Full transcript
0:00All right.
0:03Is this the clicker? Oh, that's the
0:05wrong one.
0:10Oh, here we go. Got it. Think I got the
0:13right one. All right. Uh, hey everybody.
0:16Um, I'm Dylan Gordon with CFD Research
0:19Corporation and, uh, I'm with the
0:21Invitro and Encilico Modeling Group
0:24there and it's honestly a pleasure to be
0:26speaking in front of everybody to
0:27present our data today.
0:28>> [clears throat]
0:28>> Um
0:31uh I'm here to present you know the work
0:32that we've doing for tissue chips 2.0
0:35and we are developing a uterine model to
0:37assess the effects of uh aging and
0:40reproductive health and disease in
0:42women. Uh especially considering
0:44microgravity effects on these things. Um
0:47but before starting I'd like to give a
0:48big thanks to our collaborator. Um if if
0:51we didn't have their help we really
0:52wouldn't be able to pull this off. So a
0:54big thanks to them and thank you for the
0:56opportunity to present our work. Uh I'm
0:57going to give a quick overview of what
0:59CFD research is capable of. Um I know
1:01we're running a little behind so I'm
1:02going to kind of go through this quickly
1:04but uh we we specialize [clears throat]
1:06in both in vitro and incilico modeling.
1:09Uh and some examples would be uh we do
1:11have a bloodb brain barrier on a chip.
1:13We have a cornea on a chip triculture.
1:15Uh we have a uh we've developed a lung
1:18on a chip as well as a placental model.
1:21Uh and also a bone and joint model. Um
1:24we do uh complement our modeling with
1:27high fidelity physics models including
1:30phiccoysics and phicodnamics. Um but we
1:33also we have extensive uh experience
1:35with barrier models and some of the key
1:38features that I'll point out later on in
1:40this are uh the we have some uh we have
1:42slits in between some of the adjacent
1:44channels that allow for cell cell
1:46interaction or we even have pillar
1:48designs if someone would be so inclined
1:51to have a uh monocy uh model to uh
1:56explore migration or adhesion ashes
1:58assays excuse me.
1:59[clears throat and cough]
2:01So, I know we've already been over uh
2:03what we learned with tissue chips in
2:04space, but I'll briefly um kind of go
2:06over what our key findings were. And
2:08that was that uh you get accelerated
2:10disease progression and accelerated
2:12aging in space. And these are conditions
2:14that are difficult to replicate on Earth
2:16uh just because of the fact that it's in
2:18microgravity. Um [clears throat] and
2:20what we wanted to do is we wanted to
2:22explore disease, aging and astronaut
2:25health uh with respect to um fe or with
2:29respect to women's health. Uh and in
2:31particular we'd like to study women's
2:33reproductive health or uterine aging. Um
2:36[clears throat]
2:37each of these uh can each of these
2:39aspects can impact uh uterine health and
2:41reproductive health. And we wanted to uh
2:44adapt our space flight or adapt our
2:47model for space flight to assess these
2:49effects of microgravity um while
2:51introducing a drug repurposing pipeline
2:54to see if we can either reverse or uh
2:58stop the effects of the of aging in the
3:00uterus.
3:02>> [clears throat]
3:02>> So to study this uh we wanted to create
3:05something that we call Uhura which is a
3:08micrfluidic human uterus on a chip to oh
3:11excuse me uh to study reproductive aging
3:14and we wanted to adapt this model for
3:16long-term space flight and uh that means
3:20you know and and in order to do that
3:22we'd also like to recreate the menstrual
3:24cycle. So it was uh necessary to build
3:27upon our already established triculture
3:29model that we had of this by uh adding
3:30in the mometrium. And this was uh it was
3:33necessary for us to reestablish basement
3:36membranes, flow rates, um media
3:39compositions, cell seating density,
3:42etc., etc. We had to kind of completely
3:44redo this thing to establish uh or to
3:46prepare it for long-term space flight.
3:49Uh, [clears throat] and we originally
3:51only adapted the tririculture for about
3:537 days in culture, but we want to get
3:54this out to 35 days to completely
3:57recreate the menstrual cycle. [snorts]
3:59Um, [clears throat]
4:01so I wanted to uh excuse me uh I I just
4:05wanted to give a a brief overview on the
4:08order in which we seed the cells and uh
4:11do things like increasing flow rates etc
4:13etc. So uh on day zero we uh start to
4:16establish the endometrium which is by
4:18seating the indometrial epithelial cells
4:20into the top channel there um the EEC
4:23and that's uh we allow [clears throat]
4:26them to establish for about 24 hours and
4:28then on day one we seed the uterine
4:30smooth muscle cells down at the very
4:32bottom channel and establish the
4:33myometrium. Uh after the myometrium has
4:36been established, we'll see the uh we'll
4:39go ahead and see the epithelium or the
4:40endthelium, excuse me, with the uterine
4:42microvascular endothelial cells and then
4:45eventually we'll uh see the fibiberblast
4:48last to fully establish the endometrium.
4:50Uh at the same time, we will begin what
4:53we call a ramp or gradually increasing
4:55the flow rate of media fed into these
4:57channels. we'll gradually increase that
4:59flow rate over time to uh to to a
5:02constant rate and then on day four we'll
5:05do the same thing. We'll do the same
5:07flow rate increase over time. Uh also
5:11[clears throat] as a reproductive tissue
5:13it's uh pretty important to incorporate
5:15physiologically relevant hormone cycles
5:17and in doing so we instituted a hormone
5:20dosing regimen of estradile and
5:22progesterone. Um, and we based this off
5:25of a literature review for what the
5:27serum concentrations are and down
5:29calculated or uh adapted these
5:31concentrations for use in a micrfluidic
5:33model. Uh, and then we adapted uh these
5:37concentrations to be in sync with what
5:40the crew on the ISS would be doing. So
5:42every 5 days we'll be having an increase
5:44or decrease in specific hormones and the
5:47media bags will be already uh prepared
5:50with the hormone. All they have to do is
5:51just switch them out starting at 10
5:53days, 15 days, 20 days, 25 days and so
5:56on. So finally what we have is a fully
5:59established quad culture. So we have
6:01four cell types in a micrfluidic chip.
6:03Uh as you can see here on uh you know
6:06through phase microscopy uh on the top
6:09we've got our epithelium. Uh adjacently
6:13we have our stroal fiberblast, the
6:15endthelium and then finally on the
6:17bottom the smooth muscle cells. And
6:20[clears throat] I don't know if uh well
6:21I don't know but I'm going to point it
6:22out to you. Uh here on the in these
6:25little slits you can see between each
6:27cell these are about 3 to five micron
6:29wide slits and this allows cells to
6:31communicate with each other or signal to
6:33each other should they uh need to do
6:35that. Um which we think is kind of an
6:38important aspect of our our model. And
6:41in order or in in establishing this
6:43model we also wanted to make sure that
6:44we had a good and viable model. And so
6:46we performed uh viability such as a dead
6:49all using like a new green uh contrasted
6:51with hooks uh to check our viability and
6:55in all four cell types in the quad
6:57culture we have over 90% viability. Uh
7:00so I I you know we like to think that
7:02that gives us a kind of a robust model
7:04but in uh in addition to oops excuse me
7:07uh in addition to uh checking viability
7:09we wanted to make sure that our cells
7:10were expressing the proper phenotypes
7:13and with the proper phen excuse me
7:15[clears throat] uh for the proper
7:16phenotypes we'd like to stain uh each
7:19cell type for um certain biomarkers
7:21[clears throat]
7:22that are specific to those cell types.
7:23So on the epithelium we've got uh EPAM
7:26which is an important cell adhesion
7:27molecule. Uh in the stroal fiberblast we
7:30have lamin which is a you know basement
7:31membrane secretion. In the end
7:34endothelium we've got CD31 which is uh
7:36you know an important junctional
7:38protein. And in the smooth muscle cells
7:40we have al alpha smooth muscle actin
7:42which gives us you know our
7:43cytokeleletal structure. Um in and uh to
7:47further validate the model we uh wanted
7:49to check the function of our tight
7:51junction. uh and there so what we did
7:54was uh we ran a permeability assay using
7:56fluorescent molecules like uh florosine
7:58sodium salt or 40 kilodolan tritzy
8:01dextrand ran those through either the
8:02epithelium or the endothelium and
8:05measured the permeability index which is
8:07a ratio of the fluorescent input to the
8:09fluorescent output or how how much of
8:11the uh fluoresence had migrated across
8:13the membrane and this gives us an
8:14indication of how tight our junctions
8:16are and how good our uh tight junction
8:18formation is.
8:20Um so [clears throat]
8:22[snorts] moving on from that after
8:23establishing the model and validating we
8:25needed to integrate this uh with uh the
8:28MVP that Redwire has uh developed and so
8:31what we have to do is we have to
8:32miniaturaturize our model even further
8:35which consists of our chip which is
8:38already small thankfully. We have a
8:40syringe pump which is quite large and
8:42heavy and an incubator also quite large
8:44and heavy. Um lots of tubing, CO2 gas
8:49and we have to um collaborate with
8:51Redwire to get them to integrate all of
8:53these things into a something about the
8:55size of I would say a [snorts] tissue
8:58and maybe half of a tissue box. Um we've
9:01got in [clears throat] the uh in the uh
9:03MVP we'll be able to monitor oxygen, pH
9:06and cell confluence. And uh in the
9:09integration though we do need to
9:10reoptimize the pumps. We need to
9:12optimize sensor locations, bubble
9:13mitigation. Uh you know bubbles are are
9:16kind of a problem. So we don't want any
9:18bubbles in our uh model. Um then we also
9:21have to make sure that we can uh operate
9:23within their parameters for media
9:24infusion and also uh ISS crew media
9:27collection.
9:30So speaking of uh monitoring cell
9:32confluence and health, we uh have these
9:35uh impedance sensors that we've used
9:37inside of a on a PDMS chip and we tried
9:40two separate uh experiments. These are
9:43actually wellplate experiments but um we
9:45used a slower growing smooth muscle
9:47cells seated at a slightly lower
9:49confluence or seating at a slightly
9:51lower density and monitored their growth
9:53over time. And on this uh graph here you
9:56can see we have impedance over time
9:58starting at zero days where you have
10:00kind of a lag phase and then a sharp
10:02steep growth rate and then uh confluence
10:04where you can see that the graph starts
10:06to plateau. And in addition to this we
10:09did the same thing with epithelial
10:10cells. However, we seated them much more
10:12densely and they grow a little bit
10:13faster than the smooth muscle cells. And
10:14this is over 24 hours. We see the exact
10:16same thing with the lag phase, a steep
10:19growth rate and then confluence.
10:22[clears throat]
10:24Um, so we wanted to also
10:27>> [snorts]
10:27>> uh see if we could find a way to
10:29repurpose some drugs to either stop or
10:31reverse uterine aging. And we had our
10:34biioinformatics team kind of uh look at
10:36a a public uterine data set from a GTX
10:39database. And they use a signature
10:41reversion to um kind of down select or
10:45um select drugs that look like they
10:47would be good candidates for uh
10:49reversing or perturbing the uterine
10:52aging pathway. Uh two of the candidates
10:54were boromib and palocycib.
10:58Um and it turns out that these two drugs
11:01uh can can have a um reversing effect
11:05for uh scinesscent cells. They're
11:08typically used for cancer uh treatments,
11:10but uh this is, you know, kind of why
11:13we're doing this drug repurposing. Uh so
11:15what we want to do is we want to be able
11:16to test these drugs. And what we have to
11:18do to test these drugs is we'll need to
11:21artificially sess the cells that we are
11:24going to be studying. And currently what
11:26we're using for uh scinessence is we're
11:28using hydrogen peroxide to induce
11:30scinessence into the cells at varying
11:32concentrations. And here on the left
11:34there is a um this is a beta
11:36galactoidase assay the in the green
11:39which is an indicator of scinessence and
11:42also um [clears throat]
11:44we look at uh something called nuclear
11:46morphometrics. So as you age your nuclei
11:48kind of change their morphology and we
11:50used um a pipeline from uh nature paper
11:54that allows us that allowed us to um
11:57assess the nuclear morphology and
11:59determine the level of scinessence. And
12:01that's this um chart that you have up
12:03here on the right. So in media this is
12:05the level of scinessence. The
12:06scinessence level is low. And if as you
12:09increase your hydrogen peroxide
12:11concentration you trend up towards a
12:12more scesscent uh cell based on the
12:15morphology of the nuclei.
12:17[clears throat] Um and also we want to
12:20start eventually comparing uh young
12:22uterine cells versus aged uterine cells
12:25to see if we can uh tell a difference
12:26there with uh within our model.
12:30And um our future plans are we would
12:32like to get our culture extended out to
12:34about 35 days. Uh we need to fully
12:36characterize the aging and then start
12:38exposing in ship the drugs that uh we
12:41have found as a potential repurposing
12:43candidates. Um and I'd like to give uh
12:46thanks to PI Carry German, Katenbot,
12:49Kevin Ree, Dustin and Katie for their
12:52work. Uh University of South Florida,
12:54Matt Anderson and Beamethic uh TUS Juan
12:57Yeko. He's been great giving advice on
12:59how we need to treat ourselves. Uh Sha
13:02at Ruters also been critical for us and
13:04at Redwire and Rogers, Rich Bellinger,
13:06Grant Bellinger and at Svivo for
13:08providing the chips that we use uh Gwen
13:10Fuel and Jenna Rosano.
13:12Thank you.
13:15[applause]
13:19>> Yes,
13:26>> it's a great talk. I have a quick
13:27question on the two drugs that you
13:29trying to repurpose on your invitro
13:32model. What is the dosing that you saw
13:34some efficacy? What is the
13:36concentration?
13:37>> Uh I do not have that data in front of
13:39me right now. It is a low mic I believe
13:42it is a low microar uh dosing. The it
13:45was our bioinformatics team that kind of
13:47provided us with that data. But um if
13:50you want specifics get with me after
13:52here words and I can
13:53>> I think in our experience a lot of times
13:55the dosing is way too high uh compared
13:57to the C max that we can hit in our
13:59patients. Yeah.
14:00>> Yes.
14:00>> So that's why I was asking those.
14:02>> Okay. Yeah. Thank you for the question
14:05>> once more. Yeah.
14:08>> Okay. Is this on? Um so um you I you
14:12were mentioning I believe um tight
14:15junctions and also um the potential of
14:18like studying these under microgravity
14:19if I recall. Some some of the talks
14:21blend together so sorry if I'm mixing up
14:23details. No problem.
14:24>> Um I'm particularly interested in uh the
14:27formation of like via myomatada and um
14:30how like mechan mechanical transduction
14:33might be involved in that. Do you have
14:35like the capacity to maybe comment on
14:37potential roles of mechanic transduction
14:39in uterine aging?
14:40>> I don't think I have the capacity to
14:42comment on that unfortunately. Um, so
14:44[clears throat] my role is to
14:46essentially build the model um to
14:49optimize the basement membranes, seating
14:51densities, cell types, things like that.
14:54Um, as far as your question,
14:55unfortunately, I, you know, you, if you
14:58want to get with me after this, I can
14:59take your question and forward it to the
15:01people who I think could answer that for
15:02you. But unfortunately, I not really,
15:04>> you mentioned ECM as well. Did you
15:06notice any like differential expression
15:08in um, ECM with any of the factors that
15:10you introduced?
15:11>> Well, what we did notice was um, with
15:13different ECMs, we have a uh, some of
15:16the cultures would last a little bit
15:18longer in chip with different ECMs. We
15:20used this thing called an ECM array from
15:22Advanced Biomatric to assess which ECM
15:24we thought was optimal. And I think it
15:26had about 36 different ECMs on there.
15:28You plate the cells on there for a day
15:31or two. And then you can basically
15:32assess either just with phase microscopy
15:36or you can do some ICC staining for
15:37phenotype expression to assess which is
15:40uh which basement membrane or ECM you
15:42could use. And so that's how we kind of
15:44selected the basement membrane that that
15:46we were going to use.
15:47>> Okay. Thanks. Y
15:49>> thank you Dylan for this exciting
15:50presentation.