Monthly Archives: June 2012

6.29.12 Changing medias, fixing and staining hBMSC cells on scaffold for day 1

Happy Friday! I hope you are finding ways to stay cool! I was luck enough to be working in a very cool lab (pun intended!)!

This was a busy day for me. I needed to take day 1 samples for the hBMSC  I plated yesterday for both fixing and staining to observe today as well as freeze samples to run  in a Picogreen DNA Assay at the end of the experiments. More on that later. I also needed to move the scaffolds with cells  still growing to new wells and add new media for both the human and mouse cells. It is important to the change the scaffolds to new wells since the cells are great at adhering to surfaces and since we are only interested in the number of cells grown on the scaffolds themselves, we must move the scaffolds to new wells each time we change the media.

Since the technique of fixing the cells and staining the cells both took one hour each,  I was able to change the media and freeze the samples while the scaffolds incubated. Once the stain was set it was on to the microscope to take some pictures!

Here is one of the hBMSC under the red stain for cytoplasm under 10x and here’s the same view with the green stain showing the nuclei: . Here’s a set under 4x:  and  Notice how spread out these human bone cells are compared to the mouse cells (cytoplasm on the left and nuclei on the right: and . There are definitely appear to be more mouse than human cells after the same incubation period.  We will have to wait to see the results of the DNA assay to compare the number of cells quantitatively (with numerical data) not just qualitative (observational data).

Enjoy your weekend and stay cool!

6.28.12 More fluorescence imaging and seeding human bone marrow stem cells

Good evening! Are you ready for the heat? Remember to drink plenty of fluids (water, not sugary soda!!!)  and take breaks if you are running around.

First task up this morning was to finish taking photos of the fluorescence images under the microscope. Since yesterday was my first time doing it, there was a learning curve to the technique and what I should be looking for. In addition, I needed to photograph the images using two different filters to be able to see the two types of stains used, one to stain the nucleus and one to stain the cytoplasm.

In order to understand how the fluorescence imaging works, you need to understand how we see colors (this should be review of 6th grade science). The musical group They Might be Giants wrote a song about the color spectrum: Waves are organized by their wavelength and energy on the electromagnetic spectrum. Visible light is a very thin band of wavelengths on the electromagnetic spectrum between 400 and 700 nanometers (nm). Wavelengths with higher energy have shorter wavelengths and wavelengths with lower energy have longer wavelengths.  Here’s a great website from NASA The Electromagnetic Spectrum, check it out.

Back to the fluorescence images… We used the same microscope we used to check the growth of our cells: But instead of using a regular bulb emitting visible light, we  used a mercury bulb:   and it gives of a green glow: In addition, there are four different filters which can change which wavelengths of light pass through to the sample. These filters limit which  wavelengths of light can pass through to the sample. Remember that  I stained the cells with two stains? These stains then absorbs the shorter wavelengths (called excitation wavelength) allowed to pass through the filter and and emits or allow the longer wavelength (called emission wavelength) to pass to the sample. These longer wavelengths are the ones that allow for the different colors in our sample. Check out this site for a more detailed description and diagram:  http://cse.lmu.edu/resources/MANE_Labs/Instruments/Epi-fluorescence_Microscope__EFM_.htm

So now that I understood the basics about his this imaging worked, it was time to take some more photos of the cells on the scaffold: Using the filter that allowed red light to be transmitted through my sample I took some images of the cytoplasm of the cells on the scaffold at 10x magnification: and then I switched to the filter that allowed green light to pass through the same sample to capture the nuclei stained green:   Notice the photos are in black and white? That is because the camera I used only captures the differences in intensities of the sample. This is what the image looks like on the computer screen: Once I combine the two images I can then have the computer add color to show the cytoplasm as red and the nuclei as green like this:  Pretty cool huh?

But that was only part of my work today. The rest of my time was spent on seeding the next type of cells on a different plate of scaffolds. This time instead of using mouse bone marrow stem cells, I used human bone marrow stem cells! I followed the same procedures I used to plate the mouse cells (remember that we want to keep everything the same between experiments). The only things that changed was the type of cells used and the media used to grow the cells. Since human cells produce and require different proteins than a mouse, we needed to change some of the nutrients used in the media.

In both of these experiments we will be measuring the amount of cell growth at certain periods of time, day 1 and day 7. Since we allow the time to change in our experiment, it is called the independent variable. Since we do not know the amount of cells grown, this is the factor we are measuring and collecting data on, this is the dependent variable. The dependent variable (the amount of cells) DEPENDS on the independent variable, time. One of the ways we will analyze our data from these experiments will be by graphing our results just like we do in class. Which variable is the x-axis and which variable is the y-axis? Remember y depends on x… We’ll get into more of this next week.

Next up, changing the media of the mouse cells and fixing, staining, and fluorescence imaging the human cells.

6.27.12 Changing media, making stain, staining the cells and flouorescence imaging for Day 1

Hello! It has been another busy day here at NIST in the 3D Tissue Scaffolds lab. After successfully seeding the MC3T3-E1 cells (this is the strain of mouse bone marrow stem cells that have been used in the previous experiments) we were ready to visualize some of the cells grown on the scaffold. But before we could see the cells, we needed to prepare the stains and stain the cells. Without stain the cells look like this at 10x magnification:  We want to see some more details of the cells so we used 2 types of stains: one that will stain the nuclei (“brain” of the cell) green and another that will stain the cytoplasm (the gel like material surrounding the nuclei) red. To take a quick tour of a cell check out Brainpop: Cells and Brainpop: Cell Structures. But I’m getting ahead of myself.

First up in the lab was to transfer 3 of the 18 scaffolds from the plate to the plate with the 6 control wells, rinse them with and fix the cells with a solution including formaldehyde. Formaldehyde acts as a “stop” button in the cell cycle and preserves the cells so that they will not degrade or break down. We set this plate aside for 1 hour.

Next we needed to change move the scaffolds to new wells and change the media. Before doing that we took the plate over to the microscope for a quick check to make sure the cells were growing properly and did not look contaminated. We say many cells growing in the solution and all around the walls of the well. We could not see any cells on the scaffold itself since the scaffold is made of an opaque polymer that does not let light pass through. A microscope works by allowing light to pass through the sample. That is also why samples must be very thin in order to be visible under a microscope. Once we confirmed that the cells look “happy”  (lots and lots of cells without any black stuff (mold) contaminating the wells we moved the scaffolds to new wells, added new media and disposed of the old media. Then we put the plate back in the incubator. We will change the media again on Friday.

Now it was time to make up the stains, rinse the scaffolds and control wells and stain the scaffolds and control wells for an hour. After an hour we rinsed the stain out we added deionized water to the all 9 wells and moved over the microscope to start the fluorescence imagery.

The images captured do not include color yet but they are still really neat: This is the scaffold using 4x (the scale is missing)  and here’s one under 10x with a green fluorescence filter that emits red light to show the cytoplasm of the cells (confusing, I know!)  This is the same view of the sample under 10x with a blue fluorescence filter that emits green light to show the cell nuclei: Tomorrow I will learn how to combine the two images and add color to show the two stains in one image!

6.26.12 Seeding the cells on the scaffolds

I hope you all enjoyed this beautiful weather and are ready for some heat! I was able to enjoy the morning outside and then it was back to the lab in the afternoon for “seeding the cells.” That means we took the cells we had grown in culture and transferred them to small wells with the scaffold in it. Here’s how we did it:

The first thing we had to do was remove the cells adhered to the culture flask. We did this by adding a solution that inhibited the cell membrane to stick to the walls of the flask. We then used the microscope again to make sure the cells were free-floating in the liquid. When we did check we found yes, the cells were no longer stuck to the flask but we also saw that we had allowed the cells to grow longer than we should have and the cells were “confluent.” I learned this means that there were so many cells that they were touching each other. In this photo you can see the cloudy clumps of cells floating in the medium which shows an overgrowth of cells:

Dr. Simon and I discussed whether we should go ahead with our plans to seed the cells we had or should we start fresh and grow a new culture and start to seed the cells on day 2. After checking back to see how the experiment was preformed before (remember I am the third person to perform this experiment) we decided to move forward with seeding the cells.

There were a few steps that we took in order to make sure we transferred the right concentration of cells to the scaffolds that had been used in the previous experiment (200,000 cells per mL). (This is another control in our experiment, like have the curved side of the scaffolds facing down.) First we needed to find out how many cells we currently had in solution. We did this by using a Hemocytometer:   This device is normally used to count blood cells (hemo=blood, cyto=cells) but it can be used for other cells as well. It’s really hard to count a large number of objects placed in random locations. To create an organized system for counting cells, scientists use a grid system to help you keep track of your items as you count. Using a microscope you can them count the number of cells in a portion of the grid sample and use that value to determine the number of cells in a given volume. To make sure you don’t lose track of the number you counted, you use a clicker to help you keep track: I counted 142 cells and Dr. Simon counted 168 cells in the center area. Averaging those numbers gave us a value of 160. We knew that we had transferred 10 um of solution to the device. Since there are 1000 um in 1 mL we multiplied the number of cells (160) by 1000 to get a concentration of 1,600,000 cells per mL.

Great, but we needed to use a concentration of 200,000 cells per mL (see why I say that science is a mixture of math and English?). In order to get the correct concentration we needed to dilute the solution with media by 8 fold. So for every part of solution with cells, we needed 7 parts of media. You use this same logic every time you make juice from frozen concentrate or powder mix! Too much water and the juice does not have much flavor, too little water and the juice is too tart.

Enough about math, back to cells… So we now had the right concentration of cells, now we needed to make sure the plate with the scaffold was ready. Yesterday I described how the tapered or curved side of the scaffold needed to be face down in the wells. Today I made sure 18 of the 24 scaffolds were indeed facing down. The remaining 6 scaffolds were removed from the plate stored on another plate as extras. In order for the cells to have the best chance of adhering to the scaffold, the surface needed to be wet.  Bu the scaffolds are super small and there are tiny holes, or pores in them and the media does not enter these pores easily. After we added the media to the wells, using the micropipette technique you used in the gel electrophoresis labs, we vacuumed the sample to draw air out o force the liquid into the pores:   In every experiment you not only make sure that all factors are kept the same except the dependent and independent variables but you also wan to run samples used as controls. We used controls when we preformed the micro arrays to find the contaminated salad ingredient and used those control results to show what a positive and negative should look like. In this experiment we used a second plate without scaffolds in the wells as a control. We will compare the cell growth in these wells to the cells grown on the scaffolds.  The control plate on the right and the experimental plate with the scaffolds is on the left. To make sure that all of our results are accurate we tested a total of 18 scaffolds and a total of 6 control wells. We will average our results at the end of the experiment (yes, more math!)

After vacuuming the plate, it was time to transfer or “seed” the cells: . Once all the cells are plated we labeled the plates and put them in the incubator and cleaned up.

Up next, changing the media and fluorescent staining on Day 1!

6.25.12 Making Media, Changing Media, Using the Microscope, Preparing the scaffolds

Good evening! Today I worked on a little bit of everything. First up, making more media. When I changed the media last week, I tried to place the media top on the culture plate so we needed to dispose of the media, why? The same reason that you always wipe down your work area before and after handling the cells, contamination! Luckily we had another 50 mL of media from the first batch but we wanted to make sure we would have enough media to seed the cells and run some experiments so we made up some more just to be safe.

Next up was changing the media. That meant switching out the old media from the plate and replacing it with new media. This is done to remove any wastes created from the cells as they grow, remove any dead cells, and replenish the nutrients used as the cells grow. Before putting the cells back in the incubator, we took some more images using both 4 and 10x magnification. Remember that 4x describes the strength of the objective’s magnifying power. The eyepiece also includes a magnifying lens with a power of 10x. To find the total magnification of an image you multiply the two values, so a power of 4x really means an image under the microscope is 40 times larger than viewing it using your naked eye. So what would be the total magnification of an image using a 10x object? Did you get 100 times larger?To find out more about microscopes, check out Brainpop: Microscopes (sligoms, brainpop)

In addition to taking pictures of the cells we also took pictures a ruler used to show the size of the cells. We could see one complete section of the ruler measuring 1 mm or 1ooo um (remember 1 micron = 1 x 10-6 m):  but could only see a partial section of the ruler at 10x: 

This scale was superimposed on the cell images to show the size: MC3T3-E1 cells from day  1 in culture at 10x: Compare the number of cells ans spacing between cells from day one to those taken today, day 5 of the culture under 10x magnification: . Recall that we added 1.7 million cells per 1 mL to the medium and it takes an average of 16 hours for cells to replicate or reproduce through mitosis. Even though some were dead, that still makes a lot of cells! Here’s a picture of the cells in culture at 4x on day 5:

After the microscope work, I practiced flipping the scaffolds in the plates to make sure the tapered side was facing down. In this case, tapered means not flat, jagged. In any experiment we want to make sure that all variables, called controls, are kept the same. In science a variable is a factor that can change. Since we are not testing the amount of cell growth on the tapered and smooth sides of the scaffold, we need to make sure each well is exactly the same. Other controls can include temperature, amount of buffer, and time. What other factors can be used as controls in an experiment?

Here’s a photo of the scaffold in the wells:  . Here’s a close up of the scaffold: Here’s a photo of the tampered side of the scaffold, notice it is not smooth:  And this one shows the smooth side: . So once I figured out what I was looking for, I needed to practice checking the sides of the scaffolds under the sterile hood. Why does the plate need to stay sterile? The instrument that I used, a tiny spatula needed to be sterilized using a flame: . Once the instrument had cooled, I flipped the scaffolds up to check which side was placed down:

That’s all for today, up next, seeding the cells!

6.21.12 Changing Culture Medium and Making Buffer

Good evening and happy summer solstice! Today is the day with the greatest amount of daylight all year as well as the day when the sun’s is at the highest point in the sky making all shadows the shortest.

Today I was able to take some photos of the mouse bone marrow stem cells (mBMSC)s under 10x magnification: The circular cells are dead cells. The living cells have adhered to the plate wall and you can see the cell membrane has stretched out and in some cases the cells look thread like. After taking this photos I then changed the media the cells were growing in. Remember that when liquid water freezes it expands. When the cells were originally frozen a detergent was added that poked holes in the cell membrane so that the cells would not rupture from the increased volume. We wanted to remove this detergent after 24 hours of incubation. I was able to use the automated pipette to remove the old media (all living cells were adhered to the plate so they were not disturbed) and 10 mL of new media was added and the plate was returned to the 37.2 degrees Celsius incubator.

Once that was done we mixed up a few buffers that we will need in the next experiments.The buffers were made using a powder base of PBS (Phosphate Buffered Saline)   and deionized  water. Once the buffer was mixed we tested the pH to make sure it was accurate using a probe similar to ones you used the urinalysis lab We then split the buffer into two containers.

In one container we added  a chemical that will be used to fix cells. In the other container we added another different chemical that will act as a detergent to poke holes in the cells in order to stain them.

We will let the cells incubate until Monday. We will then change the media once more. The cells will be ready to use in experiments on Wednesday of next week.  I’ll be back on Monday to let you know what we worked on then as well as post some more photos of the cells as they grow. Don’t forget to send me some ideas about what to look at under the scanning electron microscope! Enjoy your weekend!

6.20.12 Cell Culture and SEM

I hope you all found some relief from the heat today! I was lucky enough to be working in a cool lab for most of the day 🙂

The scientist I am working with, Dr. Carl Simon works in the subject area of polymers in the Materials Science division of NIST. Specifically he works on 3D Tissue Scaffolds to “develop advanced measurement tools and standards for measuring polymer scaffold properties and their impact on biological response.” This means he and his group are working on a way to compare how cells grow on different scaffold materials. Take a look at this to learn more about 3D Tissue Scaffolds.

This is  a larger sample of the  scaffold material used in these experiments: and this is scaffold material in the plate wells where the cells will added: .

But before we can add the cells to the plate, we need to culture the cells. Our goal for today was to was transfer mBMSCs (mouse Bone Marrow Mesenchymal Stem Cells) to a nutrient medium to grow cells that will eventually be added to the wells in the plate pictured above.

Even though we were working with non-primate cells, we still worked in a Biosafety Level II (BSL) lab. Biosafety level describes the level of safety from exposure to infectious agents; depends on work practices and safety equipment and facilities. A BSL I lab is suitable for work involving well-characterized agents not known to consistently cause disease in healthy adult humans, and of minimal potential hazard to laboratory personnel and the environment . A BSL II lab is similar to Biosafety Level 1 and is suitable for work involving agents of moderate potential hazard to personnel and the environment. There were quite a few safety precautions we followed to protect ourselves (including goggles and gloves) as well as protecting our culture from contamination.

Before we started the work of mixing the media, we needed to clean the area where we would be working and gather our materials: . Since we would be working with living cells, we needed to make sure all the equipment we used was sterile. Then we used an automated pipette transfer 45 mL of medium to the sterile test tubes: . Notice the red color? It is due to an added pH indicator. The liquid turns pink if the pH becomes basic and turns orange if the pH becomes acidic. Then we used the automatic pipette to add 5 mL of FBS (Fetal Bovine Serum) to the media. The FBS includes different growth factors that are necessary for the cells to grow and replicate. Finally we added an antibiotic to the media using a micropipette similar to the ones you used in the gel electrophoresis labs. Once all the ingredients had been added to the test tubes, we inverted the tubes to mix the liquids: .Then we transferred the culture medium to the culture plate and again we inverted the plate to make sure the protein in the media covered all of the plate surface: Now it was time to get the mouse cells which had been stored in liquid nitrogen: . Then we placed the cells into the hot water bath to thaw the sample for 10 minutes: . While the cells thawed, we needed to place the unused media in the fridge for storage: . We also needed to split the FBS into smaller samples to be stored in the freezer since it is not good for the proteins to be repeatedly thawed and frozen: . Once the cells were thawed, we transferred the entire sample containing 1.7 million cells into the culture plate: . Again we inverted the sample gently to spread out the cells in the media. Then onto the microscope to take a quick peek at the cells in the plate: Some of the cells had already adhered or “stuck” to the plate. Finally, the plate was placed in an incubator set at 37.2 degrees Celsius  . The last step was to clean up our station: . Up next, changing the media after 24 hours of incubation.

After starting the cell culture we moved over to another lab in a different building to take a look at a polymer different from the scaffold using a scanning electron microscope: . The sample needed to be mounted on a special tray that would be placed in a vacuumed chamber:    Once all of the air had been removed from the chamber, electricity would be applied and electrons would be used to map the different changes in height of the sample. Those differences in heights are used to make a 3D image of a super small portion of the sample and are displayed on these monitors: . Here’s an picture of the scaffold above from the SEM: The first picture shows a close up of the “struts” with a scale of 500 um ( um means micrometer or micron, and 1 um = 1 x 10 -6 m ) or 0.5 mm or 5 x 10-3 m. The last image shows a close up of one of the pores on the struts with a scale of 2.5 um or 0.0025 mm or 2.5 x 10-6 m, that’s really small! To get a sense of how small this really is, check this out: Scale of the Universe. Which object listed on this site have the same size as first image of the scaffold? Here’s another cool site that also compares the size of objects in our universe: Scale of the Universe 2.

I have the opportunity to bring in some samples to look at under the SEM this summer. Any suggestions?

Pretty cool for my second day huh?