scaffolds that you use the
27:36extracellular matrix scaffolds can be of
27:38different origin like of natural origin
27:41like physiological ones like collagen
27:43for example also non-physiological ones
27:45you might be familiar with battery
27:46matrigel which is extracted from a mouse
27:49tumor but also several labs all over the
27:51world have introduced synthetic
27:54scaffolds like polymers and nano fiber
27:56mattresses and by the way the picture
27:58that you see here the microscopic
28:00picture is a picture taken at day 14
28:03already of cultivation of primary uh you
28:06know red hepatocytes that have been
28:09cultured so for two weeks between two
28:11layers of this collagen one so this is a
28:15sandwich culture system and you can
28:17really see that the cells still are in a
28:19good shape
28:21now a more recently introduced
28:23alternative to this is the use of the
28:25decelerized liver so here and this is
28:28exemplified again by means of a red
28:30liver you will perfuse the freshly
28:32isolated liver by means of a detergent
28:35and you will end up it's pretty neat
28:37actually so in one hour you will end up
28:39with his liver as you can see on the
28:41right hand side below which is fully
28:42transparent so we got rid of all of the
28:44cells
28:45but you still have the entire
28:46extracellular matrix backbone including
28:49also growth factors and as a matter of
28:51fact this can be used then to be
28:53repopulated with stem cells which i will
28:56uh talk about in the second part of the
28:58presentation
29:00now although promising results have been
29:02obtained by following these three what
29:04we call classical anti-differentiation
29:06strategies or combinations of those they
29:08have only been successful in part and
29:10the reason for this is that they
29:12typically you know are focused on
29:15counteracting the consequences of the
29:17differentiation without acting on the
29:19true causes
29:21now over the past few decades especially
29:23with the advent of omix technologies we
29:25now have a very good idea about the
29:27actual mechanisms the causes of the
29:29differentiation and we know that there
29:31are two main mechanisms involved here so
29:33the first ones are the interaction of
29:35transcription factors with specific
29:38sequences and the promoters of the genes
29:40that are responsible for coding
29:42liver-specific proteins
29:45this is the classical cis-trans
29:46mechanism of course and among these uh
29:49transcription factors as we will see
29:51further on or for instance the liver and
29:53retranscription factor so that is the
29:55first mechanism the second mechanism is
29:57epigenetic mechanism so that is let's
30:00say changes in the chromatin structure
30:03which in turn have an effect on
30:04transcriptional activity and you
30:07probably all know that there are three
30:08main let's say determinants of the
30:10epigenome namely dna methylation histone
30:13modifications and micro rna species so
30:17each of these two mechanisms from the
30:19basis for what we call the new the novel
30:21anti-differentiation strategies
30:24and the first one again is pretty
30:25straightforward is genetically altering
30:28primary hepatocyte culture so the idea
30:30here is that during this two steps
30:32collagenous perfusion technique has
30:34already explained well there is a
30:36deterioration of the production of these
30:39liver and rich transcription factors so
30:41you want to restore this balance you
30:43want to increase the production of these
30:45transcription factors in vitro in
30:47culture and you can do this in many ways
30:49so for example by bringing them to
30:51stable over you know expression if you
30:54will which is exemplified here by means
30:57of this transcription factor called hnf
30:59for alpha hepatocyte nuclear factor for
31:02alpha
31:03the second
31:05new anti-differentiation strategy is the
31:08epigenetic alteration of primary
31:10parasite cultures and actually that was
31:12the topic of my own phd project already
31:14more than 20 years ago so here again the
31:17idea is that during hepatocyte isolation
31:20so the two-step splashiness perfusion
31:22technique and the cultivation
31:24well you mess up the uh epigenome if you
31:27will so you want to you know you want to
31:29restore this and you can do this by
31:31adding these epigenetic modifiers to the
31:33cell culture medium like trichostatin a
31:36tsa so the idea is here is that you will
31:39open up the chromatin structure in order
31:42to make them make it more accessible for
31:44transcription factors to bind and thus
31:46to produce liver specific proteins
31:50okay that was a lot of attention being
31:52paid to primary hepaticides but i did
31:53this on purpose because as i already
31:56mentioned before this is still being
31:57considered the gold standard kind of
32:00model if you will but we are still in
32:01the first group namely in the liver
32:03derived in victron models and another
32:06example here are the liver derived cell
32:08lines
32:09there are several possibilities here so
32:11for example these uh cancer-derived cell
32:14lines as you might know like have g2
32:16and well the maybe better known hip-rg
32:19cells and then the second kind of liver
32:22derived cell lines or the cell lines
32:24that basically started off as being
32:26usual common primary parasites but that
32:29have been genetically modified in such a
32:31way that they continuously grow like the
32:33wp
32:34f3 for for at the liver epithelial cell
32:38by the way the microscopic picture that
32:40you see on this slide are hip rg cells
32:42and for those familiar with hepatocytes
32:44i hope you agree with me if i would say
32:46that they pretty well resemble at least
32:48phenotypically hepatocytes
32:50the advantage as you know of cell lines
32:52in general is that you virtually have an
32:54unlimited cell supply and that they are
32:56very easy to use so you don't have to
32:59every time isolate them again like you
33:01have to do for prime hepatocytes among
33:04the disadvantages but this mainly holds
33:06for the liver derived ones is that they
33:08have like uh well very frequently
33:10apparent functionality specifically for
33:12have g2s and sometimes all genetic
33:15instability but nevertheless they are
33:16very popular also in industry so they
33:19can be used for toxicity studies and
33:20mechanistic studies
33:23and then the final example in the liver
33:24derived in digital model group are these
33:26subcellular fractions and here again
33:28there are different kind of tools if you
33:30like this s9 fraction which can be
33:32obtained by centrifugating a liver
33:34homogeneous at 9000 g or maybe better
33:37known the microsomes which can be
33:39produced by spinning a liver homogeneous
33:41at a much higher velocity so this
33:43hundred thousand g
33:45um well the advantage specifically
33:47holding for microsomes is that there are
33:50keep their functionality especially
33:52phase one by transformation capacity
33:54which again makes them very popular in
33:56pharmaceutical industry and that they
33:58are very easy to find and to buy and to
34:01make yourself actually if you have the
34:02appropriate centrifuge in your lab
34:05disadvantages well i think you can
34:07already tell by just looking at this
34:09microscopic picture on this slide this
34:11is actually a microsomal structure
34:14and of course this is very far away from
34:16this complex in vivo relevant
34:19architectural delivery that we discussed
34:21at this part of the presentation making
34:23all of this already uh also very limited
34:26in terms of interior relevance
34:28but as i mentioned before nevertheless
34:30they are very popular in industry
34:34all right so that was the first group of
34:36the liver basin virtual models so the
34:38liver derived in vitro models in the
34:40final part of the presentation i would
34:42like to shortly touch upon this more
34:44recently introduced group of liver and
34:47virtual models namely the stem cell
34:49derived in future modes and before doing
34:51so we need to go through uh some basic
34:53features some basic terminology
34:55pertaining to stem cell biology
34:58so um i assume that most of you know
35:01that stem cells are characterized by two
35:04features so they all display kind of an
35:07extent of cell renewal which means that
35:09they have the capacity to undergo cell
35:11divisions in an undifferentiated state
35:14and the second characteristic is that
35:16they also um have a kind of plasticity
35:19or a potency which means that they can
35:21differentiate into specialized cell
35:23types and as a matter of fact there are
35:26different possibilities and different
35:27scenarios that people regarding the
35:29plasticity of these stem cells so
35:31embryonic stem cells can be toney potent
35:33or blurry food which means that they can
35:35you know
35:36generate into embryonic and extra
35:39embryonic cell types or each of the
35:41three germ layer cell types as is
35:43exemplified here on this slide
35:46but then for the more adult stem cells
35:48well they can be multiple b bone or
35:50unibone which is more restricted in the
35:52sense that they can differentiate into a
35:54family of related cell types two cell
35:57types or even one cell type
36:00now i think it was back in 2007
36:04a japanese group was able for the very
36:06first time to induce the state of
36:08pluripotency in an adult cell so not in
36:11an adult stem cell but in a full added
36:13adult cell and actually they received
36:15the nobel prize for this so really
36:17showing how important this uh discovery
36:19was
36:20so there's actually this induced
36:22prerequisite stem cell uh technology if
36:24you will is schematically here on this
36:27slide
36:28i don't have the time to go through it
36:29in details but basically
36:31uh it comes down to let's say
36:34rendering an adult cell and this is
36:36exemplified here by an adult skin cell
36:39into a kind of a naive state if you you
36:41will make the cell a bit of stupid if
36:43you will so by transducing with
36:45pluripotency genes which are exemplified
36:48here
36:49so this will allow you then to you know
36:51create any kind of cell if you will and
36:54of course this opens a lot of
36:55possibilities for regeneration
36:58regenerative medicine but in particular
37:00also for in vitro toxicology and this is
37:02why that's also very
37:04important for you know us as in feature
37:06toxicologists
37:09now there are many strategies to
37:10differentiate stem cells of any kind
37:12into what we call hepatocyte-like cells
37:14and i already alluded to this so why do
37:16we call this hepatocyte-like cell
37:19now although a lot of claims are being
37:21made in literature there's not any of
37:24these approaches that allow to you know
37:27generate let's say cells that fully
37:30reflect the full repertoire of all of
37:32the in vivo relevant you know hepatocyte
37:36functions and that is why we do not call
37:37them yet hepatocyte but hepatocyte like
37:39so i'm pretty sure that people will
37:41disagree with me but this is just a
37:43reality this is just how it is because
37:45this is still a growing field if you
37:47will
37:48but as i mentioned a lot of people are
37:50working on this and there are a lot of
37:52strategies to differentiate stem cells
37:54into parasite-like cells they are listed
37:56here they may sound very very uh
37:59familiar to you because they're actually
38:01almost identical to what we have called
38:04the classical anti-differentiation
38:06strategies for primary parasites again i
38:08don't have the time to go through all of
38:10them in detail but i would say a most
38:12commonly followed strategy here is the
38:14exposure of stem cells to cytokines
38:16growth factors vitamins and
38:18corticosteroids and there are two types
38:21of exposure if you will so either you
38:23can expose the stem cells to all of
38:25these factors at the same time which is
38:27called a cocktail exposure technique or
38:29you can expose them to a sequence of
38:32these factors which is in line within
38:34vivo liver development and actually on
38:36this slide you can see again an
38:38oversimplified scheme of how individual
38:40liver development looks like
38:43so you can see it starts with endodermal
38:45cells that gradually develop into
38:47full-blown adult hepatocytes on the
38:49right-hand side
38:50what you can also see is that this is
38:52associated with a specific need at
38:54specific time points for specific
38:56factors like very early on you need a
38:58lot of fgf so fibroblast growth factor
39:01while further in the process you have uh
39:04well the cells actually need these
39:06glucose for steroids and this also goes
39:08hand in hand with the you know
39:11expression at specific time points or
39:14specific factors like for example very
39:16early on you have a lot of expression of
39:18alpha beta protein while this full-blown
39:21hepatocytes are typified by a lot of you
39:24know production of algae
39:26now this basic knowledge has served as
39:29the let's say basis
39:31uh for a protocol that is still being
39:33abundantly used all over the world to
39:36differentiate stem cells in what we call
39:38hepatocyte-like cells and this is just
39:40an example of how such a protocol looks
39:43like so it's not set in stone it's just
39:45an example but what they all have in
39:47common is that there are two main steps
39:49in these protocols so there is the
39:51expansion step which is based on the
39:53cell renewal potential of the stem cells
39:57and then there is a differentiation step
39:59which is of course fully relying on the
40:01plasticity of cells so again you expose
40:04the cells typically to all of these
40:06factors in a specific sequence in line
40:08with in vivo liver development and you
40:10can even combine this with an epigenetic
40:12strategy like for example this tsa as
40:14you can see from this protocol this
40:16epigenetic modifier so i would like to
40:19give three examples of this um so first
40:22example is retinas and primal progenitor
40:25cells that have been differentiated into
40:27hepatocyte-like cells
40:29uh using both the sequential exposure
40:31technique so where you expose according
40:33to a vivo liver development or according
40:35to a cocktail exposure technique where
40:37you're exposed to all of these factors
40:39at the same time
40:41again because of thumb restrictions i
40:43cannot go into all of the details but
40:45typically all of these readouts or the
40:47evaluation of these protocols is based
40:49on remember the individual development
40:52scheme
40:52on a number of these factors that are
40:55being expressed at specific steps in
40:58this uh procedure and in female level
41:00development so for instance the
41:02production of these liver and rich
41:03transcription factors by transformation
41:06enzymes but the most convincing as a
41:09parameter as an in vitro toxicologist is
41:12always the inducibility of
41:13biotransformation enzymes
41:15what you can see here is that surprise
41:17surprise the best results were of course
41:20of course obtained uh if you perform the
41:22sequential exposure technique as opposed
41:24to the cocktail one because you simply
41:26follow nature so it makes absolutely
41:28sense
41:29and just to show that this is also
41:31applicable to other species so human
41:33mesenchymal progenitor cells so here
41:36uh the combination was done of the comic
41:39sequential exposure technique and that
41:41combined with an epigenetic strategy and
41:43here again you could even obtain
41:46better results and then the final
41:48example is again red cells so here again
41:51sequential weather are not combined with
41:54an epigenetic strategy and what is
41:56actually shown here is that not only all
41:59of these liver you know specific factors
42:02are much better expressed if you combine
42:04sequential exposure together with an
42:06epigenetic strategy but they also become
42:09expressed at earlier time points and for
42:12instance have a look here at this hnf3
42:14beta which is being uh detected at day
42:1818 of cultivation and the common
42:19sequential exposure technique while you
42:22can already pick it up at day three if
42:24you combine this with an epigenetic
42:25strategy well you can say so what
42:28but for those of you who work with cells
42:31you know that this is pretty expensive
42:32business so if you can actually reduce
42:35the cultivation fund you will also save
42:37a lot of money of course
42:39okay to end up i would like to very
42:41shortly bring up a number of recently
42:44introduced individual models
42:46and basically what they do is to combine
42:48everything that you heard previously in
42:50this presentation so for example stem
42:52cell derived hepaticide lifestyles that
42:54are cultured in a three-dimensional
42:56configuration and a pop culture
42:58and well this is becoming a very
43:00interdisciplinary kind of effort so this
43:03is uh typically being combined with very
43:05fancy bioengineering stuff so you can
43:08you know play the cells on this chip as
43:10you can see on the left hand side or in
43:13a through bioreactor as you can see on
43:15the right hand side
43:16and uh well especially for the chip
43:18format you can combine this with
43:20microfluidics so in order to mimic the
43:22blood flow and even if you want to use
43:24this for toxicity testing you typically
43:26have this inbuilt sensor so you can
43:28really follow
43:30all the functionality and toxicity
43:31parameters in real time
43:34so some people even take it a step
43:36further they create a body or even a
43:39human on a chip and the rational of
43:40course here is that the liver is not the
43:43only target
43:44for toxic
43:45compounds
43:46basically all of our organs
43:48can serve as a target for toxicity so
43:51what you can do here is to combine stem
43:54cells you know differentiate it into
43:56that specific uh cell type or the
43:59primary cells of each of these organs
44:01and combine them on a chip and again to
44:03be uh also combined with microfluidics
44:06and inbuilt centers
44:08and this ladies and gentlemen is
44:09actually not uh anymore what you see in
44:13movies this is actually becoming reality
44:15is the bioprinted and 3d printed
44:17delivery of course all of this is now
44:19being fully let's say
44:21investigated for medicinal and medical
44:24kind of purposes but once this will be
44:27up and running this opens a lot of
44:29perspective for uh in vitro toxicology
44:32as well
44:34well at the end of the presentation i
44:35think
44:36it would be reasonable to state that
44:38today we have liver derived in virtual
44:40models that unfortunately most of them
44:43can only still be used for short-term
44:45purposes and as i explained during this
44:47presentation the main reason for this is
44:49that they are still making use of what
44:51we call the classical
44:53anti-differentiation strategy so they
44:55counteract the consequences but not the
44:58causes of key differentiation but of
45:00course if we want to use them for longer
45:02testing schemes so for weeks even months
45:05well then we have to really let's say
45:08focus on this causes of the
45:10differentiation as i exemplified here
45:12with this epigenetic strategy and then
45:15for the stem cell derived in future
45:16models well today we still have a lot of
45:19you know variability in the protocols
45:22and the sources
45:23yielding what we call hepatocyte-like
45:26cells not always with the highest purity
45:28as i mentioned this is a growing field
45:30we make a lot of progress uh it's going
45:33very fast in the stem cell field and i'm
45:36pretty sure in a couple of years we will
45:38have fully standardized protocols
45:39especially for hepatocyte-like cells
45:42yielding and i'm pretty sure convinced
45:44of this not only functional
45:45hepatocyte-like cells but full-blown
45:47hepatocytes with a lot of purity
45:50now all of this is of course very
45:52ambitious we are not there yet and an
45:55absolute requirement to achieve all of
45:57this and to be applied let's say in
45:59regulatory and industrial aesthetics
46:01is that we still need to have a lot of
46:03basic science research scientific
46:05research and of course being an academic
46:08this is exactly what you want to hear
46:10because this opens a lot of perspectives
46:12for doctoral and post-doctoral research
46:14so this is also an open invitation of
46:16course to people attending today to
46:19spend your career on helping to you know
46:22advance all of this so i'd like to thank
46:24you very very much and uh before we move
46:27into questions hopefully there will be
46:29some questions i would like to advertise
46:31already our next um
46:33webinar and here we will be switching
46:35roles so i will be sharing and then
46:37ninka will give a very exciting webinar
46:39on next generation physiologically based
46:41financing modeling which will be taking
46:43place the 25th of may well the same
46:46procedure as for the other uh into talks
46:49webinars in the sense that registration
46:51will be open tomorrow or the day after
46:54uh it's on a first-come first-served
46:56basis so please don't um you know
46:59postpone registration so it is thank you
47:02so much and i would be happy to uh
47:05answer any question you might have and i
47:07will be giving control back to you unica
47:11oh great thank you so much um
47:14and i'll stop sharing my screen there we
47:17go
47:18thank you so much uh michael for this
47:20very clear overview i i really enjoyed
47:23it the the opportunities like you say
47:26for particularly this for the liver
47:28toxicology field
47:29and what i would suggest to do now is
47:31that everyone with questions please
47:34throw them into the chat
47:36um and then
47:39and then go ahead i'll read them for you
47:41if you don't
47:42dare to speak out yourself
47:44uh if not of course i will uh
47:47i'll gladly ask them is there a first
47:49question
47:57not yet from what i said well i i always
47:59do have questions
48:00[Music]
48:02um so i i really what i really enjoyed
48:04is a clear overview of the different
48:06different types of models that you have
48:08and you started off with the liver
48:09slices and you specific specifically
48:12mentioned um that the um
48:15that you had the
48:17uh that these liver slices have their
48:19structural lobule
48:22structure still in place and that you
48:24know the next one would be your stem
48:25cells but they both seem to have a
48:27pretty short lifetime or in what cases
48:31would you use the the the slices as
48:34opposed to the hepatocytes so when is
48:37that lobular structure in the type of
48:39toxicity test that we do now
48:41uh really important
48:43so i'm not pretty sure if i fully get
48:46your question but would the question be
48:48so for what specific applications you
48:50would use a liver slice model and for
48:52which ones you would use stem cells or
48:54in general oh just in general it's like
48:56i think first hepatocyte your primary
48:58parasites and the
49:00and the slices um
49:03it really depends on the application um
49:05so for example as as you know we and my
49:08team in brussels we have a specific
49:10interest in so-called polystatic liver
49:12damage so which is due to power asset
49:14accumulation and there we know that the
49:17gold standard model is well primary
49:20parasites but even this hipaa are g
49:22cells so what we tend to advise not only
49:25for testing called static liver damage
49:27but in general for liver based and vitro
49:29modeling and using these models for
49:32predicting toxicity is try to combine
49:35two models why because they all have
49:37their disadvantages as i explained
49:40during this presentation and they all
49:42have their strengths so in order to
49:44somehow compensate for that um it would
49:48be good if you can use two models in
49:50parallel but again the model should be
49:52fit for purpose so you can not just
49:54randomly pick like a model if you want
49:57to test something what is again
50:00generally considered as the gold
50:01standard is primary hepatocytes why is
50:04that is because purely on the genotype
50:07and purely on the functionality they do
50:09reflect you know um
50:12to the maximum feasible extent in vivo
50:15capacity and that's the only reason but
50:18that does not mean that primary
50:20parasites are the you know let's say the
50:23only kind of in vitro model that should
50:24be used for all applications for liver
50:27slices for example to come back to your
50:29question
50:30if you look in literature it's being
50:32used for very specific uh kind of
50:34application like for testing cholestasis
50:37also sterosis fat accumulation and even
50:39for genotoxicity i see a lot of reports
50:42on that but apart from that you don't
50:44see it typically for many other kinds of
50:46applications so it really needs to be
50:48seen on a case-by-case basis
50:51yeah no that's a very very fair answer
50:53thanks uh thank you michelle then we
50:55also have a question uh by barbara
50:57barrett from boss f um she and it's an
51:00interesting question um
51:02if she asks to
51:03in your perspective what is the most
51:05important what are the most important
51:07steps um in the stem cell based models
51:10to get regulatory acceptance
51:12so
51:13specifically given accordance might be
51:16not easy because it's such a complex
51:18differentiation process
51:20yeah well first to be um to be clear
51:24here i mean there are many areas in
51:26which stem cells are what are already
51:28much more advanced than the hepatocytes
51:30feel like the neurotoxicity the cardio
51:33field i mean there there's no question
51:34about this even it's starting to be
51:36accepted well i'm thinking about the
51:39oecd um case now for neurotoxicity
51:42testing where we also have stem cell
51:45based models for hepatocytes we are far
51:47from being there
51:48so what would be needed is that we have
51:51a consensus of what would be the
51:53acceptance criteria
51:55i'm an editor of a number of journals
51:57and
51:58i need to be careful what i will be
52:00seeing right now so i see a lot of
52:01papers passing by and i'm sorry for the
52:04wording there was a lot of crap being
52:06published or at least trying to be
52:07published also for stem cells why
52:10because people do use the parameters
52:12that suits them best
52:14you know so for example it's very easy
52:17to show that something will be expressed
52:20at the messenger rna level but that does
52:22not mean that it is functional
52:24also what do you use as a benchmark very
52:27frequently if people use stem cells and
52:29they differentiate into hepatocyte-like
52:31cells they use as a comparison have g2
52:34cells but these are liver cancer cells
52:36and of course i mean if you compare with
52:38something that is sub-optimal it will
52:41not be very difficult to have like a
52:43better performance so to answer
52:45barbara's question i think we first need
52:47to in the scientific and the
52:49academic well scientific world as such
52:51you need to agree on this
52:53and only if we then have a full
52:55consensus and sounds criteria that are
52:59generally accepted then we can think
53:01about having regulatory acceptance but
53:04this is not yet for tomorrow i would say
53:06we are not there yet so again here there
53:09is a lot of basic scientific research
53:11that is still required
53:13great then i've got one last question
53:15from you and that is from johannes sir
53:17swimming
53:18jonas go ahead and just speak up yeah uh
53:21i'll use that opportunity to just speak
53:24as a person here um
53:25in comparison to for example the in
53:28vitro field in the cosmetics testing
53:30where you have a quiet plethora of
53:32different essays for fulfilling more or
53:34less the same purpose i would assume
53:36that the hippa
53:38yeah that the invictus world for
53:41hepatotoxicity will run a bit similar to
53:44that so wonder if you have an idea or a
53:46set of
53:47expectations which those esses need to
53:50be fulfilling of morphological
53:52characteristics metabolic
53:54characteristics transcriptional pattern
53:57uh can you envision a set of
54:00characteristics which would allow us to
54:03identify different assets from different
54:05manufacturers uh as a
54:08equivalent in vitro toxicity
54:10model for um yeah
54:12for the future
54:14yeah well again i'm not pretty sure if i
54:16fully get your question but to compare
54:19with the cosmetics field i mean we are
54:20talking about different types of
54:22toxicity that's more acute toxicity
54:24topical toxicity while the liver is
54:26typically systemic toxicity so we
54:28unfortunately don't have any in vitro
54:32at least not fully validated alternative
54:34methods available there
54:36if that were to be your question um if
54:39your question would be like
54:41when
54:42can you be pretty sure that your in
54:44vitro model is reliable and can be used
54:46for toxicity testing actually a couple
54:49of years ago we published on this uh
54:51because it was a question that we very
54:53regularly got uh i can refer to that
54:55paper it's published in arkansas
54:58toxicology together with john henchler
55:00so which is the uh associate now the
55:02editor in chief of archives of
55:04toxicology we there came up with a
55:06number of phenotypically and functional
55:08parameters that should be met by a
55:10liver-based infection model in order to
55:13be
55:14pretty sure that it is fully reliable
55:16and that you can use it for reliably
55:18predicting systemic repeated dose
55:20toxicity
55:22but again i mean there are of course
55:24some commonalities with what is being
55:26done in the cosmetics field but we are
55:28talking about very different scenarios i
55:30mean it's acute toxicity topical
55:32toxicity while the liver is more
55:34systemic toxicity
55:37yes of course yeah that answers my
55:39question i'm just uh yeah took the
55:42cosmetic field as a good example because
55:45we learn a lot with examples which are
55:47in use especially in in regulatory
55:49context thank you absolutely
55:52great so thank you so much joe thank you
55:54all for joining us for the question and
55:56i think uh that that wraps up this
55:58meeting it's one minute to one so that
56:00uh i'd like to take that last minute
56:03just to thank him what you're thinking
56:05again for for this great overview
56:08and to thank you all for joining
56:10and i hope to see you at the next uh the
56:12next meeting
56:14thanks
56:15thank you ninka and thank you elaine for
56:18uh setting all of this up and for all
56:20the logistics support so thank you
56:22everybody bye
56:34you

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