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Elizabeth Delery, PhD

Postdoctoral Fellow, Louisiana State University Health Sciences Center, New Orleans, LA, USA


My passion for science started at a very young age. My dad was a forensics investigator for the New Orleans police department, and after school I would spend time in the lab where he taught me about DNA, fingerprints, and microscopy. I was fascinated by the puzzle of all of it, and I made my life goal to be an "ist" (scientist, biologist, paleontologist, etc.). My teachers and professors encouraged my love of science and made sure to emphasize the contributions of women in science, like Marie Curie, Rosalind Franklin, and Barbara McClintock. I admired the sacrifices these women made in order to help humanity and advance their fields.

I received a scholarship to attend Randolph College where I received a bachelor's of science in Biology & Psychology with a pre-med concentration. There I learned the value of experiments and research from the best professors I have ever met and was encouraged to pursue a career in research. I was then accepted to Tulane University School of Medicine where I received my PhD in Biomedical Sciences with a concentration in Microbiology/Immunology in 2019. I completed my degree at Tulane National Primate Research Center under the guidance of Dr Marcelo Kuroda, Dr Woong-Ki Kim, and Dr Andrew MacLean. My dissertation focused on the neuroimmunology and neurovirology of HIV-associated neurocognitive disorders in a simian model, with emphasis on macrophages of the central nervous system, the choroid plexus (blood-CSF barrier), and inflammaging. One of my key projects was the creation of a primary rhesus macaque choroid plexus cell culture for use studying the effects of inflammation on the blood-CSF barrier.

Currently, I am a Postdoctoral Research Fellow at LSU Health Sciences Center where I work in the Physiology Department in Scott Edwards’s Lab studying the neurobiological interactions of alcohol, opioids and pain in rat and simian models. Away from the lab, I love to spend time with my two dogs Wishbone and Thor and going on adventures to try new food with my husband.

Learn about Elizabeth’s research

Title: Choroid plexus epithelial cell 2D and modified 3D cell culture model

Learning objectives

  • To understand history, development, and function of the blood-CSF barrier
  • Understanding the pathology and therapeutic potential of the choroid plexus
  • Designing a 2D and modified 3D choroid plexus epithelial cell model system

The choroid plexus, which makes up the blood-cerebrospinal fluid barrier in the central nervous system (CNS), lines the ventricles, produces cerebrospinal fluid, and protects the brain via a physical barrier. Perivascular macrophages line the stromal capillaries through it, and tight junctions between the apical sides of the epithelial cells regulate the microenvironment. The choroid plexus is also believed to be an immune interface between peripheral and CNS immune systems and plays a major role in the resolution of neuroinflammation by recruiting monocytes and leukocytes into the CNS. It has also been proposed to be a target of viral infection, such as a human immunodeficiency virus (HIV) reservoir, and a site of damage in cerebral hemorrhage, stroke, and hypoxia. Since the choroid plexus can allow transmigration of leukocytes, recruit myeloid cells, control diffusion of small molecules and water, and control drug permeability into the CSF, it is important to have an ex vivo culture model.

We designed a rhesus macaque 2D choroid plexus epithelial cell culture, as well as a modified 3D cell culture model, in order to study activation, diffusion, and migration through the choroid plexus. Our hope is to understand the response of the blood-CSF barrier to peripheral HIV infection, as the rhesus macaque is an ideal animal model of infection. This model can also be used to study pro- and anti-inflammatory challenges, as well as barrier properties.

Watch the webinar


Slide 1

Moderator:  Hello everyone and welcome. Today's live webinar "Choroid Plexus Epithelial Cell 2D and Modified 3D Cell Structure Model" is presented by Dr. Elizabeth Dellery, post-doctoral Fellow, Department of Physiology, Louisiana State University Health Sciences Center. I'm Christy Jewel of LabRoots and I'll be your moderator for today's event. (00:30) Today's educational web seminar is presented by LabRoots and it's brought to you by Thermo Fisher Scientific. To learn more about our sponsor, please visit thermofisher.com/CellCultureHeroes. Now let's get started.

I'd like to inform everyone that today's webinar is interactive, and we encourage you to participate by submitting as many questions as you want at any time you want during the presentation. To do so, simply type (00:00:59) Ask a Question box and click Send. We will answer as many questions as we have time for at the end of the presentation. Now if you have any trouble seeing or hearing the presentation, just click on the Support tab found at the top right of your presentation window. Or you can let us know you're having a problem by using the Ask a Question box at the far left of your screen.

This presentation is educational and thus offers continuing education credits. Please click on the Continuing Education Credits tab located at the top right of the presentation window and follow the process to obtain your credits. (01:32) I now present today's speaker, Dr. Elizabeth Delery, post-doctoral Fellow, Department of Physiology, Louisiana State University Health Sciences Center. For a complete biography on your speaker, please visit the Biography tab at the top of your screen.

Dr. Delery, you may now begin your presentation.


Dr. Elizabeth Delery:  Thank you, Christy! Hello everyone and thank you for joining me today. My name is Dr. Elizabeth Delery and today I will be presenting my Choroid Plexus Epithelial Cell 2D and Modified 3D Cell Culture model.


Slide 2

So, this is just my overview of what I'm going to be going through, just a little bit about me, a little bit about HIV, and then leading into why I wanted to look at the choroid plexus and just a little bit about that just for background, for those of you that aren't aware of it, as well as moving into how I designed the 2D cell culture and then how I created my modified 3D model.


Slide 3

So, about me. As I said before, my name is Elizabeth and I am a post-doc in the Physiology Department at Louisiana State University Health Sciences Center. Currently, I study the neurobiology of alcohol, opioids and pain.

I attended Randolph College for my undergraduate studies, where I majored in biology and psychology, with a pre-med concentration. (03:02) It was while I was here that I was first introduced to research, as you can see from the little blue square in the bottom left. I worked in the Biology Department, where I studied soil characteristics of Listeria innocua survival and growth. In the Psychology Department, I actually looked at various exercises and social activities on cortisol levels in expression.

So, I really decided that—undergrad really introduced me to research and wanting to figure things out.(3:34) So, my professors encouraged me to go on and pursue my Ph.D. And I was accepted to Tulane University School of Medicine Biomedical Sciences Ph.D. Program, and I completed my Ph.D. at Tulane National Primate Research Center. My dissertation was actually on the choroid plexus and HIV pathogenesis and I just graduated this past spring. In my free time, my husband and I like to go on adventures and we have two very rambunctious dogs, Wishbone which is on the left, and then Thor, who's the little goofball on the right.


Slide 4

Also, in my free time, I run an Instagram page where I like to talk about science. I like science communication because I like to help fight the internet pseudoscience that's out there, while showing that science is fun and get people excited to learn about it. (4:25)

I also want to break the scientist stereotype that we're always in the lab and that we're really boring and that we don't have lives outside of science. And then I also like to promote inclusion and STEM, whether it's race, sex, orientation, disability, et cetera. And here are some examples of some goofy posts I posted. The one on the left, I was talking about eyeballs and how we see with some super goofy eyeball glasses. And the other one was a maternity photoshoot I took with my dissertation, because it took me about nine months to write. So, let's start talking about my research.


Slide 5

So, just in general, a little background on the HIV epidemic. In 2017, there were 36.9 million people living with HIV in the world. They had around 1.8 million new diagnoses and just shy of one million deaths from HIV-related disorders in 2017. So, It's still a very pertinent global health issue.


Slide 6

In the United States, D.C. has the highest percentage of new cases of HIV in the United States. And then Louisiana—which is the state that I currently live—is actually in fourth place, with 22.1 new diagnoses per 100,000 people. So, it's still very close to home and a very important issue for me, living in Louisiana.


Slide 7

So, some primary obstacles to curing HIV is the presence of long-lived viral reservoirs. Now these reservoirs can persist despite the use of long-term combination anti-retroviral therapy, which is the drugs we use to treat HIV. And that's combination, two or more drugs taken at the same time. The brain is one of these long-lived viral reservoirs. (06:14) And since my background at Randolph was biology and psychology degrees, I really wanted to focus on the brain and figure out what was going on and why there's issues with the brain being a reservoir.


Slide 8

So, when HIV infects the brain, it leads to a series of disorders known as HIV-associated neurocognitive disorders. There are three different levels and here's the list of diagnostic criteria on how to identify.

As you can see, you have asymptomatic, which is more of the mild—it has the prevalence percentages in the center column is around 30% and that's in combination anti-retroviral-treated HIV-positive individuals. You also have mild, which is a slightly more severe type and then the most severe is actually HIV-associated dementia. (07:02) Collectively, these disorders are known as HAND, that's just a little name for them, acronym for them.

So, HIV is believed to enter the central nervous system when infected monocytes or macrophages are unknowingly trafficked across the central nervous system, via a trojan horse or trojan herds model. A trojan horse model argues that a few infected  monocytes and macrophages enter the central nervous system and they establish a latent infection in the brain. (07:35) While the trojan herd model argues that there's little—like there's a surge of infected monocytes and macrophages into the central nervous system late in infection towards the progression to AIDS. Either way, when HIV enters the central nervous system, it can lead to these disorders.


Slide 9

So, pathologically, HIV when we're looking at  the actual size of brain tissue, neuropathology includes lesions, perivascular cuffing, leukocytic infiltrates and the presence of multi-nucleated giant cells, which are these giant cells that are a result of macrophages fusing together and chronic inflammation. Whether it's a failure of division or whatever, they just make these gigantic cells.

And as you can see on the left, I just have an example of an H&E of one that two of the vets at the Primate Center took. It's kind of towards the top area, you see this big circle with a lot of blue nuclei around it—or purple circles of blue nuclei around it, and that's your multi-nucleated giant cell. (08:36)

And for a better example on the right this is an immunofluorescent image of a multi-nucleated giant cell, which is pretty much dead center in the picture. And it's a green circle with a blue DAPI nuclei around it. The green is CD163, a macrophage marker, because again, these cells are formed from macrophages.

So, in doing a literature review, to learn more about HIV-associated neurocognitive disorders, I began to focus on the choroid plexus, which is the blood cerebrospinal fluid barrier, CSF barrier, a place where HIV-infected cells can enter the central nervous system. (09:11) And it's been identified in the literature as being one of the reservoirs. It's also been identified as being an area of productive infections, as well.

So, the choroid plexus lines the lateral third and fourth ventricles of the brain, which is like the space surrounding the brain. As you can see kind of like the inner side of the brain—I can't really point, but you've got the area in the top part, if you can see right below "arachnoid" you see the choroid plexus in the center of the skull. (09:42) Here's a better picture of it, how it lines the actual ventricles. And it produces the cerebrospinal fluid that protects the brain and it's composed of these fenestrated capillaries inside a neuron-devoid stroma surrounded by a single layer of epithelial cells.

So, that's how you really identify it. It kind of looks like this curly broccoli almost, that's got  a single layer of epithelial cells on the outside, and that's really how you can find it on a slide. (010:14) It's super, super easy to spot once you know what you're looking for.


Slide 10

So, I've got some more pictures, so you can get a better idea of what it looks like. This is kind of a 3D representation of it. As you can see again—I described it like broccoli, it doesn't really look like broccoli—but you've got these protrusions that come off of it. So, it's this 3D structure that's kind of floating inside the ventricles. It's attached but floats out into the cerebrospinal fluid that it produces.

And then here is a better example of the single layer of epithelial cells, followed by—you have the inside, the fenestrated capillaries on the inside. It's got the endothelial cells surrounding the capillaries.(010:51) So, for something to get into the central nervous system, it has to first cross the endothelial cells of the fenestrated capillaries. It has to encounter any immune cells inside of it—so macrophages or microglia—and then it will travel out through the choroidal epithelium.

It allows for the diffusion of water and sodium through to maintain water homeostasis in the brain. And it can also allow the movement of larger molecules into and out of the brain. (011:21) Like I mentioned before, cells can traffic through the choroid plexus in basically normal immune surveillance, but you can have increased movement in times of brain damage. It can also prevent drugs or allow drugs to enter the central nervous system, because it has multidrug resistant proteins to either block or allow drugs from entering the central nervous system.


Slide 11

So, just for a brief history of the background of the choroid plexus. It was first identified by Herophilus way, way, way back then in B.C. and he called it a "choroid concatenation." Galen later—in like 130 to 200 A.D. first gave it its true name, "choroid plexuses." It was discovered in 1543 by Vesalius. The choroid plexus produces cerebrospinal fluid. But it really wasn't until the early 1900s that you started to get the detailed structure and morphology. (012:19) With increased scientific technology and microscopes, we were able to actually really see more about the structure and how it's set up in the brain.

If you want to know a little bit more about this, just some background on it, Shane Liddelow has two really great review papers that are actually really interesting reads out there, just the development of the choroid plexus and the CSF Barrier, as well as just the different barriers of the brain and just how we've learned about it over time. So, those are both very, very great review articles, if you'd like to go back and read more about this topic.


Slide 12

Now I've been boring you about the background research. You really want to know why, why the choroid plexus and why we wanted to focus on this. The choroid plexus is an adult stem cell niche. It's one of the locations that's been known to create neural progenitor cells. It's the interface between the blood and the cerebrospinal fluid or CSF. It allows a transmigration of monocytes and leukocytes into the central nervous system, and it's responsible for immune signaling. It can recruit monocytes and leukocytes in infection, hemorrhage and stroke. (013:26)

So, if you have brain damage, it can actually signal out to the peripheral immune system and request additional cells to come help out with any damage in the brain. It's also responsible for the diffusion and transport of drugs, as I previously mentioned. And like I said from some of our earlier work, it's a reservoir for HIV.

Since HIV-associated neurocognitive disorders are the result of infected cells entering the central nervous system, whether it's via trojan herd or trojan horse model, having a choroid plexus epithelial cell model would allow us to study the barrier properties at one of the last lines of defense between the blood and the cerebrospinal fluid in the central nervous system. (014:10) So, that's kind of what got me interested in the choroid plexus and studying it and figuring out why. So, like I said, we sought to figure out a way to create a choroid plexus epithelial cell culture.


Slide 13

So, when you're designing a primary cell culture, you have to keep a lot of things in mind. You want to know what cells you're looking for. You have a couple different types—epithelial-like, fibroblast-like, lymphoblast-like, and those types all have different needs in culture.

You also need to know the type of substrate, whether they can just sit on the plastic petri dishes or if they need gelatin, collagen or Matrigel. You also have to use sterile conditions when you're using your cell cultures to prevent bacterial and fungal contamination, which can completely ruin your culture.(014:58)

You also need to know what type of food or media the cells need, in order to grow wand survive. These can include things like different amino acids, carbohydrates, vitamins and minerals, growth factors to help promote growth and even hormones. And also, you need to have culture conditions, like when they're in an incubator, what concentration of gases do you need, pH, osmotic pressure and even the temperature the cells need to grow. So, there are a lot of different factors to think about.

Now if you're curious about this and you want to learn more, Gibco has some great background stuff. (015:31) They have the Cell Culture Basics Handbook and an online interactive lab, which are both free and awesome. And I actually used both when I first started out trying to figure out how to design my own cell culture. So, to get practice, before you actually get in a lab and do stuff, I do highly recommend these resources.


Slide 14

So, since we knew that we wanted epithelial cells, their polygonal in shape and need a substrate to attach to. So, we looked at the three different types. These are just some goofy pictures I got. We have gelatin. This is a picture of jello, because it's the closest thing—gelatin is just sort of this clear, boring goo. So, I figured a picture of jello was a lot more fun. You also have collagen, so here's a picture of collagen matrix. And then here's a picture of the actual matrix to symbolize Matrigel, which is the more extreme, really nice substrate with which to grow cell cultures. (016:22)

So, since our lab already does a lot of cell culture work—we grow both brain microvascular endothelial cells and astrocytes—we kind of had an idea of what media we're going to need to grow brain cells.


Slide 15

And after a lot of playing around with different types of media, like DMEM and M199, we settled on M199. We added some additional L-glutamine which is a...losing my thought—an amino acid energy source for the cell, and you need that for them to grow. M199 also has Earle's salts and a phenol red pH indicator, so you could follow the pH in the cells and see if the cultures are going well. (017:05) So, that's why we chose M199.

Then we also added an anti/anti which is an anti-mycotic and anti-bacterial thing to help prevent the growth of contaminants in our cell culture, which again, would completely ruin them. And then we also used fetal bovine serum. We used exosome-depleted fetal bovine serum to have a really pure FBS to add to our cultures, to help the cells grow.


Slide 16

There's a lot of trial and error involved in this culture. And I killed several different cultures before we actually got them fully started, once we figured out the ideal substrate and growing conditions and the media and additional supplements we needed to grow. And about the first week after plating the cells, after a digestion, we started to see the cells attach and colonies begin to form. And as you can see on the left, you've got colonies of about five cuboidal-like cells that are forming as a little baby colony. (018:03) And on the right, it's a little bit harder to see, but you have a slightly larger colony. There's a lot of interanimal differences depending on the feeding density with which you plated the initial cells, how long it took them to attach, like 24 to 48 hours, the collagen substrate, could all affect how quickly these cultures grew.

So, in two to three weeks, you start to see these colonies expand and spread out. Again, they're polygonal cuboidal like cells, which are definitely epithelial-like in nature. (18:33) So, it's kind of what we were expecting to see in our cultures.


Slide 17

It took about four-to-eight weeks, again depending on animal variation, feeding density, things like that when we first started, but within four-to-eight weeks, the plates began to become confluent. We were worried a little bit about fibroblast contamination, as you will with most cell cultures. So, if we saw that it started to kind of look like fibroblasts growing in the cultures, we treated with something called cis-4-hydroxy-D-prolineor cis-HP, which has previously been published to be a fibroblast treatment. (19:08) So, we use that if necessary to take care of fibroblast contamination. So, once you reach confluence, the next step is to see, hey, this is a primary cell culture. Am I able to subculture this and keep dividing it and make more wells of it?


Slide 18

So, we were able to successfully subculture. You can kind of see on the top left how there's that like little corner. That's because this is a collagen-coated little disk that we put inside the wells, that we grow the cells on these disks. And then we get to fix them and then stain them to confirm the identity. Because yes, these cells are epithelial-like in nature, but we can't conclusively say that they're choroid plexus epithelial cells at this point.


Slide 19

The next thing we had to do, again, validate them and confirm their identity. Now we can do this by finding literature reported markers for the cells of choice. So, we used klotho, which is an anti-aging hormone. Transthyretin, which is a binding protein that "TRANSports THYroxin and RETINol" and the capitalized letters are how it got its name. We also used vimentin and phalloidin. Vimentin is an intermediate filament protein and phalloidin stains F-actin. (20:21) So, those are both kind of like structural proteins, to you know, really show that we've got this cuboidal and polygonal-like cells.

We also used zonula occludens-1 or ZO-1 which I identify as tight junction proteins. Now if you remember from the choroid plexus pictures from way back, the choroid plexus is also known for having tight junctions between the single layer epithelial cell around the outside. So, ideally we would like to see ZO-1 in these cultures. We also wanted to look at focal adhesion kinase which identifies cell-to-cell adhesions. (20:51)

You also have to—to do a true stain to say, "Hey, what I've got is correct," you also want to do isotype controls, to make sure there's no non-specific binding of your antibodies to anything in your cells. So, you always do isotype controls, to make sure that the staining you're seeing from all of the above antibodies are true. You also want to exclude other cell types when you're confirming your identity, like neurons, astrocytes, microglia and even macrophages. Now thankfully for us, there are no neurons in the choroid plexus, so that ruled out that cell type. But you also have astrocytes, so we stained for GFAP. (21:27) Microglia, which we looked for Iba-1, as well as macrophages for CD163.

So, this is how you would go through to confirm, to say that, "Hey, we've got the cells of the shape that we want and the morphology that we want. We think these are it. Let's confirm, by making sure it stains for true markers." The markers are true staining, as in it doesn't bind to non-specific binding in the background and then you also exclude other cell types. And at that point, you're kind of like narrowing it down to say, "Hey, this is the cell type that I want, and I've got the correct type."


Slide 20

So, here's an example of our klotho staining and again, klotho is the anti-aging hormone. I've got the legend on the right, listing the different colors. I didn't think about this ahead of time, but I do apologize if you are red/green color blind, because I put klotho and phalloidin in red and green. So, I do apologize about that. But klotho is the red stain that is perinuclear to the blue DAPI and then phalloidin again is the structural protein that kind of just shows the shape of these cells. And as you can see, all of the cells here were positive for both klotho and DAPI—sorry, phalloidin and DAPI. (22:34) So, you do see the cells and they are positive for klotho and phalloidin, which does kind of give you the shape and also show you that they are positive for a marker of the choroid plexus.


Slide 21

And next, we look at transthyretin and DAPI. It's a little bit more of a fainter stain on the screen, but again with the isotype controls this was true staining. And as you can see here, the cells were positive for transthyretin which is in green and then the DAPI in blue. So you can see the kind of again, cuboidal cells that are positive for transthyretin, with their little DAPI nuclei in the center.


Slide 22

Then we looked at the structural proteins. Again, you've got the vimentin and the phalloidin. Phalloidin again is in blue, so this is kind of the blue squares. The phalloidin staining and the DAPI in the center is the nuclei circles, and the vimentin is red. So you can kind of see the structure and the shape and even the (D actin) and the filaments in these cells, which is super, super cool.


Slide 23

Here was my combined image, with both the transthyretin, vimentin, phalloidin and DAPI. It's one of my favorite pictures, because you have that nice cuboidal cell in the center of the picture. So, you've got all the positives for the stainings that we're looking for, plus it's got the shape. It's really looking like we had identified choroid plexus epithelial cells.


Slide 24

So, the next thing we wanted to do was look at ZO-1 or the tight junction proteins. And as you can see, I had to increase the brightness just so you could see it just a little bit better on the screen, since it is tinier with the PowerPoint. You can see the DAPI nuclei in the center and as you can look around at a lot of these cells, you see these bright red kind of dots. If you're able to zoom in, you see these nice lines and this is indicating tight junctions that are forming between the cells. That's ZO-1 staining. (24:14) So, these cells had formed tight junctions with each other gain indicating choroid plexus epithelial-like features.


Slide 25

And then again, we looked at focal adhesion kinase. So, if you kind of look to the bottom left, you can see a really great example of two—you see those little like red spikes coming off the cells, and that's actually where they've formed adhesions or are trying to form adhesions with adjacent cells. So, as of right now, they are all positive for all of it.

So, using all this knowledge that we have the right morphology and we have all these positive stains, it's really looking like we have choroid plexus epithelial cells and it's at this point we can definitely say that the cells that we got from the choroid plexus and are positive for all of these markers, negative for our isotype—I'm sorry, the staining for our isotype controls and it was negative for astrocytes, microglia and macrophages, that we do know that we've got choroid plexus epithelial cells.


Slide 26

So, this is our 2D culture. Now there are some differences between 2D and 3D cell culture and a lot of our other presenters—if you want to go back and look at some of their presentations, they really did talk about the differences between 2D and 3D culture. So, I did want to give you a little bit more of a background on the differences between 2D and 3D. So, 2D is simple, easy to do and cost effective. However, it doesn't really mimic in-vivo, and the cells don’t really interact—like they only interact with the plastic and the edges of the plate. They don’t really interact as much with each other in the matrix. (25:37) They also don't have gradients or a microenvironment.

Now 3D is a little bit harder to do, complex, a little less cost effective but it's a better in-vivo representation. It can show cell-to-cell interactions and extracellular matrix interactions. And you can also look at gradients and the microenvironment. So, this is a really popular model to use in cancer biology and tumor biology, because you can create these little like organoids and actual tumors in culture. (26:02)

Since the choroid plexus has a lot of different cell types going on, and we were really just more interested in the barrier properties in that single layer of epithelial cells, we kind of wanted to do a modified 3D. I'm calling it 2.5—because we wanted something simple and cost effective, but we wanted to be able to represent, you know, the in-vivo representation of the barrier properties of the movement and the gradients across this choroid plexus epithelial cell barrier.


Slide 27

So, we decided to work on this modified 3D and we used Transwell inserts in these petri dishes, where we actually plated cells in these Transwell inserts in the center, and we could add media on the outside wells and the inside wells. Because I kind of at this point wanted to see diffusion properties and whether media could move across...if we could form a barrier and if the media could move across that barrier. And as you can see, I did a little bit of a  lower level of media on the outside well and the inner well, you can see how the media is a little bit higher, because I added more into that center well. (27:03)

As you can see here a little bit—I didn't take a really good picture—you can see how the media levels have actually leveled out. So, there was movement as media crossed and they leveled out. So, both the media in the inner and outer walls were at equal levels, indicating that there was permeability and diffusion of this liquid across. I really wanted to know how good these barrier properties were, and you can't really do that by just looking at a microscope at these Transwell inserts. You can't really look at the properties of this barrier.


Slide 28

So, that led us to working with our next thing. This is the xCELLigence Model. I did just want to clarify at the very beginning that Thermo Fisher and Gibco do not endorse xCELLigence or its owner ACEA Biosciences. This stuff is presented because it's how I used the models, how data was analyzed and it's a very specific device. And you'll see as I'm going forward on why I just had to present this data in this system, but I did want to clarify that in the very beginning.

So, xCELLigence is a real-time label-free cell monitoring system. (28:05) So, you've got these little 16-well plates, and they've got electrodes in the bottom of these plates. The picture on the left is a schematic of one of the wells. Now you can run an electron flow through the well and they'll move from the negative to the positive terminal, and when there are no cells present, there's no impedance. So, you have a nice unimpeded electron flow. When you add cells moving to the right picture, and the cells attach and adhere to the substrate, you start to get an impeded electron flow, as the cells attached make it harder for the electron current to move from the left—the negative terminal to the positive terminal on the right. (28:44) So, that's the model design with it.


Slide 29

And one of the readouts that you'll see from the xCELLigence device, which is taking this data and using the initial cell index with which you plated, it will calculate—using the impedance, too, it can calculate the cell index and the increases you'll see based on the cells you've added. So, this is an example of what a trace would look like on the device. So, when you first add the cells, you'll see this rapid increase in the trace, as the cells begin to adhere and impede the electron flow. (29:17) You'll start to have a slower increase due to the cell proliferation, kind of in the middle, especially if it's a slower growing cell line. And then you'll get a plateau once the cells have reached confluence and really blocked the electron flow through the culture. And that is your baseline cell barrier.

You can then add different experimental treatments. In this case, they added an apoptosis inducer and as you can see here, when you see that stark drop in the trace, it's a decrease due to cell death or detachment. (29:46) And that can actually show you barrier properties. At the top when you have the plateau, you've got a nice, tight barrier. When you start to see the traces drop off, it's indicting that there is damage to the barrier. Whether it's due to cell death or detachment, something is disturbing the barrier properties.


Slide 30

So, here is an example of one of my models. This is just an example (inaudible) and again, it's real-time. It's super nice feature that I could put the cells in, let it grow over time, go home and sleep, come back and work on it the next day. So, you can see at the bottom on the Y axis, this is done in hours and this is about 160 hours. So, we went out several days with this specific experiment. And as you can see, there's an example of the plateau when our experiment reached confluence. (30:32) And then here, these little spikes are when I paused the machine to pull out the plates, to add whatever experimental conditions. So, those are what those little peaks are moving forward.


Slide 31

So, here is a zoomed in picture of our barrier and our confluence. So, as you can see, you see that rapid cell growth, as you go from the zero when there are no cells attached and adhered to, rapid growth when they are adhering. And then you have that plateau, which is a really great, nice plateau showing that the cells had attached and adhered to the bottom of the plate. And you have some nice barrier properties here and we actually have created a usable barrier.


Slide 32

So, our next step we were kind of curious, so when HIV—going back to the data I talked about before—with HIV, when it first enters the central nervous system, there are some arguments about trojan herd, trojan horse. But either way, the infected cells cause a weakening of the choroid plexus barrier, which can allow more movement of infected cells into the choroid plexus and into the central nervous system. (31:38) TNFαis a pro-inflammatory cytokine that can be secreted by both macrophages and even endothelial cells.

If an HIV-infected monocyte crosses the fenestrated endothelium inside the choroid plexus, a normal immune surveillance, and triggers the release of TNFα or even encounters a macrophage inside the choroid plexus, which triggers the release of TNFα, we wanted to see if that danger signal could then weaken the choroid plexus epithelial cell barrier. (32:04) This could be one explanation for how infected cells enter the CNS and trigger that increase in movement in cells across the choroid plexus barrier. When you have more infected cells and more damage in the brain, especially the weaker your barrier, you're more likely to have a more severe presentation of HIV-associated neurocognitive disorders. That's why this was really important to us to look at the pro-inflammatory challenge.

So, there's a lot going on in this picture, but the blue and the pink lines at the very top, those are the choroid plexus plus just media control measurements. (32:37) Now as you can see, it stays around zero. I baselined this out, since that is our control. And the red and green were cell cultures where at the very beginning, on the left of the graph, I added TNFα, the pro-inflammatory cytokine. As you can see, both the red and green traces start to drop off.

If you think about our graph from a few seconds earlier with the concept of xCELLigence, when you start to see that decrease in trace, it indicates that there is cell detachment or death, but a damage to the barrier function. (33:06) So, TNFα did indeed damage our choroid plexus barrier properties. And this little spike here around...it looks like 90 hours, 90.4? It's a very tiny screen. It's just a blip where it blips up and drops off again, the more times you challenge with TNFα the worse the barrier properties become. So, the more pro-inflammatory challenges you have, the barrier really gets damaged. So, we did indicate that a pro-inflammatory cytokine could affect our choroid plexus epithelial cell barrier.


Slide 33

So, the next thing we wanted to do, we wanted to repeat the experiment with a pro-inflammatory challenge, but then we wanted to follow it with an anti-inflammatory challenge. We decided to use steroids since steroids are anti-inflammatory and we wanted to see if we could reverse the damage caused by the inflammatory cytokine TNFα. So here again, this is a really busy graph, but the pink and blue traces in the very beginning on the left side—so, you have the line dividing the graph—if you look to the left side of that, the left side is a TNFα challenge. (34:14)

So, on the top part on the left, you see the pink and the blue and those are the choroid plexus-plus...well, choroid plexus-plus, meaning it's just choroid plexus at that point in this graph. And right below it, in the red and the green, you see the choroid plexus-plus, TNFα. Now once you move past that divider line in the middle and you move to the right, you actually have four different conditions, because we took half of the control wells and half of the TNFα wells and we added dexamethasone, a steroid. (34:45)

Now as you can see, the top picture is pink, which is choroid plexus and dexamethasone. Blue is just the choroid plexus plus media. Green is the choroid plexus plus the TNFα and dexamethasone and then red is the choroid plexus plus TNFα. And as you can see here, again, red is the worst. That's the choroid plexus plus the pro-inflammatory cytokine with no steroidal treatment. But when you look at green, after that middle divider line, once we started adding dexamethasone to those wells, you actually see an increase. (35:15) With each subsequent administration of dexamethasone, you actually see an increase in the barrier properties on the cell index of these wells. And again, the dexamethasone was helping to repair the barrier properties of these wells.

Again, blue, that's going to be kind of our baseline and another interesting thing, when you just look at the baseline of the choroid plexus plus just dexamethasone—just to rule out any other effects of dexamethasone on the cells by themselves—you do see an increase in barrier function.(35:44) So, dexamethasone did slightly help tighten the barrier of just plain choroid plexus cells without TNFα, but it's still really interesting to note that dexamethasone was able to repair some of the barrier function in TNF-damaged cells and affected cell.

Possibly, this is one explanation why there is—like with the TNFα, why there is some weakness to the blood brain barrier or the blood CSF barrier and why infected cells could actually trigger the weakening of the barrier. (36:19) And this is promising, because maybe this steroidal treatment or even any other kind of anti-inflammatory treatment might be helpful in patients suffering from HIV-associated neurocognitive disorders.


Slide 34

So, for some conclusions, HIV is still a pertinent global health crisis. This is due to the long-lived viral reservoirs in which the brain and the choroid plexus are one. The choroid plexus is really important, because it's an immune interface with the monocyte trafficking and barrier dysfunction seen in HIV-associated neurocognitive disorders, as well as some other diseases, too. So, it was really important to study the choroid plexus epithelial cell barrier. (37:00)

And I forgot to mention this way, way, way at the beginning, but the reason why my culture is important is because we did this in rhesus macaques—those are primates—and they're one of the ideal models of studying HIV in animals. And so, while they do have some rat and pig choroid plexus epithelial cell cultures out on the market, rhesus macaques are the better model for this specific study with HIV. (37:29) So, that's why we set about to create this choroid plexus epithelial cell barrier.

Again, choroid plexus epithelial cells are the last line of defense, especially in HIV infections. So, we were curious in creating an epithelial cell barrier. And so, we created a 2D choroid plexus epithelial cell culture. We validated that by the pathology, the choroid plexus epithelial cell markers and tight junctions.


Slide 35

Also, the choroid plexus epithelial cell modified 3D culture allows us to study the barrier functions of the choroid plexus in real-time. We were able to see that the pro-inflammatory TNFα impairs choroid plexus epithelial cell barrier, potentially explaining why we have increases in infected cells into the central nervous system. Also, the anti-inflammatory dexamethasone returns barrier function after pro-inflammatory challenge.


Slide 36

So, some future directions, I want to continue to work on this modified 3D cell culture model, especially in terms of HIV and actually look at HIV's effects on this model. These are other applications to other diseases, other conditions, other viruses, bacteria, et cetera. I also want to study the permeability more and see maybe drug diffusion, things like that. This work is in-revision in Frontiers. So, I'm super excited about that.


Slide 37

And then hopefully, from your learning objectives you are able to understand the history, development and function of the Blood-CSF Barrier, the choroid plexus, and understand the pathology and therapeutic potential of the choroid plexus. It's a really understudied region of the brain, and it's obviously my favorite part of the brain. So, I do hope you can appreciate it a little bit more now. And then also, too, how to design a 2D and modified 3D choroid plexus epithelial cell model system.


Slide 38

And I'd like to acknowledge everyone at the Primate Research Center who really helped out with this project, Confocal Core, things like that. Dr. MacLean's lab, who was my PI for my PhD, as well as my Mercelo Kuroda and Woong-Ki Kim who were advisors on my Ph.D. I'd also like to thank Thermo Fisher and Gibco Scientific, for selecting me for this honor and thank you so much for Chelsea and Christy, for all of their hard work in getting this together and making sure I met my deadlines in turning things in.I'd also like to thank my current PI at LSU and then all teachers and professors I've had in my training, who've really gotten me to this point in my career. So, thank you.


Slide 39

Again, thank you guys for your time and if you guys have any questions, I will be happy to take them.


Moderator:  Thank you, Dr. Delery for your informative presentation. We will now start the live Q&A portion of the webinar. And if you have a question you'd like to ask, please do so now. Just click on the Ask a Question box located on the far left of your screen and we'll answer as many questions as we have time for.

Now those questions we are unable to answer live today and those submitted during the on-demand period will be answered via the email address you provided at the time of registration. Okay, Dr. Delery, let's get started. Our first question, what kind of neural progenitors does the CP produce?


Dr. Elizabeth Delery:  That is an excellent question. So, there's been a lot of papers out recently talking about neural progenitor niche found in the choroid plexus. And that's still kind of a newer field, like they're not really sure what types—they're leaning more towards like radial glial cells, like glial and astrocyte cells. Like one paper from a group in Japan talked about how they were able to take these neural progenitor stem cells from the choroid plexus and implant it into a damaged spinal cord. (41:01) And these cells actually differentiated into astrocytes.

So, they were thinking it turns into either astrocytes or glial cells, depending on the signals from the surrounding area and like danger-associated molecular patterns, things like that can actually trigger the differentiation into different cell types, depending on the need from the central nervous system. Does that—is that question—there are a lot of really good papers out there, too, if you're interested in reading more about it. (41:28) They also argue there's age-dependent markers that can affect this and that these neural progenitor stem cells decrease with age. And a really good paper is "Age Dependent Niche Signals from the Choroid Plexus Regulate Adult Neural Stem Cells" by Vargas et al, if you really want to read more about that. So, next question?


Moderator:  Yes, thank you Dr. Delery. Is it barrier function or cell population that you are looking at?


Dr. Elizabeth Delery:  So, originally we were looking at cell population. We wanted to look at the epithelial cells and if that led to more barrier function, to see—since that's like the last line of defense between pathogens and the central nervous system, they first have to cross the fenestrated capillaries. And since we're discussing this, I do want to apologize quickly.


Slide 40

There were some hiccups in the slides, especially when it's showing the choroid plexus, just so you guys are able to see what it looks like. So, with this question, we're talking about barrier function, let me pull that up, so you can see it, too. There was just a glitch in loading the animations. So, as you can see here, it's lining the ventricles of the brain. And then also, here's an example of the fenestrated capillaries and choroid plexus—and again on the next page—which looks more at the barrier function. If you look right here...next.


Slide 41

Okay, so here, you see the fenestrated capillaries and then choroidal epithelium. Now for a pathogen to get into the central nervous system, it's got to first cross the fenestrated capillaries in the center, go through the stroma and pass through all the immune cells of the stroma, and then exit the choroidal epithelial cells into the CSF in the ventricles. So, that's why we started looking at barrier function more, because it's kind of like the last line of defense before moving into the actual central nervous system. (43:12) But we are kind of hoping to use this more as like the true 3D model and make more of a stromal-like layer on top, but we're still playing around with that. So, hopefully we'll be able to do that in the near future. Next question?


Moderator:  Yes, now this question is two parts. How do you control for contamination? And if your plates got contaminated from necropsy,what did you do?


Dr. Elizabeth Delery:  Great question. Okay, so necropsy is actually a super—it's basically an animal version of an autopsy, that's when you're dissecting the animal. And it's a very, I'd say, dirty environment. While we do wear PPE and we are protected and we have a dedicated suite for the necropsy of the primates, you have multiples pathologists and vets in there, pulling out different tissues for different testing types. And it's possible to get fecal matter from one area of the body up into the choroid plexus, when you're trying to pull it out of the brain. (44:10) And so, it's very easy to become contaminated with bacteria. So, we controlled for contamination by one, having as sterile of an environment as possible, wearing the proper PPE and extracting the choroid plexus first before doing other parts of the necropsy, for collection of tissue.

We processed it in a sterile hood and made sure we sterilized it every time we used it. And then we also, if it seemed like it was getting contaminated, we added additional antibacterial or antimycotics like Primocin and Fungizone, if we really needed to supplement the media to make sure contaminants didn't grow. So, that's a great question.


Moderator: Now Dr. Delery, how is the choroid plexus taken?


Dr. Elizabeth Delery:  Perfect. So, like I mentioned with necropsy, they cut the skull open and you're able to pull out the choroid plexus. It looks like this kind of like red string inside the brain and back in the back by the occipital lobe, you actually will see part of it and a pathologist—we have veterinary pathologists who are skilled at the brain and different parts of the body, and they were able to pull out the red string of the choroid plexus and put it into a tube of (inaudible). Next question?


Moderator: Yes. Now why not study (TEERs) in order to study barrier function, in terms of resistance the endothelial layer poses to systemic circulation?


Dr. Elizabeth Delery:  Okay, so that's an excellent question. We looked at the epithelial layer rather than the endothelial layer and basically, I was a graduate student, so I used what we had available to us. And at the time, the Primate Center did not have a (TEERs) set up. So, I used what was available to us and what we had supplies for. But it's a great question on a technology I could use in the future, should I have access to it. (45:59) But I was basically using previously studied models that my PI had used and equipment he had already, because I didn't really have time to write a grant to get more funding from the NIH for more equipment. But that is an excellent suggestion for future testing.


Moderator:  Dr. Delery, can you elaborate on why you couldn't use human choroid plexus?


Dr. Elizabeth Delery:  That's awesome, okay. So, you can't use human choroid plexus because the only samples I get—I don't really know anyone out there who would voluntarily submit to an open brain surgery where we would remove pieces of their choroid plexus. And they have, they do actually have, I believe, a human line out there, however, it was taken from a patient who had cancer in his choroid plexus. And that can affect growth rates of the cells and behavior of cells. (46:49) So, we didn't want to use that, just because it might have different properties than a true normal choroid plexus since it was cancerous. So, that's why we stayed with the next best model, the primate model.


Moderator:  Thank you Dr. Delery. Now after extracting the choroid plexus, do you dissociate it into individual cells before culturing or do you put the whole string in the media?


Dr. Elizabeth Delery:  Excellent question. So yes, actually we did, we mechanically dissociated it. We chopped it up with a scalpel until we had little pieces of it. And then we also used a digestion Collagenase/Dispase solution, to help break it up a little bit more. It was easier...if cells attached in clusters, because we didn't dissociate it really well, they actually grew a little bit better. But if you put whole choroid plexus chunks in there, it also just didn't grow well. So, it was kind of that fine line in between. (47:46)

And we do have a paper coming out that's currently in-revision, that will have a more detailed methodology, too. So, if you're interested in that and you want to follow-up with me, it should hopefully be out in the next couple of months or shoot me an email. It was on the very first slide. Let me pull it back up right now, actually, so you guys can get it. You're more than welcome to submit your questions through Thermo Fisher and Gibco or email me and ask me any questions.


Slide 42

Or if you just want to say, "Hey, send me the paper when it gets published," and I would be more than happy to do that.


Moderator:  Thank you, Dr. Delery. Now we've got some great questions coming in. We have time for a few more. And let's go with this one, it's come in several times. How many replicates were done with the xCELLigence studies?


Dr. Elizabeth Delery:  Oh, awesome question. So, for xCELLigence, we're lucky in that it's a 16-well plate. And as you remember from some of the pictures—let me pull up, so I can show you right now...


Slide 43

Dr. Elizabeth Delery:  You can see four lines here and obviously 16 divided by four is four. So, we had four separate wells for each condition and they were averaged together to produce these lines.


Slide 44

Kind of when you see this—you see the long vertical lines here, that's actually the standard deviation on the graph.


Slide 45

They were a little bit messier for some of the other ones, which is why I wanted to do just the lines themselves. But that's four replicates for that one right there. And I know replicates are kind of an iffy subject, because do you mean replicates of animals or do you mean replicates of studies? We repeated the TNFα studies and the dexamethasone studies with a couple other animals, too. I believe it was three with TNFα and two with dexamethasone. So, in addition to multiple wells for each animal, we did have more than one animal to practice it in. (49:23)

Again, a lot of people who work with mice are going to be upset by the fact that we were only able to do five primates for all of these studies. We've done more since then, but just for this, the paper topic, we did about five primates, just because they are a select resource and more scarce than mice, and also way more expensive to house for a year than mice. So, we used our available resources as an appropriate (n) for primate studies.


Moderator:  Thank you. Now our next question, how are you using this in your current studies?


Dr. Elizabeth Delery:  Oh, that's awesome. Okay, so I've kind of moved a little bit from the HIV studies. I'm now studying neurobiology of alcoholism and inflammasomes and opiates in the brain. And actually, as you know, alcohol and opiates have to cross the central nervous system or cross the blood brain barrier and blood CSF barrier to enter the central nervous system. And so, I'm working that into my PI's grants and also submitting one of my own grants later this year, to start looking at the choroid plexus more in relation to other conditions and other disorders.


And having a primary cell culture that allows me to do additional testing on it is fantastic. And we have some frozen cultures, too, that we're going to work with in the future. So, I'm excited to see if I can expand on these barrier properties, to study opiates, alcohol, things like that.


Moderator:  Thank you, Dr. Delery. We're excited to see this, too, and we'd love to know what do you hope to accomplish with this?


Dr. Elizabeth Delery:  So, ideally—so, I know this is a really tough topic for a lot of people, but animal research. You know, if you're familiar with the US IACUC laws, the goal is to reduce, replace and like limit the number of animals we use, replace it with more simple models. Because we want to make sure that they have the most humane care and we're not causing undue suffering or anything like that. And while I realize it's a necessary step in pharmacological testing, you know, disease modeling, things like that, I would like to create ways that we limit the number of animals that we use. (51:22)

And so, I hope that this culture can be used to study conditions ex vivo, that would also limit the number of primates that would have to be experimented on. Whether it's drug delivery or the effects of drugs on the blood CSF barrier, et cetera, I'd love to use this model to help, one, humankind increase quality of life, but also limit the amount of animals we use in studies. So, that's my hope for this model going forward.


Moderator: Thank you, Dr. Delery. Now do you have any further comments for our audience?


Dr. Elizabeth Delery:  No, thank you guys so much for tuning in today to watch this and again, please reach out to me on my email. Again it's listed right here, too, and then social media.


Slide 46

I would love to know things you want to learn about, things you want to talk about. If you're watching and you're applying to grad school soon, feel free to let me know if you need any help or advice going forward or picking a dissertation topic or a PI or whatever. I'm still fairly new to the post-doc game, so not too far away from the Ph.D. that I can't provide advice if you guys need it. And again, just get out there and love science, love what you guys do and feel free to reach out to me and connect with me on Instagram or LinkedIn. (52:37) I believe I'm the only—or like the top Elizabeth Delery on LinkedIn right now. So, feel free to reach out to me on social media.


Moderator:  A big thank you again to Dr. Elizabeth Delery for her time today and her important research. I'd also like to thank LabRoots and our sponsor Thermo Fisher Scientific for underwriting today's educational webcast. Now before we go, I want to thank our audience for joining us today and for their interesting questions. Again, just a reminder, those questions we did not have time to answer today and those submitted during the on-demand period will be addressed by Dr. Delery via the contact information you provided at the time of registration. (53:14)

This webcast can be viewed on-demand and LabRoots will alert you shortly via email when it's available for replay. We encourage you to share that email with all your colleagues who may have missed today's live event. Thanks for joining us today and we hope to see you again soon. Bye-bye.

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