News in Translational Research: Too Much Information?

Although today’s blog has little to do with tissue engineering specifically I thought that it would be a great opportunity to discuss last Saturday’s article in the New York Times, linked below:

New York Times: Genes Now Tell Doctors Secrets They Can’t Utter

This article discusses the quandaries faced by researchers and doctors who discover things about their patients which are not covered by the consent forms. Instances include: discovering that a patient has a gene which has been linked to a significant risk of a particular cancer, discovering that the patient sample contains the HIV sequence (though whether a transient or permanent infection can’t be determined without further testing), or negatively, discovering a patient doesn’t have a gene that they thought they had, and therefore could avoid breast surgery.
It seems that doctors and researchers tend to go to bat with ethical boards to fight to let patients know when they discover that a patient can take preventative action. However, scientists are less certain about what to do when they discover the patient has a risk of a disease that they can’t be proactive about. And what if the patient specifically stated they never wanted to be contacted? If the establishment contacts the patient and asks if he want to reconsider, then that implies that there is something he ought to know.
As a scientist a few levels removed from the clinic, I generally don’t have these sorts of worries. However, a few months ago, some of my transplants became tumorigenic. These transplants had been seeded with cells donated by an actual patient (at my level we are just given a number identifier for the patient). As I am not involved in the clinic, I spoke to the relevant people, and down the chain all the cells were pulled and marked as not suitable for transplantation. The tumors could have been formed from anyone of multiple factors, chemical sterilization treatments of the scaffolds, contact with tumorigenic mouse cells transplanted in the same mice etc. And a repeat of the experiment with slightly different conditions yielded no tumors, however, that ugly incident is probably the closest I’m going to get to bench-to-bedside effect.

Thesis Writing: Is a Thesis Just a Long Paper?

University of Oxford Notes of Guidance: Preparation and Submission of a Thesis

These notes of guidelines offer very little in way of succor to the poor graduate student trying to tackle the daunting task of writing a thesis. A scientific thesis can be construed as an extremely long scientific paper with the same components, however there are many important distinctions.

  • I vs. We: Peer-reviewed publications use the royal “we” to infer all the authors on the paper (with the general knowledge that some people contributed more than others depending on their placement in the author list). Thesis writers use the term I and take personal credit for all the work that they have actually done. Of course, due credit is given to work previously carried out by the lab or done in collaboration with other researchers.
  • “As described previously”: A term often used in peer-reviewed journals and accompanied by a reference to either a previous paper from the same group or a different group they stole the method from. This chain of “as described previously” especially accompanied by the term “with a few minor alterations” can go back multiple papers to the point where the technique that one is actually looking at can’t be determined by tracing it back. In a thesis the entire procedure is detailed in nauseating depth. This is incredibly important because a thesis is supposed to be reproducible, even more so than a peer-reviewed publication. Additionally, the specificity of protocols allows the protégé of the student, and there often is one in academic labs, to easily grasp what was done.
  • Literature section: Literature sections in peer-reviewed papers are often called background, and are meant to bring the reader into the specific content within a few short paragraphs. In a thesis the literature review starts out much wider in scope and waxes lyrical for many pages, citing many references (more like a review and less like an article), discussing all components in the wider scheme and then bringing them together to discuss the niche topic.

So far I’ve made a draft each of the Methods and Literature Review sections. This week’s task is to tackle the results section, bringing all the different graphs together and organizing the figures.

 

Wish me luck!

Technique: Freezing cells

Just this past week our lab bought two new -80°C freezers. (In contrast, a home freezer is generally -18°C). We use these freezers to store biological samples, including cells, when we’re not storing them in liquid nitrogen, a frigid -210°C.

Why do we freeze cells? Logistically, we freeze cells because we get our cells from living specimens who donate valuable cells and aren’t infinite sources. Also, although I pointed out how cells expand exponentially in my post on August 11th, they only do this near the beginning of their lives and undergo a process known as “senescence” at later age when they stop growing, unless they are true stem cells. Additionally, cell behavior and differentiation have been shown to change with continued expansion in a Petri dish, until cells might not be a good representation of their origins. Therefore, if we get a higher number of cells that we need at low passage (see the August 11th entry), we freeze them. For example, in our lab we never use our bone marrow stromal cells above passage 5 for transplants, and above passage 10 for any studies at all.

Mouse and human body temperatures are 37°C, and therefore, that’s what the incubator temperatures in our lab are always set at, to allow cells to grow at a normal rate, without causing them undue stress. As cells get colder, all of their biological processes slow down, but importantly, do not die (or proliferate). Therefore, freezing provides an excellent way of keeping cells exactly the way they are for long periods of time.

“But”, I hear you say, “surely the ice crystals that form during freezing will puncture and kill all of the cells?” That’s exactly the reason why we can’t freeze cells in normal growth medium (liquid) but instead using “freezing medium”. This typically contains 10% DMSO (dimethyl sulfoxide) which actually helps protect cells from rupturing, and is known as a “cryoprotectant”. However, we can’t use just DMSO because it is actually toxic to the cells. Therefore, the rest of the solution is generally made up of components that one would find in normal growth medium (e.g. FBS [foetal bovine serum]). There are also many different proprietary freezing mediums on the market.
The Procedure:
1) Make, or buy, freezing medium: I use 10% DMSO, 90% FBS
2) Trypsinize and centrifuge cells as if you were passaging them
3) After removing the media/trypsin solution, resuspend the cells to 1×106/mL (or as desired, but not more than 4×106/mL) in freezing medium
4) Portion the cell solution into freezing vials, 1mL per vial
5) Place vials in an isopropanol chamber (a special freezing apparatus that slows down the freezing process to avoid shocking and killing cells) and place container in a -80˚C freezer
6) The next day, transfer to a normal container box, and, if desired, transfer the cells in liquid nitrogens

News in Tissue Engineering: Tracheas

The trachea is a vital human body part. It is a tube that connects the larynx to the lungs, allowing humans to breath. Tracheal collapse happens as a result of some heart conditions, Cushing’s syndrome, and some respiratory conditions, including lung cancer that has spread. Without a trachea it is impossible to breath. There are extreme options, such as having a tracheotomy in order to avoid having to breathe through the mouth, but these are very invasive and uncomfortable procedures. Tissue engineering has been applied to the trachea over the past few years in an astonishing way, and will continue to do so, setting new levels of treatment.

Science Daily, 2008: First Tissue-Engineered Trachea Successfully Transplanted

In this article we see that the scientists utilized two of the three components I talked about in my August 8th post, i.e. cells and scaffold. The scaffold, in this case, is a 7cm section of decellularized trachea from a donor. The donor was deceased – it is not possible to donate a trachea section while still alive. 7cm is a really large area to consider in terms of modern tissue engineering, as much research is done on a much smaller scale. The term decellularized means that the tissue was stripped of all cells to make it less immunogenic to the recipient. However, the physical structure of the organ is retained and, importantly, its biological activity, as the proteins that make up the structure are kept and still active. This scaffold was then seeded with cells that had been collected from the recipient, i.e. autologous cells, so that there would be no graft/host response. Seeding the cells is important because it provides the organ with a dynamic, living presence instead of just implanting an empty construct.

New York Times 2011: Synthetic Windpipe is Used to Replace Cancerous One

In this article we see that a similar approach of seeding autologous cells on a tracheal scaffold was taken. However, in this instance the scaffold material is different, instead of being a donated natural scaffold, it is a synthetic polymer material. This has several associated pros and cons. The pros of using this approach are that it doesn’t require a donor and associated organ processing logistics, but, more importantly, the scaffold can be shaped to fit the recipient exactly using modeling and advanced fabrication techniques, whereas donor tracheas are often not a good fit. The major con of the system is that the scaffold is inert and not biologically active and covered in normal tracheal proteins the way that the previous scientific group described. In order to overcome this issue, the group at the Karolinska Institute also grew the cells on the scaffold in a bioreactor, but added in several growth factors that they hoped would induce the cells to differentiate as desired, and start the biological pathways that would continue to affect how the cells behave even after transplanted.

Thesis Writing: Psychology of a Thesis

How does one begin to write a thesis? More importantly, perhaps, how does one keep up the strength to write the entire thesis through to its bitter/sweet end? These questions loom over all graduate students from Day 1 of their course. Some days the worry stands out more, on other days it’s possible to get distracted by actually doing the research and forget that one day it all needs to get written up. Some days one thinks about the writing process and tackles some small aspect of it, on other days the worry is there but we push the worry deep within us, very much like the proverbial ostrich with its head in the sand. There is, however, no denying that thesis writing causes grad students much stress. This was demonstrated by Izawa et al. in 2003, who wrote a paper showing elevated stress marker levels in students just prior to thesis submission.

Episodic stress associated with writing a graduation thesis and free cortisol secretion after awakening

There is an abundance of advice on the web for students about to embark on a thesis project. This includes advice to students by universities, such as these pages from Dartmouth and Yale:

Dartmouth: Writing a Thesis

Yale: Writing a Thesis

And, touchingly, there is this advice from the University of Oxford to advisors of students writing theses:

Oxford Learning Institute: Writing a Thesis

Then, depressingly, there are many companies that make money off students who use their services, because they don’t have mentors who have told them not to. Companies, such as one site I saw while researching for this blog post (but won’t deign to put a link to here), even claim that they can specifically help you write your Oxford thesis (with poor grammar, going by the standard of their website). But, more importantly, such companies not only take advantage of students who are psychologically vulnerable, they also damage the integrity of those students.
On a brighter note, there is a light at the end of the tunnel! Even though graduate students slave away at writing and research, one day that writing and research is all complete. Then, we stand at the exit from our cave, gazing out into the real world (hopefully with degree in hand). Some yearn to return to the days of flexible hours: sleeping in some days and pulling all-nighters and sleeping in the lab on other days. Some yearn to return to our graduate student stipends: not that the money was all that great, but rather that we could be paid to be students (who knew?!). But the important part is that eventually, despite our raised cortisol levels, most graduate students do finish….

Well… about 55-65% of them finish, as shown by the PhD Completion Project:

Council of Graduate Schools: Ph.D. Completion and Attrition

Noteworthy: Silk Scaffolds

BBC: Silkworms Could Aid Breakthrough in Tissue Engineering

Every Monday I plan to talk about something in the news and how this relates to the field of tissue engineering. This week I’ve posted a link to a video about the creation of silk. Textile specialists have long looked at silk because it is a naturally produced material, from either spiders or silk worms. However, the first review of the scientific use of silk as a biomaterial wasn’t published until 2003. This is yet another example of just how rapidly the field of tissue engineering is progressing and how rapidly it is growing. In 2003, just 189 papers were published, according to the scientific publishing database PubMed, compared to 438 in 2011.
Today, it’s very evident that silk scaffolds are being tested around the world, from the USA, to Germany, to China, to Australia, and everywhere in between. Silk is, however, very rarely used on its own, and when it is, it does need to be chemically treated to change its mechanical, and sometimes biological, properties. Silk from various spiders, for example, has to be processed to remove an outer coating which is biologically toxic. Much more common is to find silk that has been modified, either by adding adenovirus to stimulate a biological process, or through the attachment of various proteins etc. that will give it various properties. Crosslinking of fibers strengthens the network and allows it to hold its shape better. And very commonly silk will be mixed with synthetic polymers such as polyacrylamide, whose biological properties are much easier to control to get reproducible results.
What the group did at the Institute of Materials Research and Engineering in Singapore accomplished in making colored silk worms that can generate fluorescently colored silk is very interesting because, as stated in the video, it makes it easier to do fluorescence imaging of the scaffold. From a biological standpoint it is interesting not just because of the new product it produces, but because it raises the possibility of incorporating many different characteristics into the silk, such as mechanical properties, or modified biological moieties (both would come from modifying the structure of the silk fibroin molecules during production by the silkworm). Previously, all materials had to be modified post-extraction and purification, now they can be modified before production even starts. It’s the biological equivalent of producing spinach-flavored pasta instead of taking plain pasta, cooking it, and adding spinach.

Technique: Passaging cells

Every Saturday I plan to write about a technique I’m using/have used in my project. I’ll explain what the purpose of it is, why I want the data it produces, and how to perform it. Even, and especially, if you don’t work in a science lab, I hope you still won’t just dismiss the entry as nerd-speak, and instead, read through. I promise to keep the entry concise and not assume you know anything more than high-school science.

Yesterday I “passaged my cells”. Passaging cells is the first thing any person starting in a cell culture lab learns. Cell culture is the process of growing cells in the lab. The cells can be from any source: animal, vegetable, or mineral – just kidding, minerals don’t have cells. I’m lucky in the almost all the cells I’ve worked with are adherent. This means that if you take a solution of cells and place it in a plastic dish, the cells will attach and grow on the bottom, instead of floating around. They key word here is “grow” because cells divide and multiply over time. Everyone, for example comes from a fertilized egg, a single cell, which divided in two, and those two divided, and those four divided, etc. until finally there’s you (very simplified and some major assumptions taken with that analogy, but the concept applies). Cells growing in a dish are no different. They multiply. Exponentially. This means that they’ll run out of empty space in the dish since they’re only growing in 2D and not on top of each other. When they completely run out of space we call this “confluent” because they look like a single layer. When cells get confluent they often change their behavior, because they touch each other a lot more, so we generally want to avoid this. We also want to avoid confluency for the sheer reason that if cells are growing in 2D and run out of space, they will stop growing, and generally, as scientist, we always want more cells, so we need to keep them growing. We prevent cells from becoming confluent by taking them off the dish they are currently in, and placing them in a dish with bigger surface area, so their density is decreased and they can expand again. This process is known as “passaging”.

The Procedure:
1) Remove cell dishes from the incubator. We keep our cells at 37°C (human body temperature).
2) Remove the media (growing solution) from the dish and rinse with salt solution.
3) Add trypsin to the dishes to cause them to detach from the plastic (a few minutes at 37°C).
4) Remove the cell solution and add normal growth media to stop the trypsin reaction.
5) Count the cells (optional – but helps decide how big of a dish to replate them in).
6) Centrifuge the cells to make a cell pellet
7) Remove the liquid from the top, this ensures all trypsin is removed
8) Add in new growth solution to cells and place in the new, larger dish, or use as desired.