A Summer in a Tissue Engineering Lab

Updated: Oct 28, 2019

Amanda Zheng



You are drinking hot chocolate and it spills. Your hand jerks away in pain! Recognizing pain in situations of danger is a normal response of the nervous system that can help your body prevent further injury. The hot spill triggers the nerves in the affected area which relay information from skin to brain and brain to hand to generate evasive movement. The system typically works pretty well except in the cases where the nerves become damaged.


PERIPHERAL NERVE DAMAGE

Peripheral nerve damage affects over 20 million Americans due to factors such as diseases, such as HIV and diabetes, and old age [1]. Often, it is not possible to directly suture the nerve stumps together, necessitating the implementation of alternative methods. The current gold standard for treatments are autografts (grafting in nerve segments from one part of the body to the site of injury) and allografts (grafting in nerve segments taken from a donor)[2][3]. However there are many drawbacks to the current procedure such as the infection risks, incompatible matches, finite nerve donors, and limited functional recovery[4][5]. These implications have severe consequences, resulting in the death of 20 patients on the organ wait list per day. To counteract this, tissue engineers have been hard at work finding ways to grow cells artificially in the lab, such as implementing the use of artificial nerve conduits, that can alleviate the need for human nerve donors.


THE FIGHT FOR NEW SOLUTIONS

I’ve been interning this summer at a tissue engineering lab to learn the basics of the trade. The postdocs and graduate students in the lab have been conducting extensive researches ranging from creating organ simulations on chips, engineering multifunctional nanocarriers, and seeding cells on silk to observe their growth [6][7][8]. Though this may already seem incredible, other experiments in the field prove that creativity can be applied anywhere towards future ingenuity. One new technique was composed of running electrical simulation through neuron cells in order to mimic the charges induced in real signal processing and allow the neuron cells to grow in a more natural environment [9]. Other approaches to growing successful neuron cells consisted of altering the composition of the supporting scaffold material such as hydrogel scaffolds, which provide a 3D network composed of hydrophilic polymers that can hold large amounts of water to simulate living tissue[10]. Similarly, decellularized sheets from the extracted extracellular matrix of existing cells are used to simulate cell growth in a lab using the most natural material possible [11]. Another strategy specific to neuron cells is to combine neurotrophic factors, a family of biomolecules that promote the differentiation of neurons, directly to the scaffold solution for easier absorption [12]. To further promote the cell growth, recent studies are coating the scaffolds with materials such as graphene oxide because of its superior properties and ability to promote adhesion, proliferation, and differentiation of various kinds of cells [13].

Around the lab, there were no whole organs to be seen, instead the lab benches are full of partially completed projects and samples, giving a sense of continuous productivity around the bustling place. There are immunofluorescence microscopes to view the literally nanoscale cells and fibers from the experiments. Incubators are used for creating the ideal environment to store the cells.

The cells are in some ways similar to newborn babies, both being miniscule, fragile, and extremely demanding. To keep the cells alive, they must constantly be kept in the incubator with the right conditions. Their medium (nutrients) needs to be replaced every 2-3 days to ensure good nourishment. The cells have to be handled under a 100% sterile biosafety cabinet requiring all tools to be sprayed with alcohol (to kill bacteria and other contaminants) before entering the pressurized area. However, seeing the proof of life in the cells under the microscope was so rewarding, it made all the hassle completely worth it.

My personal project was to grow nerve PC12 cells on aligned or random fibers to observe the difference in the neurite growth on the cells. These fibers are made with a polymer solution (PCL or polycaprolactone) going through a process called electrospinning, in which the polymer solution runs through an electric field to create nanofibers. The nanofibers are spun onto a microscope cover glass slide, on which we then seed cells. The random scaffolds are created by letting the machine electrospin freely onto the cover glasses on top of aluminum foil. On the other hand, to create the aligned nanofibers, coverglass slips were temporarily secured to a rotating disk. The fast rotating speed of the disk caused the fibers to follow along the direction of the rotation and therefore create parallel fibers in one direction. The metal on the syringe needle of the electrospinning machine and the aluminum foil are both connected to an electrical source where kilowatts of electricity run through the contraption [13].

The purpose of these scaffolds is to mimic the natural environment of the cells, more specifically the extracellular matrix. They support the cells to regenerate the diseased or damaged tissues. Ultimately, the purpose of the scaffold is to implement the cell and scaffold complex into a site of injury for tissue repair and regeneration [15]. By seeding PC12 cells onto the random and aligned scaffolds, it was discovered that the aligned pattern of thicker diameter fibers was the most ideal conditions to promote the growth of longer, more effective neurons due to the nature of neuron cells to flourish in aligned environments.

This discovery can be used to cultivate functional neuron cells in hospitals for the twenty million people suffer from nerve damage around the world. Though my cell filled summer is not as hot as crowd filled beaches, I’m happy that my work contributed slightly toward a solution to alleviate those suffering.


References.

[1] Ratini, Melinda. (3/16/18) Nerve Pain and Nerve Damage. WebMD Medical Reference. https://www.webmd.com/brain/nerve-pain-and-nerve-damage-symptoms-and-causes#1. Retrieved: 8/3/18

[2] Ghasemi-Mobarakeh, L., Prabhakaran, M. P., Morshed, M., Nasr-Esfahani, M. H. & Ramakrishna, S. (2009) Electrical stimulation of nerve cells using conductive nanofibrous scaffolds for nerve tissue engineering. Tissue eng. Part A 15, 3605–3619. Retrieved: 8/5/18

[3] Kehoe, S., Zhang, X. F., Lewis, L., O’Shea, H. & Boyd, D. (2012) Characterization of PLGA based composite nerve guidance conduits: effect of F127 content on modulus over time in simulated physiological conditions. J Mech Behav Biomed Mater 14, 180–18. Retrieved: 8/5/18

[4] Chalfoun C, Wirth G, Evans G. (2006) Tissue engineered nerve constructs: where do we stand? J Cell Mol Med. Retrieved: 8/5/18

[5] Wiberg M, Terenghi G. (2003) Will it be possible to produce peripheral nerves? Surg Technol Int. Retrieved: 8/5/18

[6] Yang, Y., Hu, Y., Wang, H. (2016). Targeting Antitumor Immune Response for Enhancing the Efficacy of Photodynamic Therapy of Cancer: Recent Advances and Future Perspectives. Oxidative Medicine and Cellular Longevity. 11 pages. Retrieved: 8/9/18

[7] D. Wang, C. Chen, X. Ke, N. Kang, Y. Shen, Y. Liu, X. Zhou, H. Wang, C. Chen, L. Ren. (2015) Bioinspired Near-Infrared Excited Sensing Platform for in Vitro Antioxidant Capacity Assay Based on Upconversion Nanoparticles and Dopamine-Melanin Hybrid System. ACS Applied Materials & Interfaces. Retrieved: 8/9/18

[8] Tourlomousis, F, Chang RC. (03/2016) Numerical investigation of dynamic microorgan devices as drug screening platforms. Part I: Macroscale modeling approach & validation. Biotechnol Bioeng. Retrieved: 8/11/18

[9] Lynch, KJ, Skalli, O, Sabri F. ( 20/04/2018). Growing Neural PC-12 Cell on Crosslinked Silica Aerogels Increases Neurite Extension in the Presence of an Electric Field. J Funct Biomater. Retrieved: 8/3/18.

[10] El-Sherbiny, Ibrahim M. and Yacoub, Magdi H. (11/10/2013). Hydrogel scaffolds for tissue engineering: Progress and challenges. Global Cardiology Science & Practice. Retrieved: 8/3/18.

[11] Junka R, Yu X. (01/12/2016) Novel Acellular Scaffold Made from Decellularized Schwann Cell Sheets for Peripheral Nerve Regeneration. Regenerative engineering and translational medicine. Retrieved: 8/3/18

[12] Lee K, Silva EA, Mooney DJ. (2/6/11) Growth factor delivery-based tissue engineering: general approaches and a review of recent developments. J R Soc Interface. Retrieved: 8/9/18

[13] Gardin, Chiara, and Piattelli, Adriano, and Zavan, Barbara. Graphene in Regenerative Medicine: Focus on Stem Cells and Neuronal Differentiation. Trends in Biotechnology. Retrieved: 8/3/18.

[14] Laura A. Smith, Xiaohua Liu, Peter X. Ma. (01/01/2008). Tissue Engineering with Nano-Fibrous Scaffolds. Colloids and Surfaces B: Biointerfaces. Retrieved: 8/3/18.

[15] Sharma, B., and Elisseeff, J.H. (01/2004). Engineering structurally organized cartilage and bone tissues. Ann. Biomed. Eng. Retrieved: 8/7/18.

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