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Novel Approaches to Cellular Transplantation from the US Space Program

Research in the treatment of type I diabetes is entering a new era that takes advantage of our knowledge in an ever increasing variety of scientific disciplines. Some may originate from very diverse sources, one of which is the Space Program at National Aeronautics and Space Administration (NASA). The Space Program contributes to diabetes-related research in several treatment modalities. As an ongoing effort for medical monitoring of personnel involved in space exploration activities NASA and the extramural scientific community investigate strategies for noninvasive estimation of blood glucose levels. Part of the effort in the space protein crystal growth program is high-resolution structural analysis insulin as a means to better understand the interaction with its receptor and with host immune components and as a basis for rational design of a "better" insulin molecule. The Space Program is also developing laser technology for potential early cataract detection as well as a noninvasive analyses for addressing preclinical diabetic retinopathy. Finally, NASA developed an exciting cell culture system that affords some unique advantages in the propagation and maintenance of mammalian cells in vitro. The cell culture system was originally designed to maintain cell suspensions with a minimum of hydrodynamic and mechanical sheer while awaiting launch into microgravity. Currently the commercially available NASA bioreactor (Synthecon, Inc., Houston, TX) is used as a research tool in basic and applied cell biology. In recent years there is continued strong interest in cellular transplantation as treatment for type I diabetes. The advantages are the potential for successful long-term amelioration and a minimum risk for morbidity in the event of rejection of the transplanted cells. The pathway to successful application of this strategy is accompanied by several substantial hurdles: (1) isolation and propagation of a suitable uniform donor cell population; (2) management of host immune rejection; (3) protection from the autoimmune component of the disease; and (4) anatomic placement of the engrafted cells that permits timely response to blood sugar levels as well as effective release and deployment of insulin. Bioreactor technology may provide some critical advances for surmounting some of these scientific hurdles. The NASA bioreactor is a horizontally rotating cylinder that is completely filled with culture medium. Gaseous exchange is maintained by a concentric cylinder of permeable silicon. In slow rotation (15-25 rpm) particles of small mass such as cells and tissue aggregates remain suspended in the rotating body of fluid. This novel approach suspends cells without stirring thus, allowing objects of different size mass to colocate and interact in a very low shear environment. In fact, analysis of the forces acting on individual cells in the rotating bioreactor reveals that the cells are continuously falling through the fluid medium. The conditions in the bioreactor permit assembly of cells into aggregates. three-dimensional tissue growth, synthesis of intercellular matrix,10 differentiation, and some sinusoid formations that may serve as a surrogate vasculature for larger tissue segments.

Pellis, Neal R.↗

Pressure probe study of the water relations of Phycomyces blakesleeanus sporangiophores

The physical characteristics which govern the water relations of the giant-celled sporangiophore of Phycomyces blakesleeanus were measured with the pressure probe technique and with nanoliter osmometry. These properties are important because they govern water uptake associated with cell growth and because they may influence expansion of the sporangiophore wall. Turgor pressure ranged from 1.1 to 6.6 bars (mean = 4.1 bars), and was the same for stage I and stage IV sporangiophores. Sporangiophore osmotic pressure averaged 11.5 bars. From the difference between cell osmotic pressure and turgor pressure, the average water potential of the sporangiophore was calculated to be about -7.4 bars. When sporangiophores were submerged under water, turgor remained nearly constant. We propose that the low cell turgor pressure is due to solutes in the cell wall solution, i.e., between the cuticle and the plasma membrane. Membrane hydraulic conductivity averaged 4.6 x 10(-6) cm s-1 bar-1, and was significantly greater in stage I sporangiophores than in stage IV sporangiophores. Contrary to previous reports, the sporangiophore is separated from the supporting mycelium by septa which prevent bulk volume flow between the two regions. The presence of a wall compartment between the cuticle and the plasma membrane results in anomalous osmosis during pressure clamp measurements. This behavior arises because of changes in solute concentration as water moves into or out of the wall compartment surrounding the sporangiophore. Theoretical analysis shows how the equations governing transient water flow are altered by the characteristics of the cell wall compartment.

NASA Discipline Number 40-30↗

Additive Manufacturing, Design, Testing, and Fabrication: A Full Engineering Experience at JSC

I worked on several projects this term. While most projects involved additive manufacturing, I was also involved with two design projects, two testing projects, and a fabrication project. The primary mentor for these was Richard Hagen. Secondary mentors were Hai Nguyen, Khadijah Shariff, and fabrication training from James Brown. Overall, my experience at JSC has been successful and what I have learned will continue to help me in my engineering education and profession long after I leave. My 3D printing projects ranged from less than a 1 cubic centimeter to about 1 cubic foot and involved several printers using different printing technologies. It was exciting to become familiar with printing technologies such as industrial grade FDM (Fused Deposition Modeling), the relatively new SLA (Stereolithography), and PolyJet. My primary duty with the FDM printers was to model parts that came in from various sources to print effectively and efficiently. Using methods my mentor taught me and the Stratasys Insight software, I was able to minimize imperfections, hasten build time, improve strength for specific forces (tensile, shear, etc...), and reduce likelihood of a print-failure. Also using FDM, I learned how to repair a part after it was printed. This is done by using a special kind of glue that chemically melts the two faces of plastic parts together to form a fused interface. My first goal with SLA technology was to bring the printer back to operational readiness. In becoming familiar with the Pegasus SLA printer, I researched the leveling, laser settings, and different vats to hold liquid material. With this research, I was successfully able to bring the Pegasus back online and have successfully printed multiple sample parts as well as functional parts. My experience with PolyJet technology has been focused on an understanding of the abilities/limits, costs, and the maintenance for daily use. Still upcoming will be experience with using a composite printer that uses FDM technology to print plastic while laying an internal filament of Kevlar or carbon-fiber inside the printed material. It has been incredible being exposed to this range of technologies and I feel very fortunate to be ready for virtually any kind of printing technology I come across in the future. Design work played a part in my internship this term as well. Working with Hai Nguyen, I was able to design a set of testing tips and a test frame for use with an Arcjet. The testing tips will be made of several different materials that will possibly be used in the heat shield of the Orion space craft. These designs included technical drawings that were presented to the fabrication shop. The frame design was created from 80/20 (a popular brand of frame construction equipment) and included an order form with pricing for fabrication. An independent design was also done for the virtual reality lab. This design was to create a hand-controller based on a previous design. This final design was sent directly to a 3D printer without technical drawings. Overall, my design work has given me experience with using 80/20, helped improve my CAD (Computer Aided Design) proficiency, and increased my knowledge of how to set up technical drawings for fabrication. The final role I have played in this internship has been to assist with testing of the inflatable technology materials working Khadijah Shariff. I began the internship assisting with permeability testing with the initial plan to continue the testing independently after training. Unfortunately, the testing apparatus suffered a technical failure and had funding pulled which cancelled that portion of the project. Further testing with inflatable technology continued with tensile testing of various stitching methods. This testing took place over a two-days and concluded successfully. Final testing was to be more tensile testing but of straps used to connect various inflatable sections. Unfortunately, the needed grips for the tensile tests could not be located and put the testing on hold. It is possible this round of testing will still take place by the end of the internship if the grips can be found. Overall, this portion of the internship has helped me become familiar with one kind of permeability test as well as a popular tensile/compression testing machine. Finally, I also had the chance to be trained using a metal lathe for making very small tips for a soldering iron. These tips will be used to melt threaded brass inserts into 3D printed plastic. Working with James Brown, I was able to successfully machine a brass rod down to as little as 0.064 inches plus or minus 0.001 inches. It was very rewarding to learn how to best use the machine and become familiar with a skill that will undoubtedly be used again in the future. I have been told by several professional engineers that learning to use a lathe and mill will be invaluable skills in the field. This knocks 50 percent of that goal off and I look forward to learning the mill at some point in the future. As is apparent with this list of projects, my internship was not focused on a single over-arching goal. Instead, I was able to gain experience in a myriad of very different areas. I feel like my time here was spent bouncing from one project to the next. Though sometimes difficult to switch gears, it was very rewarding to be a part of so much in so little time. My career and education will be positively impacted by what I have learned at JSC. My experience with 3D printing has improved my ability to handle many issues that may come up in the future with multiple different technologies. The design work I took part in, especially creating technical drawings, will help me better present designs to any engineer or shop I will encounter. My testing experience has helped me become familiar with a popular kind of tensile test machine that will likely be similar to the kinds I will encounter in the future. Finally, my experience with fabrication has given me a rare opportunity, as an engineer, to take part in the fabrication of a part. This experience will help me better tailor my future designs for the manufacturing process and has given me an appreciation for detailed/delicate machining work. My experience at JSC has been successful and will continue to assist me for a long time within the engineering field.

Zusack, Steven↗

Dynamics of Single Chains of Suspended Ferrofluid Particles

We present an experimental study of the dynamics of isolated chains made of super-paramagnetic particles under the influence of a magnetic field. The motivation of this work is to understand if the chain fluctuations exist and, if it does, how does the fluctuation affect chain aggregation. We find that single chains strongly fluctuate and that the characteristic frequency of their fluctuations is inversely proportional to the magnetic field strength. The higher the field the lower the characteristic frequency of the chain fluctuations. In the high magnetic field limit, chains behave like rigid rods without any internal motions. In this work, we used ferrofluid particles suspended in water. These particles do not have any intrinsic magnetization. Once a magnetic field is applied, a dipole moment is induced in each particle, proportional to the magnetic field. A dipolar magnetic interaction then occurs between particles. If dipole-dipole magnetic energy is higher than the thermal energy, the result is a structure change inside the dipolar fluid. The ratio of these two energies is expressed by a coupling constant lambda as: lambda = (pi(a(exp 3))(chi(exp 2))(mu(sub 0))(H(sub 0))(exp 2))/18kT Where a is the particle radius, mu(sub 0) is the vacuum magnetic permeability, H(sub 0) the applied magnetic field, k the Boltzmann constant and T the absolute temperature. If lambda > 1, magnetic particles form chains along the field direction. The lateral coalescence of several chains may form bigger aggregates especially if the particle volume fraction is high. While many studies and applications deal with the rheological properties and the structural changes of these dipolar fluids, this work focuses on the understanding of the chain dynamics. In order to probe the chain dynamics, we used dynamic light scattering (DLS) in self-beating mode as our experimental technique. The experimental geometry is such that the scattering plane is perpendicular to the magnetic field. Therefore, only motions in this plane are probed. A very dilute sample of a ferrofluid emulsion with a particle volume fraction of 10(exp -5) is used in this experiment. We chose such a low volume fraction to avoid multiple light scattering as well as lateral chain-chain aggregation. DLS measures the dynamic structure factor S(q,t) of the sample (q is the scattering wave vector, t is the time). In the absence of the magnetic field, identical particles of ferrofluid droplets are randomly distributed and S(q,t) reduces to exp(-q(exp 2)2D(sub 0)t). D(sub 0)=(kT/(6(pi)(eta)(a)) is the diffusion coefficient of Brownian particles (where Xi = (6(pi)(eta)(a)) is the Stokes frictional coefficient of a spherical particle in a fluid of viscosity eta). If interactions or polydispersity can not be ignored, an effective diffusion coefficient is introduced. Formally, D(sub eff) is defined as: D(sub eff) = - q(exp -2) partial derivative of (ln(S(q,t)) with respect to time, as t goes to 0. D(sub eff) reduces to D(sub 0) if no interactions and only a few particles size are present. Therefore, we can use DLS to measure particle size. The particle radius was found to be a=0.23 mu m with 7% of polydispersity. In this case, if we vary the scattering angle theta (and so q) we do not have any change in the measured diffusion coefficient: it is q-independent. When a magnetic field is applied, particles aggregate into chains if lambda > 1. We first studied the kinetics of the chain formation when lambda = 406. At a fixed scattering angle, we measured diffusion coefficient D(sub eff) as a function of time. Experimentally, we find that D(sub eff) decreases monotonously with time. Physically, this means that chains are becoming longer and longer. Since we are only sensitive to motions in the scattering plane and since chains have their main axis perpendicular to this plane, the measured diffusion coefficient is the trans-verse diffusion coefficient. We can relate D(sub eff) to the mean number of particles per chain N(t) at a given time and to the diffusion coefficient of an isolated particle D(sub 0) as D(sub eff)=f(N(t))D(sub 0). Since f(N) is known from other recent work, N can be expressed as a function of the time. We found a square root dependency: N(t) proportional to the square root of t. As expected for very low volume fraction, this behavior is characteristic of a diffusion-limited aggregation as suggested by several authors and by our previous work. In this study, we focus on the dependence of the effective diffusion coefficient on the scattering angle and the magnetic field strength. After the magnetic field is applied (lambda = 406) for a long time, typically 6 hours, kinetics of chain formation becomes very slow. Chain size does not vary much over the next hour period. Thus, we can perform different interesting experiments. First, at a fixed magnetic field, we measure the effective diffusion coefficient as a function of the scattering angle (from 5 to 130 deg). Our results show that the measured diffusion coefficient increases linearly with the scattering angle: D(sub eff) proportional to q. If we do the same experiment for different lambda values, D(sub eff) depends on lambda as D(sub eff) proportional to lambda(exp -1/2). We also find for different lambda values that the same asymptotic D(sub eff) value is obtained when q approaches zero. The angle dependency of D(sub eff) suggests that an additional motion exists besides chain drifting. Chain size is constant during experiment, which was verified by measuring the same diffusion coefficient at the beginning and at the end of the angle switching. If chains are rigid, D(sub eff) is independent of q. Therefore, we found that D(sub eff) not only measures the motion of the entire chain but also its internal fluctuations. These internal motions are the fluctuations of the particles in the chain. To understand the q dependency of D(sub eff), let us look at the probing length used. In our study, the characteristic length scale probed is l=2pi/q which is in the range of 0.9<l/a<20. When l is much larger than the particle radius (l/a going to infinity) we are mainly sensitive to the center of mass diffusion of the chain. D(sub eff) is then the diffusion of the entire chain and depends only on N but not on q and lambda. The value D(sub eff) allows us to obtain the number of particles per chain N. In the opposite limit (i.e. l/a < 1), we are sensitive to motions on the size of individual particles. The main contributions to the measured diffusion coefficient come from internal motions of the chains (i.e. particles' fluctuations inside the chain). We investigated also the effect of the chain size on D(sub eff). For the same value of the magnetic interaction lambda and different chain sizes, we found that the slope of D(sub eff) versus q decreases when N increases as a power law D(sub eff) proportional to N(exp -0.7). This is an important behavior because this means that the longer the chain the slower its internal fluctuations besides the whole chain motion.

Cutillas, S.↗