Newly Formulated Cell Media Will Change the Way We Study Cancer

cell culture media bioprint bioprinter

The first synthetic cell culture medium was formulated 60 years ago by an American physician named Harry Eagle. As a pathologist, Dr. Eagle needed a way to keep cells alive longer in a laboratory setting in order to study their growth and behavior. His formula, better known at EMEM (Eagle's minimal essential medium), is composed of a mixture of sugars, vitamins, salts, and amino acids and as its name implies, is the bare minimum of nutrients needed in order to keep cells alive ex-vivo.

Since its creation, Eagle’s medium has become an essential consumable in labs worldwide where it is used by researchers to study animal cells. However, the formulation hasn’t changed much since making its debut in 1959 and recently scientists have begun to wonder if feeding cells the bare minimum of nutrients is skewing the results they are obtaining in lab.

Thinking of EMEM as Gatorade (which it essentially is), you can imagine what would happen if you tried to subsist on a diet of Gatorade alone. Your body wouldn’t behave normally under such harsh conditions so why do we expect your cells to be any different?

In 2012, a researcher by the name of Saverio Tardito set out to create a more relevant cell medium.

“The vast majority of biomedical researchers use cell culture media that were not designed to reproduce the physiological cellular environment but were formulated to enable the continued culture of cells with minimal amounts of nutrients and serum”.

Improving the metabolic fidelity of cancer models with a physiological cell culture medium, Science Advances

His final concoction, called Plasmax, is a mixture of approximately 60 nutrients and metabolites found in the human blood. In their paper, published in Science Advances, Tardito and his colleagues at Cancer Research UK Beatson Institute compared Plasmax with traditional cell culture media and found that cells cultured in Plasmax behaved in a more physiological manner.

By studying Plasmax in conjunction with cancer cells, Tardito and his team concluded that their newly formulated medium can improve the degree to which in-vitro models behave as they would in-vivo and ultimately provide better models for cancer research.

As we enter the renaissance of tissue engineering, we are deepening our understanding of the complex organisms that make up the human body. In order to develop novel drugs, better study disease, and regenerate tissue, it is imperative that we develop more relevant models in the lab that mimic the geometry, environment and diet that cells exist on in the body.

Read the full article here.

Allevi Author: Tohoku University Images Cell Activity in Hyrdrogels

tohoku university allevi bioprinter bioprinted hydrogel imaging

One of the most rewarding “AHA” moments of bioprinting is seeing your cells proliferate within a 3D tissue. As 3D bioprinting becomes more widely adopted within the fields of tissue engineering and personalized medicine, it is important that researchers have the ability to monitor cell activity within in a 3D structure AFTER the print is finished.

Our most recent Allevi Authors have tackled the method of electrochemically monitoring a tissue in their new paper out in the Analytical Sciences Journal titled, “Electrochemical Imaging of Cell Activity in Hydrogels Embedded in Grid-Shaped Polycaprolactone Scaffolds Using a Large-scale Integration (LSI)-based Amperometric Device”.

In their paper, researchers from Tohoku University in Japan use their Allevi 2 bioprinter to print PCL scaffolds as a support material for photocured hydrogels. They then used an amperometric device to electrochemically monitor the living cells. Through their study, they were able to determine that electrochemical imaging is a great way to monitor cell differentiation and will be useful for evaluating the viability of thicker bioprinted tissues.

Congratulations to the Tohoku University researchers on their findings!

Allevi Author: 3D Bioprinting a Spinal Cord

neuron neuronal allevi 3d bioprinter bioprint.jpg

People often ask us, “what is it that a bioprinter can do really well?”, and we tell them that it’s the ability to print and pattern living cells. Your cells are incredible organisms; they understand the environment around them and communicate with other cells to perform specific organ functions. This is why a bioprinter is such an amazing tool - it empowers you to control the geometry and placement of multiple cell types which allows cells to mimic the environments that they are used to in the body. But some cells are more finicky than others… induced pluripotent stem cells and neural cells for instance are difficult to keep alive and difficult to control.

That’s why this next #AlleviAuthor from University of Minnesota really blew us away with their new paper titled “3D Printed Stem-Cell Derived Neural Progenitors Generate Spinal Cord Scaffolds” and published in Advanced Functional Materials, wherein they used Allevi bioinks to 3D bioprint a spinal cord using induced pluripotent stem cells and oligodendrocyte progenitor cells (OPCs).

Successfully bioprinting multicellular neural tissue is a huge win for the field of regenerative medicine as it would allow damaged tissue to rebuild functional axonal connections across the central nervous system, essentially healing damaged connections. This technique will hopefully help develop new clinical approaches to treat neurological disease, such as spinal cord injury.

You can access the full paper here to learn more.

Allevi Author: Lattices vs Sheets for Cardiac Tissue Bioprinting

There are so many variables that go into creating viable 3d bioprinted tissues; bioink selection, print geometry, cure times, rigidity, flexibility, degradation time and cell viability to name a few. Not to mention, each of these parameters needs to be analyzed and perfected for every cell line in the body. As a community, we are still figuring out the perfect protocol for each organ system.

In a new paper out this week titled “A Comparative Study of a 3D Bioprinted Gelatin-Based Lattice and Rectangular-Sheet Structures”, our newest Allevi Authors tackled one of these lingering questions, “What is the best print structure for cardiac tissue, lattice or sheet?”

Researchers at University of Texas El Paso and University of Texas at Austin used their Allevi 2 bioprinter and furfuryl gelatin to study and compare 3d bioprinted lattices vs sheets. Through their comparison, they discovered that the lattice structure was more porous with enhanced rheological properties and exhibited a lower degradation rate compared to the rectangular-sheet.

Further, the lattice allowed cells to proliferate to a greater extent compared to the rectangular-sheet. All of these results collectively affirmed that the lattice poses as a superior scaffold design for tissue engineering applications.

Read the full paper here to learn more about the rigorous testing and analysis the team conducted during their study.

How to Pick Materials for Use in 3D Biofabrication

We’ve talked about what bioprinting entails in general. We’ve touched on the types of cells that we can incorporate into bioprinted constructs. We’ve also discussed the potential of this technology to create novel chimeric structures and compared 3D bioprinting to plastic 3D printing. It’s now time to talk about another critical component in all of this: biomaterial formulations that will provide the scaffolding for the cells of our engineered constructs.

A recent paper titled, “Engineering Hydrogels for Biofabrication”, published in Advanced Materials in 2013 did an outstanding job discussing the state-of-the-art in the field of biomaterials. In this post, we attempt to summarize their work in the context of our own efforts at Allevi.

Malda et al.  25th Anniversary Article: Engineering Hydrogels for Biofabrication.   Adv. Mater.   2013 , 25, 5011–5028

Malda et al. 25th Anniversary Article: Engineering Hydrogels for Biofabrication. Adv. Mater. 2013, 25, 5011–5028

Biomaterials form the scaffolding which supports cells and growth factors during their delivery to damaged tissues and organs. As mentioned in previous pieces and in what will be the subject of a piece coming out later in the week, the 3D environment of the cells is vital in helping maintain cell morphology, differentiation state, and activity. Unfortunately, the 3D scaffolds that have been used in most existing work offer limited spatial control of the cells, ECM, and growth factors encapsulated within them. This is where the additive manufacturing technique of 3D bioprinting comes in to offer the potential of much greater spatial regulation and the ability to design more physiologically accurate tissues. That said, we are still limited in the resolution we can achieve with 3D printing techniques - something which we, at Allevi, are striving to change.

Some factors must be considered when attempting to increase the resolution, i.e. the minimum distinguishable structure size, we can achieve: the viscosity of the solution being printed, the size of the nozzle of the printer head, the speed at which the solution is dispensed, and how the solution undergoes the change from liquid to gel state.

Cells experience shear stress as they are ejected through the nozzle and work remains to be done to determine the maximum shear stress cells can withstand while remaining viable. This will help us determine how small the size of the nozzle can be through which cells are being extruded. Shear thinning is an interesting material property of some hydrogels in which the shear causes the polymer chains to elongate and reorganize into a less entangled conformation, thereby reducing the viscosity of the gel as the shear is applied. In essence, the more the shear, the more easily the gel can flow.

An important consideration is how we can crosslink gels to form stable structures that retain their 3D conformation. Harsh chemical treatments and heat are not feasible when considering gels encapsulating cells, since cells are unable to tolerate these conditions. Increasingly, the focus has been on using ionic crosslinking and photocrosslinking to promote gelation. These techniques will be featured in future posts.

So having considered some of the parameters important in choosing a biomaterial, let’s discuss some commonly used bio-ink. There has been a tendency to favor naturally derived polymers such as alginate, gelatin, chitosan, collagen, fibrin, and hyaluronic acid. These polymers have bioactive signals which allow for improved cell viability and proliferation potential. However, the biggest challenge remains the variability between different batches of naturally derived polymers, making it difficult to control experiments with them.  This has led to the use of synthetic polymers such as poly(ethylene) glycol (PEG), which allow the creation of highly precise, stable 3D structures but offer lower cell viability due to the absence of bioactive signals. Such signals (peptide sequences and growth factors) must be incorporated into these networks to try and recapitulate the characteristics of natural polymers.

Combinations of synthetic polymers, such as PEG, with rigid fibers such as those of polycaprolactone (PCL) may allow for improved structural stability of the constructs. An important point to note is that the degradation process of these two different polymers must be finely tuned so that the hydrogel construct dissolves away as cells start depositing ECM and proteases and other enzymes degrade the gel components, while the fibers retain their position for longer to continue offering the mechanical support to the cells.

So what are some of the challenges we face when it comes to picking the right bio-ink? Well, we have yet to design one which mimics the ECM closely enough. We need to balance the need for structural stability with the requirement for cells and signals to communicate with each other via diffusion and other transport mechanisms through this matrix. We also need to be able to reproducibly manufacture and assess these constructs (could a 3D bioprinter be the answer to this?) and design a range of materials matching the enormous diversity of scaffolds that support the many different cell types in our body.

Pretty simple, right?