THE JOURNEY FROM CELL CULTURE TO BIOPRINTING AND BEYOND
One of the most important laboratory methods to study physiology and disease effects in animals including humans is animal cell culture. At the outset, it seems incredible, since all that is put into the cell culture bottle is a carefully controlled nutrient solution, growth serum and a few microlitres of cells disrupted from their respective tissue associations/organs. The cells soon feed on the nutrients, multiply and form a mat known as a monolayer. Alternatively, they can also be grown in suspension. However, this requires much skill and patience since even a little success entails many failed sterility checks, contaminated media (an unforgivable sin in a cell culture laboratory) and other disasters that just creep in at the most critical moment.
Life for a Microbiologist is just as harsh in the 2000’s as it was at the time of Dr.Robert Koch! Healthy cell cultures could be injected with different viruses and their Cytopathic effect studied. They could be treated to various physiological and chemical tests to test or study cellular response and survival under different conditions; especially with response to different drugs. The expression of specific genes can also be studied. Conventional cell culture protocol demands that a “cell line” of the respective cell type be maintained for continued studies. These subcultures need a careful balance, in terms of the right concentration of serum and other nutrients.
There are many other challenges in animal cell culture. In the restricted environment of the cell culture bottle, there is the constant buildup of undesired metabolites. This corresponds to an inability to cycle nutrients in, and waste metabolites, out of the system; requiring regular subculturing. Another challenge commonly encountered with conventional 2D cell cultures done in bottles, was the manner in which the cell layer just kept tearing apart under the slightest stress. This problem basically occurs since the cells really did not have any other source of anchorage to build their structures other than the nutrient filled glass/plastic culture bottle. Cellular behavior in a 2D dimension also raised serious questions about whether it truly represented an actual “in-vivo” 3D environment.
There have been many efforts at increasing the interaction between the nutrients (growth factors being studied) and the cells such as the MLM (3D cell culturing by magnetic levitation) method, where a combination of nanoparticles and magnetic fields encourage cells in culture to be levitated and interact with each other. Supportive structures known as Bio-scaffolds (which is somewhat similar to helping a creeper vine grow using a guiding rope), have led to the dawn of 3D tissue culture. Basically, scaffolds for cell culture serve as structural support and also serve as the medium through which nutrient supply is carried out much in the fashion of blood vessels carrying nutrients. This would mean mimicking a near perfect biological tissue system. Scaffolds also allow for complex and more intricate cell networks. 3D scaffold structures are available with several commercial companies now and have shown unique promises with cancer research where it has been shown in lab grown cancer cells on 3D scaffolds offered more accurate “in-vivo” cellular behavior in the lab (http://www.nature.com/drugdisc/news/articles/424870a.html#b7).
However, nature still eludes us in developing a completely successful scaffold structure that is equal to that of the natural Extracellular matrix which connects and serves as a bridge between several different types of tissues. This is why synthetic scaffolds or scaffolds derived from other animals (such as pigs) are required. Whenever an animal based scaffold is used, there crops up the problem of immune response. To overcome the problems of immune response, an interesting process calledDecellularization is now being used, which can even be applied to whole organs. In a radical procedure, the structure/organ is washed in a detergent solution to remove all the cells followed by an enzymatic treatment to remove genetic material. The resulting structure has no cellular identity, but retains the internal and external shape of the original organ, thanks to a network of connective tissue called the Extracellular matrix. This had made us understand that there is a ghostly exoskeleton for every organ, a shape giver that serves as a scaffold for cells to grow on.
One of the most noted pioneers in the application of decellularised organ structures is University of Barcelona’s Professor Paolo Macchiarini. His pioneering success with transplantation of lab grown personalized Trachea’s has made giant strides in the world of organ transplantation. The first patient to receive the tracheal transplant was a 30 year old woman. At the outset, the procedure involved taking a seven centimeter tracheal segment from a transplant donor and decellularising it over a period of six weeks. Now, all that is present is a protein segment with the shape of a trachea which can basically be considered as a Bio-scaffold. In a parallel process, stem cells extracted from the patient’s bone marrow was cultured in the lab to reach a sufficient population and made to mature into chondrocytes (cartilage cells which make up the trachea). Now, the decellularized trachea was seeded with these chondrocytes on the inside. Similarly, Epithelial cells were seeded on the outside of the structure to replicate the lining. These processes were done separately in a bioreactor made especially for this process; for about a month before successful transplantation to replace the lady’s left main bronchus. A similar procedure was also used to save the life of a 10 year old child successfully.
Perhaps the most incredible example of Decellularized scaffold being developed besides the successful trachea is the development of laboratory grown hearts ! Dr.Doris A.Taylor, now of the Texas Heart Institute and her team have developed a process for whole organ decellularization and then reseeding them successfully with cells taken from rat and pig hearts. The heart is beating and this means that the day is not far when surgeries similar to those done for the tracheal transplant would be done for the heart as well! This was originally developed at the University of Minnesota’s Centre for Cardiovascular repair. You can read more about it from their page.
Decellularized heart work at Texas Heart Institute
Such advances take place largely due to our understanding of cell differenciation and tissue structure, leading to the formation of organs. Stem cells are at the heart of such procedures owing to their innate ability to differentiate into virtually any cell type under the right conditions. This development has made it possible to extract adult stem cells from a person and differentiate it into the desired cell type in a laboratory. Using specialized micro-bioreactors, these cells can be encouraged to multiply over a 3D scaffold (synthetic or animal-based). Advances in converting stem cells from the patient and converting them to a cell of needed functionality is central to these developments.
Stem cells have been the rage for quite some time owing to their regenerative properties of being able to grow into virtually any cell type under the right conditions. Interestingly enough, even stem cells may not be absolutely necessary in the near future; thanks to the Nobel peace prize winning work of John Gurdon and Shinya Yamanaka who have developed techniques to convert adult cells back to stem cells! These techniques have heralded a completely new and unprecedented era in Biomedical technology/Medical treatment. This has been termed as Regenerative medicine and defined in Wikipedia as:
Regenerative Medicine refers to a group of biomedical approaches to clinical therapies that may involve the use of stem cells.Examples include the injection of stem cells or progenitor cells (cell therapies); the induction of regeneration by biologically active molecules administered alone or as a secretion by infused cells (immunomodulation therapy); and transplantation of in vitro grown organs and tissues (Tissue engineering).
A parallel and most fascinating story of regeneration medicine involving Decellularization being applied would be from the work of Dr. Stephen Badylak, a Research Professor in the Department of Surgery and director of Tissue Engineering at the McGowan Institute for Regenerative Medicine at the University of Pittsburgh. He has created a regenerative medicinal powder popularly hailed as “pixie dust”! It is actually dried decellularized cell powder prepared from cells taken from the lining of a pig’s bladder. This serves as an extracellular matrix performing as a support structure for the regeneration of tissue. Already, it has been used to regrow a cut finger and part of a heel. This revolutionary application might eventually replace the need for bone and skin grafts. You can read more here and here.
With the way this field is growing in strength and popularity, and the added advantage of Biotechnology’s greater control over Stem cells; it would only be a matter of time when whole organ regenerations would become as normal as going to the hospital for a Dialysis (an extremely complicated but routine procedure). Scientists have extrapolated on the 3D Bioscaffold principle to build entire artificial joints in rabbits using just cells.For a complete detailed history on the types of lab grown organs out there, you can read Jennifer Walsh’s article on Business Insider.
While these cell culture and organ culture events seem incredible, it would not have been possible if not for the absolute / ultimate engineering feat of all; the Cell. It is the biological cell’s innate ability of adhesion, growth that makes it possible to accomodate technological intrusions into it’s assemblage. Or perhaps even that too is about to change ! It is a common natural observation that mussels cling to each other on slippery sea rocks despite the relentless lashing of waves. Scientists at Penn State University’s Department of Bioengineering, have been inspired by the Bioadhesives found in Mussels to develop sutureless wound treatment using what they have termed as “injectable citrate-based mussel-inspired bioadhesive (iCMBA)“.
In the field of Biodegradable scaffolds, the Wake Forest Institute for Regenerative Medicine (USA) and Dr.Anthony Atala‘s team seem to dominate the field in developing protocols rapidly for regenerating several different organs and anatomical parts including ears, digits and urinary bladders using biodegradable scaffolds. Their efforts are largely made possible thanks to the development of biodegradable materials such as Polyglycolic acid (PGA) composite matrices for making scaffolds. Back in 2006, they had created a revolution by growing a new urethra (the tube that empties urine from the bladder) for five young boys (10 to 14 years old) from Mexico from the their own cells. The procedure involved culturing cells in the laboratory from small tissue patches taken from each boy’s bladder. The cultured cells are then coated onto a biodegradable scaffold. The cells are allowed to adhere and cover the urethra shaped scaffold. Once ready, they were surgically transferred to each of the boys. The biodegradable scaffold eventually degrades away leaving a pristine urinary tract made from the boy’s own cells! What is even more interesting is that the urinary tracts of these patients are working normally even after a follow up period of six years. You can read the entire report on Lancet here and it’s feature on Time here.
Dr.Paolo Macchiarini and his pan-european team have successfully used a synthetic Bio-scaffold in the case of a 30 year old male patient whose only option was a complete tracheal transplant due to damage caused by tracheal cancer. In his case, the cells were seeded onto a plastic scaffold, instead of using the decellularization process (which involves finding a donor trachea first). The transplant had been successful. However, what is even more important to realize here is that this is the first successful transplantation of a fully synthetic windpipe retrofitted with biological cells. You can read more about it on ScienceDaily.
Confocal fluorescence micrograph of threedimensional nanoES/neural tissue with neurons stained red, nanoelectronic network of same size as axons and dendrites stained blue/green. Courtesy of Charles M. Lieber, Harvard University. Source: http://cmliris.harvard.edu/assets/Nano_Today1.pdf
It is interesting to also note that Bio-scaffolds have been getting smaller and sophisticated. Already, an amazing offshoot development is taking place at Harvard University’s Wyss Institute for Biologically Inspired Engineering. They have combined Microfabrication techniques with biological tissues/cells to come up with “Organ on a chip“! These are actually tiny but exacting representations of organs/organ systems with respect to their physiological response and other cellular mechanics. Specific cell types are combined with microelectronics to create what is essentially a Bio-MEMS system (Biomedical Microelectromechanical system). A good report on their technology is available here and here.
However, Bio-MEMS is currently limited by an “isthmus” between electronics and biological cells. There is a clear demarcation between both worlds although the scale may have been significantly brought down. Cells grown with such scaffolds or on chips will still have to be connected as a layer to receive electrical signals from a separate system. However, this is also going to change. Research teams led by Harvard University’s Prof.Charles M.Lieber and Boston Children Hospital’s Daniel S.Kohane have developed the first amalgamation of living tissue and electronics proclaimed widely as the first “Cyborg tissue”.In effect, a scaffold was created by incorporating nanowire based electronic devices with polymers. The resulting mesh like scaffold is comfortable enough for insertion of cells which can then penetrate and grow. What happens as a result of this growth is a seamless integration of biology and electronics!!! The system has just come alive since the biological cells will grow into the system and in effect fuse seamlessly with the electronics !!! The work has been featured in Lieber’s research page on Nano Bio-Interface (includes a gamut of other interesting paranephilia) as :
Cyborg tissue: We are pursuing the development of novel biomaterials that seamlessly integrate nanoelectronic device arrays with synthetic tissue. This highly interdisciplinary work involves implementation of new ideas for 3D nanodevice arrays interconnected on the scale of natural tissue scaffolds, together with 3D cell culture and advanced measurement techniques to create tissue that is ‘innervated’ over many length scales.
Now, 3D cell culture methods, incorporation of Bio-MEMS and even nanowires may seem impressive for the science enthusiast or from an R&D perspective for a major Pharmaceutical company, that foresees the potential these methods have in reducing time spent on animal testing. However, what good does it hold for the average person in the society? While science fiction stories and fantasies are abound with such a merger, the real current applications lie in the study of diseases and development of medicines. Such integrative scaffolds can in effect detect and measure electric activity of the cells or cellular network in response to particular stimuli/specific drugs.
While all of this has been happening, a silent revolution in industrial prototype development has heralded a new age for regenerative medicine and for the cell to be delivered not as a solution or a coating but as precise structural units to make up a whole structure. We all realize by now that cells are the building blocks of any given organ. Now, if these cells can be precisely placed onto a scaffold much akin to placing bricks to make buildings, then we have a biological organ with greater stability and hopefully, extended functionality. Originally hailed as a remarkable innovation for prototype development and for some movie magic, the 3D printer has come of age for Biology and more importantly whole organs, in the brave new world of Bioprinting! But it took some very creative or rather a lateral line of thought to come up with the idea that cells could in fact be printed with an inkjet printer ! For this unique thought, we should probably thank Professor Makoto Nakamura, now of the University of Toyama Facility of Lifescience and Engineering, Japan. The professor realized that cells were just about the same size as the liquid ink droplets that inkjet printers dispensed on paper. His initial trials on printing cells in 2002 with a home printer from Epson proved cloggy. Eventually, in 2008, Prof.Nakamura developed the first 3D structure made out of cells using inkjet technology. His hallmark has been in the printing out of Bio-tubes similar to blood vessels made of cells at a speed of three centimetres every 1.5 minutes. You can read more of his story here.He developed a Bioprinter device and eventually hopes to print out entire organs; the heart being his favourite ! Speaking of Bioprinters, one of the most well known is the NovoGen MMX Bioprinter from a company called Organovo which was jointly developed in collaboration with another company called Invitech. At the outset, the Bioprinter uses a Hydrogel like material onto which it deposits these cells. The basic technique relies on arranging the cells in this hydrogel layer by layer close enough to allow the natural adhesive forces between the biological cells to stick onto each other, thereby forming a complete layer. You can read more about the process here and here. The Bioprinter gained special fame when it printed out a Kidney during the course of Dr.Anthony Atala’s revolutionary talk at TED on “Growing new organs“.
Speaking of offshoots, there are already several movements and start-ups cropping up from the pioneers who have heralded this era of regenerated organs. Already, a start-up called Modern Meadow for Meat grown in the lab is doing the rounds. Bioprinting is improving rapidly with the companies involved mergering to form larger enterprises in order to improve their technologies. The most noticeable being that of Organovo’s link up with Autodesk for improved bioprinting software and Zenbio for 3D cell culture solutions. The former link-up would fill the gap in Bioprinting software deficiency and the latter would result in new tissue culture assays made to order for therapeutic, diagnostic and pharmaceutical 3D applications.
As for Bio-scaffolds, improvements are being made parallel to advancements in 3D printing technologies. This also involves improvements in the area of Biomaterials which degrade faster and present less immunological response on transplantation. Then there are sudden technological leaps which leave you wondering “why didn’t I think of this before !?!”. One such leap which has sprung forth is the World’s first 3D pen which can extrude heated and cooled plastic material as per the drawing drawn with it. This is what 3Doodler has to say about it:
“Have you ever just wished you could lift your pen off the paper and see your drawing become a real three dimensional object? Well now you can!”
I can already imagine the day when researchers and doctors can simply draw out tissues onto wounds using this pen. Surgeons would have a “healing wand” in their hands! Of course, it isn’t the same as drawing out plastic structures. However, what this means is that the technology has arrived to eventually rebuild human body parts. These advances are also at the cusp of a much awaited alternative to organ transfers.
Dr.V.R. Manoj currently teaches Environmental Sciences and Engineering to Engineering grad students in Velammal Engineering College,India. His multi-disciplinary qualifications include Environmental Sciences/Biotechnology, Microbiology, Plant Tissue culture and Bioinformatics. He has worked in Cell culture and in the Renewable energy industry. He began the Indian chapter of Humanity+ (formerly the World Transhumanist Association). He was an IEET Affiliate Scholar for 2010-2012, and continues as a contributor. Much of his written work can be accessed at his Blog (Cybofree : http://www.cyborgfantasy.blogspot.in )