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Vivek Kamat, PhD

Postdoctoral Associate, Florida International University, Miami, FL, USA

Vivek Kamat, PhD

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Biography

Dr Vivek Kamat received his PhD from the University of Pune (India) in 2018. During his doctoral training under the supervision of Dr Kishore Paknikar and Dr Dhananjay Bodas at Agharkar Research Institute (Pune, India), he mastered the size-controlled synthesis of a variety of nanoparticles and the process of soft lithography fabricating complex microstructures using natural and artificial polymers. His work involved the size-controlled synthesis of drug-loaded nanoparticles and assessing their biological effect on cancer cell lines. Further Dr Kamat worked in the area of tumor microenvironment specifically understanding cellular response and growth in confined volumes. He has experience in modeling and simulating biological system Insilco using COMSOL to study fluid dynamics, particle transport, and heat transfer. Dr Kamat has experience working in interdisciplinary areas involving cell biology, nanotechnology, electronics, and microfabrication.

Dr Kamat joined the laboratory of Professor Shekhar Bhansali at Florida International University Department of Electrical engineering (FIU, Miami, USA) where he is working on understanding circulating tumor cells using electrical and optical methods to investigate cell-cell interactions and tumor microenvironment. He is also involved in integrating artificial intelligence and machine learning to understand and predict cancer progression. He has active collaboration with Professor Kalai Matee at the Herbert Wertheim College of Medicine (FIU) working on transcriptomics analysis of cells growing in continuous flow conditions in microfluidic devices and along with his research he is involved in active teaching at the undergraduate level at FIU College of Engineering.

Learn about Vivek’s research

Title: Cancer on a chip: A microfluidic 2D and 3D cell culture system for studying cellular microenvironment

Learning objectives

  • Understand the difference between conventional and microfluidic cell culture
  • Learn the process of fabricating microfluidic cell culture chips and the requirements to establish a successful cell culture
  • Carrying out biological assay in microfluidic cell culture systems to understand cellular function

At present cancer research focuses on three major areas: cancer diagnostics, drugs development, and next-generation therapies. About 90% of the in vitro research relies on traditional two-dimensional (2D) monolayer cell culture systems. 2D cell culture systems fail to accurately recapitulate the structure, function, physiology of living tissues, and this means that the results from studies involving efficacy of new drugs, gene expression, metabolic pathways, and cell proliferation do not correlate to actual in vivo scenarios. In contrast, a 3D cell culture system promotes many biological relevant functions that are not observed in 2D. The primary reason for this is attributed to two reasons: 1)In vivo cancer cells experience limited diffusion of oxygen, nutrients and signaling molecules in a dynamic way, which the 2D fails to mimic. 2)Cellular interaction, function, growth and signaling all occurs in a highly complex 3D architecture with the influence of extracellular matrix and other regulatory factors, which cannot be recapitulated in 2D systems.

To achieve this dynamic coordination, microfluidic cell culture systems can be employed that can provide a continuous flow of nutrients, exchange of gases and other regulatory factors in a well-controlled manner. Such a system is ideal to mimic the in vivo environment of cells. Coupling microfluidics with 3D cell culture system will allow study of cellular functions such as proliferation in dynamic systems, cell-cell interaction and cellular response to the external environment in a much more realistic environment. Further, microfluidic systems give an opportunity to study cellular interaction by fabricating microstructures and artificial scaffolds to study cellular movements and the underlying mechanobiology. Using the microfluidic approach better drugs and therapies can be developed which can be easily translated to in vivo systems and hence can bridge the gap between in vitro and in vivo systems.

Watch the webinar

(00:00)

Slide 1

Moderator:  Hello everyone, and welcome to today's live webinar, "CANCER ON A CHIP: A microfluidic 2D and 3D cell culture system for studying cellular microenvironment," presented by Dr. Vivek Kamat.

I'm Christy Jewel of LabRoots, and I'll be moderating this session. Today's educational web seminar is presented by LabRoots and brought to you by Gibco at Thermo Fisher Scientific.  Now, let's get started. 

(00:32)

Today's event 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 them in the Ask a Question box and click Send. We will answer as many questions as we have time for at the end of the presentation. If you have trouble seeing or hearing the presentation, use that Ask a Question box and let us know you’re having a problem. 

This presentation is educational and thus offers continuing education credits.  (01:04) Please click on the Continuing Education Credits tab, located at the top right of your presentation window, and follow the process to obtain your credits.

I now present to you today's speaker, Dr. Vivek Kamat, post-doctoral associate, Florida International University. For a complete biography on our speaker, please visit the biography tab at the top of your screen. Dr. Kamat, you may now begin your presentation.

(01:31)

Dr. Vivek Kamat:  Thank you for the introduction. Good morning, my name is Dr. Vivek Kamat. Firstly, I'd like to thank Gibco and Thermo Fisher Scientific for giving me this platform to share my work. I would also like to thank Jessie for giving this opportunity to talk about microfluidic 2D and 3D cell culture systems for cancer research.

(02:01)

Slide 2

To start with, I would like to share some background information and geographical insights about my education, workplace and academic career. I was born in the City of Pune, also known as "Oxford of the East," located on the Western Indian state of Maharashtra. The city is blessed with vibrant, rich culture and, also, the highest number of research organizations in India.

(02:23)

Slide 3

I did my graduation and post-graduation in biotechnology from Garware College and carried my doctoral work at Agharkar Research Institute and received my Ph.D. in 2018. After which I immediately joined the group of Dr. Shekhar Bhansali at Florida International University, in the Department of Electrical Engineering.

(02:43)

Slide 4

So, to begin with, here are the cancer statistics obtained by World Health Organization, which shows the prevalence of cancer throughout the globe. In the image, it can be seen that premature mortality rates are significantly higher and is the number one leading cause of death in 48 countries—which are indicated by the blue color—is the second leading cause of death in countries marked with light blue color. And countries with fourth and fifth-leading cause of death are marked in orange and red color. It is expected that that until 2020 there will be 18.1 million new cases with 9.6 million deaths due to cancer, and therefore, cancer research is one of the major priorities in developed and developing nations.

(03:29)

Slide 5

So, currently, the major challenges faced are early detection, cancer prediction, new drug screening and post-cancer treatment. So, to address this challenge, the first experimental step starts on carrying out in vitro cell culture assays. But there is a need to develop better models to study cancer, as we all know that the magnitude of radiation which we receive during performing in vitro to in vivo transitions.

So, we need better systems, because to study cellular functions and behaviors in dynamic conditions which mimic in vivo scenarios, understand the tumor microenvironment in a realistic way and study various co- and multi-culture systems, which would eventually bridge the gap between in vitro and in vivo systems, generating realistic data in primary stages of investigation.

(04:25)

Slide 6

So, let us see how dynamic cellular microenvironment works. Dynamic system is combination of biochemical, physical and physicochemical factors which constitutes cells' microenvironment. As seen in this schematic, cell experiences blood flow, shear stress, pressure drop, stretching, interaction with physical barriers and collectively imparting physical forces on the cell. Further, there is concentration gradient, exchange of gases that are chemical signaling molecules and extracellular molecules which constitutes the biochemical factors. And lastly, physicochemical factors such as temperature, pH collectively interact and affect the cells and yet in such a dynamic system, cells are able to replicate and perform its designated function.

(05:12)

Slide 7

But in traditional dish based or conventional assays, these functions such as sensing, replication, exchange of materials might be altered, as cells do not experience a dynamic environment. It is known that cell exchange, morphologies in cells—I mean, I'm sorry, it is known that cell changes morphologies when exposed to substrate deformation, but cells in culture plate do not experience these forces and eventually lose these properties. Therefore, studying dynamic system for studying cellular microenvironment without compromising natural properties and behavior of the cells is crucial. So, how can we study such dynamic systems? And one of the answers is microfluidics.

(05:59)

Slide 8

So, what is microfluidics? Microfluidics is study and manipulation of small volumes of fluids in channels and chambers with dimensions of tens of microliters. Microfluidic cell culture attempts to develop devices and techniques for culturing, maintaining, analyzing and experimenting with cells in micro-scale volumes. Now using this microfluidic technique, we can actually make devices to carry out 2D and 3D cell culture systems. In the following image, shows a microfluidic chip comprising of inlets for media, a culture chamber in which cells can be grown, an outlet for removal of spent media. So, such chips can be used to culture cancer cells for assessing various different cellular activities.

(06:43)

Slide 9

So, now let us see what are the differences between in vitro culture and microfluidic cell culture system. In in vitro system, the cell substrate is transparent, stiff and has low gas permeability, while microfluidic systems, the substrate is soft and is flexible and also has high gas permeability. In microfluidic systems, cells can be handled and cultured, ranging from single to thousand cells which is actually not possible in a plate-based assay. Conventional cultures is carried out in flask, dish and plates, which require larger volume, as compared to microfluidic devices which can work with volumes as less as 60 nl.

Also, nutrient in traditional culture are in great excess and media needs change after every 48 hours, while in microfluidic system, media turnover is faster and some reports indicate more glucose consumption. One peculiar thing about microfluidic cell culture is the proliferation rate and in conventional assays, the proliferation rate is typically between 18 to 24 hours. But in microfluidic system, there have been reports where it has been shown less, as well as faster proliferation rates of the cells.

(07:57)

Slide 10

So, how do we culture cells in microfluidic systems? Culturing cells in microfluidic devices require a strong understanding of certain fundamental principles spanning disciplines such as physics, chemistry, biology, and engineering. So, it's more of an interdisciplinary kind of a study and there are certain criteria which need to be followed. Firstly, the developed systems should closely mimic conditions that exist in vivo and the person handling should have good command on cell culture techniques to translate methods from macro- to a microscale. And this is usually the most challenging part. Also, another criteria is the person should have a strong background in designing and microfabrication techniques.

(08:42)

Slide 11

So, let us see what are the steps involved in establishing a successful microfluidic culture. So, the first step, obviously, is the fabrication of the device. This is typically carried out by techniques such as photolithography and soft lithography, after which you have to select the type of cell which you are going to investigate. So, it can be cancerous, non-cancerous stem cells and based on their properties such as adherent or non-adherent, we might need to fabricate a different or (new kind @09:05) of microfluidic chip.

Then we need to perfuse media into the chip, which is usually done using syringe pump or peristaltic pumps.  The chips are then monitored using microscopic technique. We usually use live/still microscopy in our lab. And at last, the spent media is collected, which contains all the extracellular components and dead cells.

(09:30)

Slide 12

So, let us see, the first step which is the fabrication process, in details. So, microfabrication process is divided into two parts. One is photolithography and the other is soft lithography. So, as the name suggests, photolithography is a technique which is used to etch surface using light and this is usually a UV source. Basically, in photolithography, we have a mask, which is nothing but a stencil on which the design of the microfluidic chip is etched on a silicone substrate which is called a "wafer". A layer of photoresist is coated.  In our lab we use SU-8 for this.  After which the mask is aligned on the substrate and UV is exposed.

Once exposed for a definite time, the UV light passes through the stencil of the mask and erodes the photoresist, at that point creating design on the mask onto the substrate. After etching, the final baking and development is carried out to obtain the design on the substrate. Further, PDMS, which is known polymer, which is called polydimethylsiloxane is added to the substrate and allowed to cure. Once PDMS solidifies, it is peeled off and the design gets replicated on PDMS.

In case of soft lithography, the mold is first prepared and then PDMS is just added into the mold and cured.  PDMS is a room temperature vulcanizing rubber that solidifies and mixed with curing agent, and this process can be accelerated by applying heat. So after curing, the PDMS is peeled and we are left with design pattern on the PDMS.

(011:08)

Slide 13

Further, this is the process of photolithography. So, as I explained earlier, in the first step, the silicon wafer is removed from the clean room. It is kept on the spin coater. In the third step, the photoresist such as SU-8 polymer is added and is spun at desired RPM to achieve a specific thickness, and then it is pre-baked.  After this step, the mask is selected. Step 6 and 7 is aligning of the mask using a mask aligner. In Step 8, UV is exposed. And finally, in Step 10, the exposed surface is washed and then we have the desired geometry on the wafer.  Now this acts as a mold and PDMS is added on the wafer which is allowed to cure and the microfluidic chip is peeled. So, in the last image you can see the design of the microfluidic chips and the channel.  

(012:03)

Slide 14

In case of soft lithography, the process is quite simple. And I would like to share one-step protocol which we use in our lab to design circular microchannels and chambers. The protocol describes using agarose powder, which is readily available, which you can mix with water and allow it to boil. We then cut the tip of syringe, aspirate this agarose powder and allow it to cool. Once cooled to room temperature, push the agarose out from the syringe to get cylindrical agarose shape, cut the agarose into desired height and pass the copper wire through the agarose cylinder.  Then we place this entire setup into a box and you just add PDMS to it.

We keep the entire setup at 70 degrees for three hours, which is the curing process and later once PDMS is solidified, you just remove the chip and boil it in water. The agarose will melt off, and you can simply pull off the wire, having circular channels and chambers, and this can be used to culture cells.

(013:06)

Slide 15

So, there are certain parameters which need to optimize for successful cell culture. And these parameters are first is the flow rate. So, flow rate can be set using syringe pump or peristaltic pumps. Usually, flow rates can be set to mimic actual flow condition in blood vessels and capillaries, which range from one to 2000 μl per minute.  Second is the temperature and CO2 control. Cells require physiological temperature and CO2 control. Therefore you can use a PID controller, with heating plate, coupled with CO2 controller. Or there is also an automated onstage incubator with CO2 systems, which are available, which can be mounted on microscope to perform live/still imaging. And lastly, we also need to monitor the cells for which you can use inverted microscope or even lens microscope.

(013:57)

Slide 16

So, using these techniques we attempted to culture cells in the 2D platform, in the first image, you can see the microfluidic chip, which was fabricated using photolithography. The chip consists of inlets and outlets, which are interconnected to small chambers, which are 100 μm in height and 3 mm in diameter.  Media containing DMEM/F12 supplemented with 10% FBS was used for the experiment and we selected MDA-MB-231 cells for our study.

After the chip was fabricated, the chip was coated with poly-L-lysine and fibronectin to allow cells to adhere. Then MDA-MB-231 cells were inoculated in the channel and allowed to incubate for two-to-four hours. This is crucial since cells need some time to adhere to the substrate and we cannot immediately start a flow rate. After two-to-four hours, syringe with the media is placed in the pump and set at a desired flow rate and connected to the chip.

We need to take care not to set the flow rate too high because that would end up leading formation of bubbles, which is a major challenge in microfluidic 2D and 3D cell culture systems. Further, we incubated the system at 37 degrees Celsius with 5% CO2. We monitored the cells by recording time lapse imaging of the cells after every three minutes for 12 hours at different flow rates.

(015:31)

Slide 17

And I have a sample video, which shows the migration of the cells. The flow rate was 3 μl per minute and from our data, we came to a conclusion that flow rate has negligible effect on cell migration. In this time lapse, it is seen that cells are flowing in flow conditions but they're not affected by flow rate. Rather, we can also see some cells which are migrating in the opposite direction of the flow. Also, it was noted that cell proliferation was much slower, although these findings are still under investigation, but are initial proof of concept.

(016:07)

Slide 18

Further, we also found that cells do tend to adhere to the side walls of the microfluidic chip as compared to the central region. This is probably due to low pressure at the side walls, as compared to the central region, and also, due to high surface roughness at the edges, which provide better contact points for the cell. Another recent investigation which we are working on is testing of different drug loaded nanoparticles on cancer cells. In our finding, post six hour treatment cellular death and cell shrinking was observed. But strikingly after 12 hours, we do observe some cells trying to reestablish and proliferate which might be useful to explore drug resistance and even diffusion of drug in continuous flow. This might help develop better drug molecules to treat cancer.

Although these studies look simpler, there are several challenges which are encountered in microfluidic systems, the major being controlling evaporation rate and generation of bubbles. So, in the Image C, you can see that you usually end up having these bubbles in the microfluidic chips, which is a major challenge. But overall, if these problems are taken care, it's easy to establish a 2D or a 3D cell culture system.

(017:28)

Slide 19

Now when we come to a 3D environment, everything changes. So 3D tumor models are most ideal to study cancer.  Abnormal growth of cells lead to tumor formation and you know tumor comprises of several cells in clusters and this makes it challenging to work with. So, as in the schematic, tumor cells experience non-uniform exposure to nutrients and oxygen, there is difference in proliferation gradient along with altered cell signaling. Also, exposure to drug is limited because most of the cells are on the surface and the cells at the core of the tumor do not get exposed to the drug molecules. But technically, this is what is in real conditions, right. So, how do we develop tumor models in continuous flow system?

(018:15)

Slide 20

So, our first step of study was to investigate different scaffolds, which will serve as a 3D architecture for the cells to grow. So, we selected gelatin, cellulose, PDMS, and one novel scaffold which is a carbon-based scaffold for our study. The study is still under investigation, but I would like to share some very primary study results.

So, the average pore size of the scaffold ranges from 5 to 50 microns, which depends on the synthesis protocol and how we make the different scaffolds.  I have incorporated an image of the scaffolds and primary studies we did using a fixed cell plate assay, where we get it out different biocompatibility studies and also MTT assay. So, initially, we tested the biocompatibility and it was found that these scaffolds are pretty biocompatible, like we just did the studies on NIH and MDA-MB-231.

(019:10)

Slide 21

Further, these scaffolds were incorporated into microfluidic chip, which was designed in such a way which would hold the scaffolds in the cell culture chamber. So, the chip comprises of inlet and outlet, which a single large chamber—and this chamber was somewhere around 40 mm by 10 mm—in which the scaffold was placed prior to bonding with the glass slides. Then the medium inlet was connected and using the same protocol described earlier, we were able to culture cells on these scaffolds.

In the following image, you can see that we have this experimental setup where we connect the media to the chip, but this setup is usually on an on-stage incubator under the microscope or also, you can place this entire setup in a sealed incubator.

(020:04)

Slide 22

So, one of the technical problems which we were facing during our studies was that when we stain the scaffolds, we usually end up with dye, which remains back in the scaffold, and it is very difficult to image them. So, as you can see, when we tried using gelatin and PDMS scaffold, we were able to see cells growing in voids of the scaffolds and they established small, tumor-like structures. Nevertheless, we were able to establish these cells in the scaffolds.

(020:38)

Slide 23

We also observed similar results when we used PDMS scaffolds and also cellular scaffolds. As you can see that we have these tumors—tumor spheroids which are growing on these cellular scaffolds after three days of incubation.

(020:57)

Slide 24

Lastly, the scaffold which right now we are working on is the carbon based scaffold. And in the first image you can see that it has an interwoven network of these fibers along and we tried growing cells in these fibers, and we were pretty successful in growing cells up to 14 days. As you can see in the image, we ended up with tumor mass of cells ranging from 100 to 200 μm in diameter. At present, we are working to characterize this scaffold and tumor bodies and I've not included that data in this presentation.

(021:38)

Slide 25

So, to summarize, microfluidic cell culture microchip was successfully fabricated using photolithography.  A simpler method for fabricating was explored using soft lithography. We did not see any influence of flow rate on cell migration. Cells prefer to grow in the region of low pressure along the side walls of the chip in the 2D microfluidic chip. And the 3D cell culture was established in continuous flow by incorporating different biocompatible scaffolds.  Lastly, we were successfully able to culture cells up to 14 days which eventually turned out to be tumor cells.

(022:23)

Slide 26

Lastly, I would like to acknowledge my mentors. Dr. Shekhar Bhansali for providing me the opportunity to work in his lab. Dr. Dhananjay Bodas and Dr. Kishore Paknikar, for his excellent mentoring and support during my Ph.D. which eventually made me an independent researcher. My colleagues and friends, Dr. Natalia and Dr. Ehnaz for their scientific input and help for this work. And lastly Isabella and Dennis, who are working with me on this project and we are exploring newer possibilities every day.

(022:56)

Slide 27

Thank you. And you can reach me out on these contact listed. Any questions?

(023:14)

Moderator:  Dr. Kamat, thank you for your presentation. Okay, so let's get started with our Q&A. Just a quick reminder to our audience on how to submit questions. Just click on that "Ask a Question" box located on the far left of your screen. As a reminder, any questions that we are unable to get to today, Dr. Kamat will be answering them via email. So, don't worry if we don't get to your question right away. Okay, let's see. Our first question is why not use serum-free medium culture conditions?

(00:23:43)

Dr. Vivek Kamat:  Hello. Yeah, can you repeat the question?

(023:50)

Moderator:  Absolutely. Valerie is asking why not use serum-free medium culture conditions?

(023:59)

Dr. Vivek Kamat:  Okay, yeah, so basically when we started doing the studies, I never tested with serum-free media. Because usually the thing is when culturing cells in these kinds of microfluidic devices, I usually end up with less cell viability if I start using media free of serum. So, usually media supplemented with 10% FBS is the most ideal condition and in that kind of a condition, we usually get cells proliferating in a much better way, rather than using media free of serum.

(024:37)

Moderator:  Thank you. Now we have a question coming in from the University of Michigan. What is the size of organoid when you finished 14 days scaffold culture?

(024:50)

Dr. Vivek Kamat:  Yeah, so after 14 days of the culture, when we measured the size of the scaffold, when we measured the size of the organoids, we were able to get somewhere around 80 to 150 μm size organoids. So, that was it yeah.

(025:12)

Moderator:  Thank you. Here's our next question. When performing cell migration studies, what was the seeding density of cells in the chip and how were the flow rates optimized for the study?

(025:27)

Dr. Vivek Kamat:  Okay. So, when performing the migration study, we basically seeded (inaudible@25:30) in the microfluidic chip. And to optimize the flow rate, basically we started from 3 μl/minute and went up to 100 μl/minute, which is similar to the flow rate observed in arteries and veins So, in that way we were able to see the cells migrating against the flow rate, also. And that was the condition in which we set up the experiment.

(026:06)

Moderator;  Now, Dr. Kamat, do you have experience with co-cultures of cells?

(026:13)

Dr. Vivek Kamat:  We haven't tried co-culturing cells yet. But soon, I'll be working on a co-culture system with astrocytes and neural cells using the same PDMS or the carbon-based scaffold.

(026:31)

Moderator:  We have another question coming in here. You mentioned the scaffold had an approximate core size of 50 um, however, some of the spheroids you observed were 100 um in diameter. Does this suggest that the scaffold was flexible enough to stretch, allowing a larger spheroid?

(026:55)

Dr. Vivek Kamat:  So, in the initial studies, microscopic studies, which I have observed although the core size is smaller, usually the cell tends to cling on these scaffold or these groups, and usually we see at some point that the scaffold is flexible. In the case of PDMS, we did observe the scaffold to be quite flexible and cells growing within the scaffolds.  And then we also see some of the cells growing outside the scaffold, making a larger spheroid kind of a structure.

So yes, the scaffolds are flexible, but that also depends on type of the scaffold. So, we did not see that in case of carbon-based scaffold, but in PDMS scaffold, we did see that the scaffold is quite flexible and we usually get large spheroids.

(027:49)

Moderator:  Thanks for that question, Jeffrey, and we actually have another one from him. We're going to go onto this one. Did you observe significant non-specific cell binding to the tubing interfaced to the PDMS chip?

(028:04)

Dr. Vivek Kamat:  Yeah, that's a good question, actually.  So, it usually ends up that we do see a lot of cells which bind to the tubing. But not a significant amount of cell, but yes, cells do tend to adhere to the tubing, as well as to the channels, the inlet channels, specifically at the inlets and outlets. So, to give a rough estimate, around 5 to 10% of cells, seeded cells will usually end up into the tubing and into the inlets and outlets of the chip.

(028:38)

Moderator:  Thank you, Dr. Kamat. And here we have another question. Have you investigated the effect of different culture media on the growth and metabolism of cancer cells grown ex vivo?

(028:53)

Dr. Vivek Kamat:  No, not really, actually. I have not tested different type of medias yet on the cancer cells. But in one of my previous studies, we do observe changing morphologies when we use different kind of media in continuous flow cultures. But I haven't used – I haven't done a complete study using different type of media yet.

(029:17)

Moderator:  Okay, and our next question coming in here. We have so many great questions coming in. As a reminder to our audiences, questions that Dr. Kamat is unable to answer today, due to time constraints, he will be answering them via the contact information you provided at the time of registration. Alright, let's go on to our next question. How do you make a 3D structure in a microfluidic device?

(029:42)

Dr. Vivek Kamat:  Okay. So, basically, we are basically making these scaffolds which are of this three dimension and then incorporating them in the microfluidic device. So, there are several protocols in which the device itself, the scaffolds are built inside the device. But before binding it to the glass, we do incorporate the scaffolds into the microfluidic device. So, basically, we are not making the structures in the device.  The structures are made separately and then incorporated in the device by bonding it, either plasma bonding it or by other methods. So, in case of PDMS, usually, we make these porous scaffolds and then we incorporate it in these smaller devices. And then we carry out cell culture studies. So, it's kind of easy in a way that we can later on just open up these devices and remove these scaffolds, so we get the entire structure intact, but with cells which are growing on the scaffold. So, it's quite easy to extract this scaffold from the devices. And that's how we work.

(031:00)

Moderator:  Thanks, Dr. Kamat. Now what are the chances of contamination and other troubleshooting?

(031:05)

Dr. Vivek Kamat:  Oh yeah, actually that's a good question. So, we usually end up with the...the chance of contamination is...it depends on the handling and also on the conditions. Usually you would not get the contamination if you have properly sterilized the media, the microfluidic chip and other, you know, devices and tubing and other things.  But usually, if the tubing and the connectors are not taken care of, then we usually end up with contamination and also other troubles. The major troubleshooting is, as I mentioned, we usually end up with bubbles, which is where the major kind of issue is when using microfluidic devices. And also, sometimes evaporation rates. So, if the flow rate is too less, it usually evaporates the media or something like that, and then that's another challenge. So, it takes a lot of time to optimize these systems, but once it is optimized, it gives real good results.

(032:18)

Moderator:  Now, let's go back to the topic of scaffolds. Now I have a few versions of this question coming in. Was the scaffold treated prior to the experiment?

(032:27)

Dr. Vivek Kamat:  So, what we usually do is we treat the scaffolds with poly-L-lysine for cell adherence. So after the scaffolds have been prepared, we usually wash them in 70% IPA and then two or three washes with CVS and then try to keep the scaffold in deionized water for at least four-to-six hours. And after that, we treat it with poly-L-lysine for cell adherence.

(032:58)

Moderator:  Now, Dr. Kamat, was any viability assay performed and if not, which are the assays to characterize the tumor bodies?

(033:09)

Dr. Vivek Kamat:  So, actually, we did try to study the cells under the microscope. So, basically, other assays which can be performed would be latex staining or you know, ATP generation assay.  And also, one can go for Ki67 based assays, which is a proliferation marker in tumor cells. So, basically fluorescent-based assays can be carried out to study cell viability and proliferation.

(033:43)

Moderator:  Thank you, Dr. Kamat. Now we have time for one more question. Which drug loaded nanoparticles were used in the study and how was the study carried out?

(033:55)

Dr. Vivek Kamat:  So, this is one of the studies which I've been trying in my group. And basically, we are making different nanoparticles which are natural polymers, using chitosan-alginate nanoparticles. And what we do is we synthesize these nanoparticles in the range of 100 to 150 nm, which are encapsulated with the anticancer drugs such as doxorubicin or paclitaxel.  And then we tried to study the effect of these nanodrugs on cancer cells in continuous flow culture. So, basically, using these nanoparticles to study how does cellular uptake or how does the activity, drug activity and interaction with the cell takes place in a continuous flow kind of a culture?

(034:54)

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

(034:58)

Dr. Vivek Kamat:  So, yeah, I mean, this field is quite challenging and interesting, and we can do a lot of studies using microfluidic cell culture systems. Further study, it's possible in the near future to translate all the in vitro studies to microfluidic systems. It would be really great and would give us a great depth of understanding in studying these cellular microenvironment and cellular interaction at the more dynamic level, rather than using the traditional in vitro assays. So, thank you everyone.

(035:37)

Moderator:  I would like to once again thank Dr. Vivek Kamat for his time today and for his important contribution to cell culture research. I would also like to thank LabRoots and our sponsor Thermo Fisher Scientific for underwriting today's educational webcast. Now before we go, I'd like to thank all of you for joining us today and for your great questions. Again, questions we did not have time for today and those submitted during the on-demand period will be addressed by our speaker via the contact information you provided at the time of registration. This webcast can be viewed on-demand. LabRoots will alert you via email when it's available for replay, and we encourage you to share that email with your colleagues who may have missed today's live event. You will now be redirected to the registration page for our upcoming webinars on "Choroid plexus epithelial cell 2D and modified 3D cell culture models," presented by our next Cell Culture Hero, Dr. Elizabeth Delery. Dr. Delery will be presenting live on July 31st at 9 a.m. Pacific Time. We hope to see you there! Bye for now and have a great day.

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