Friday, December 13, 2019

User Interaction Design for Genetic Engineering: Let's Replace Agar with Bagels

Agar plates are a staple of most labs.
They hold moisture well and can have a reasonable distribution of nutrients, chemicals and antibiotics that you may need to grow your organisms.

The problem is for the uninitiated, agar plates aren't intuitive. Without an autoclave, melting all the agar can be a strange process of uncertainty. Streaking out organisms is a mess and the agar is usually so soft most people starting their forays into genetic engineering just destroy the plate.

I have been thinking alot about how to make genetic engineering and lab protocols more intuitive. User interaction design is an important characteristic of making a technology widespread. And let's be honest science is not designed with the user in mind. Have you ever given someone a pipette for the first time and seen them try and use it? lol Fuuuuckkkk you science.

Science is so poorly designed that even PhDs struggle to learn and use new equipment, techniques and protocols. This is compounded by the problem that scientists are notoriously against change. If it works it doesn't need to be changed because generally scientists want to focus all their efforts on doing experiments so they can publish papers. There is no incentive in science to design better ways of doing science.

Running The ODIN, I get to see where people struggle the most when trying to learn genetic engineering and one of the biggest problems is making and using agar plates.

Agar is not expensive so I'm not looking for something cheaper. If you are still using molecular biology grade agar I applaud you for being a dumb ass. You can purchase 1kg of agar on amazon for $40 or 1kg of "molecular biology grade agar" for $146 both work exactly the same.

What you really need is something that is easy to use, it doesn't shatter into a million pieces if you touch it and is intuitive for most people.

To me alternatives would be something people are familiar with. That we use in our everyday life. I'm just winging it here and not proposing these as alternatives but trying to think differently about science.

I chose a bagel

I wanted to see if I could grow some genetically modified bacteria and propagate the cultures. I know this seems insane and I think I went off the deep end on this one but I think that is sometimes what is required.

First, I wet all the bagel with either water and ampicillin or LB ampicillin (LB is a nutrient media that helps bacteria grow). Using antibiotics like ampicillin is a standard method in genetic engineering to select for the genetic modification. I took some ampicillin resistant GFP engineered bacteria and streaked it on each of the bagel pieces. The fluorescence from GFP makes it easier to track the bacteria through the experiments. Streaking bacteria on a bagel is tough as the surface was soggy and uneven and inoculation loops aren't really made for that. Still I managed to get a little bacteria on.

I put the materials in ziplock type plastic bags. I imagined this would hold the moisture in. I then incubated them at 37C overnight.

The bags did not hold moisture well and the materials seemed to dry out a bit. I think this will be one of the most important things to think about in the design.

Though the light conditions of this image are not comparable to the original taken before the experiment it definitely seems like there is less fluorescence than when I started, I imagine because the bagel dried out. ugh.

Still, its seems like the bacteria survived and so I decided to break off a piece of bagel and use it to inoculate some liquid media and see if I can propagate the culture.

I used a centrifuge to spin down the culture and the bacteria that grew in the culture are fluorescent meaning the bagel inoculation worked.

After 48 hours of having the fluorescent bacteria on the bagel there doesn't appear to be contamination of any kind and the bacteria are still fluorescing.

I don't think bagels are the future of genetic engineering but I don't think agar plates are either.
Most everything that is done in genetic engineering and molecular biology is a complete kludge. The fact that I need like 5 different pieces of equipment to put DNA in bacteria is insane.

Am I really insane for doing this or are the people who continue to do science in such a kludgey way insane?

Tuesday, December 3, 2019

Using My Own Blood Serum For Human Tissue Culture

Growing human cells/tissue in culture is one of the foundations of modern medicine. This typical involves taking human cells from an individual or purchasing them obtained from one of many companies that cell(lol) them.

Human cells have long been assumed difficult and technical to culture but recently I have built out protocols and a class that allows people to culture human cells with minimal equipment and experience in their own kitchen even. I am constantly trying to use the most simplistic and cost effective techniques and materials to lower boundaries in science and thus increase innovation. I want anyone to do experiments with human cells if they want. This adds value for all humans not just Biohackers.

What if all you needed to grow human cells was yourself? Your own blood.

Generally, when growing human or animal cells in culture the liquid media used to grow the cells contains Fetal Bovine Serum (FBS). Fetal Bovine Serum is literally the serum(non-red blood cell part of blood) from fetal cows. After a cow is slaughtered they remove the fetal calf and puncture its heart to extract the serum before they kill it. It is pretty barbaric and doesn't scale well. I mean unless of course we want to farm fetal cows for the sole purpose of extracting their serum?

Why do scientists use FBS instead of serum from other animals? This is not really known. Most people say it is because FBS contains less antibodies than other serums and so is less reactive to the cells in the culture but I am skeptical as to how many animal serums have actually been tested head to head. Scientists do this often where they do something only because other people did it with very vague reasoning why. Scientists tend to be very skeptical of change. In fact, there are serums from adult Chicken, Goat, Horse, Pig, Rabbit and many others. You don't read about these often or at all in the modern scientific literature.

I wondered if fresh serum from my body would work in human tissue culture? I am a trained phlebotomist(true story) so I decided to draw my own blood and use a centrifuge to separate the red blood cells from the serum.

The hardest part is always drawing my own blood. I usually use my median cubital vein on one arm and draw the blood with one hand but thinking about it now I should probably use a vein on my leg so I can use both hands at the same time, oh well, next time? From that blood draw, I obtained around 6mL of my serum. Probably about a third of the blood was serum so if we use the maximum blood donation number of 500mL you can get ~150mL of serum from yourself in one sitting. I should probably say don't try this at home. Drawing your own blood isn't the easiest and you can hurt yourself. Again, I am a trained phlebotomist.

I wanted to grow up some human cells with the serum but I wanted to compare it to FBS and Newborn Calf Serum (NCS), which as the name implies is from newborn calves(I don't know if any calves were hurt in the salvaging of that serum but I assume not). NCS is much less expensive monetarily and much less expensive morally and ethically.

For the experiment I used 3 replicates of HEK 293 cells for each media formulation in a 12 well tissue culture plate and did the experiment twice. I used 10% serum in each case. Above are example pictures. As you can see from the picture that Josiah Juice (my serum) performed as well or better than FBS qualitatively. NCS performed fine though cell growth seemed consistently slightly less than FBS and Josiah Juice. Still NCS seems like a great inexpensive alternative to FBS as NCS costs around 10 times less than FBS! Caveat, these cells were grown up in FBS before being transferred to other serums in the experiment so they are probably under-performing. I imagine that NCS and Josiah Juice cells would do better after the cells acclimatized to the serums on the second or third splitting.

This is pretty fucking cool. Run out of FBS and in a pinch you can use your own serum. But seriously this experiment is more to prove a point.

The biotech and science industry is truly fucked because everyone is doing what everyone else is doing and hoping that somehow their dogged go-getty attitude is somehow going to lead to innovation. What people need to be saying is "Fuck what everyone else has done. Maybe they just did what was readily available to them. I am going to figure shit out instead of copy for a vague reason so I can do my research as fast as possible to try and publish a paper no one cares about."

The explosion of human tissue culture and lab grown meats cannot rely on FBS forever as there is an extremely limited supply that cannot scale. A whole company or industry could be built around supplying a low cost scalable FBS alternative that works just as well or better. Fetal Bovine Serum alone has a market size of $700 million. This will only grow.

Maybe growing human meat using human serum is the answer or maybe that is just my next project.

The nitty gritty...

For working in human cell culture I mostly focus on HEK 293 cells. This is a human embryonic kidney cell line that was modified to be "immortal". These immortalized cells are robust and are great for people to learn cell culture techniques. Obviously, conditions that can be applied to HEK293 cells cannot be applied to _all_ cell lines. However, I think it would also be foolish to think that they can't be applied to many other cell lines.

I culture human cells in a non-CO2 environment. This is because you don't actually need CO2 for tissue culture. The reason people use CO2 is mostly because they use DMEM which contains bicarbonate and CO2 is required to buffer the media. Honestly, I don't get it? Still trying to figure out why people use this contrived method.

I use L15 media. This media is not buffered with bicarbonate so no CO2 is required. In the media, I generally use Ampicillin (100ug/mL), Streptomycin(100ug/mL) and Gentamycin(50ug/mL). This generally prevents most any bacterial contamination that can happen in a non-sterile setup. Yes, that is correct. No sterile hoods or sterile areas are used in my human cell culture. With a little experience and by sterile filtering(0.22uM) the media I rarely experience contamination of cultures.

Briefly, HEK293 cells were grown to confluency in L15 with 10% FBS and the above antibiotics. The cells were washed in PBS and then incubated in a 0.25% Trypsin 0.02% EDTA solution for 5 minutes to removed the adherent cells. The same volume of cells were then added to each well. Each well contained 10% of each serum plus L15 and the above antibiotics. Cells were incubated at 37C with no CO2.