BIOTECHNOLOGY 3 OVERVIEW - Mains Associates · 2002-09-06 · Biotechnology is the application of...

id you ever stop to consider that some of

the foods in your refrigerator are products of biotech-

nology? Biotechnology is the application of knowl-

edge concerning biological systems to the production

of consumer goods and services. Foods like cheese,

yogurt, and beer are all products of biotechnology in

its most basic form — harnessing existing biological

processes, such as bacterial fermentation, to produce

goods for human consumption.

The term biotechnology probably also brings

to mind genetically engineered bacteria, plants, and

animals. It is this facet of biotechnology that allows

farmers to plant crops that can withstand certain

herbicides or diseases and helps researchers to

develop bacteria that can produce human insulin,

which is essential for the treatment of diabetes, and

drugs to dissolve blood clots, reducing the risk of

heart attack and stroke.

Although most biotechnology research bene-

fits the medical and agricultural fields, this kind of

work also supports a broad range of manufacturing

industries. Processes that use biological components

or that mimic biological systems can be used for a

variety of purposes, including creating new materi-

als, removing contaminants, and improving the effi-

ciency of chemical reactions. For example, microbes

are used to process sewage at city wastewater treat-

ment plants and to produce alcohol-based fuels for

motorized vehicles. Bacteria that can break down oil

ANNUAL REPORT 2001-20024

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BIOTECHNOLOGY 3

OVERVIEW

Farmers have benefit-ed from biotechnologyby being able to growhigh-yield crops thatare resistant to herbi-cides and disease.Harnessing biotech-nology has allowedthe agricultural indus-try to produce moreon fewer acres.

Biotechnology hasenabled scientists toturn to naturalsources for pollutioncontrol. Bacteria havebeen geneticallyaltered to perform avariety of environmen-tal clean-up tasks,including ingesting oilslicks.


and petroleum have been discovered, and

researchers have genetically altered these bacteria

to create microbes that can feed on oil slicks.

Biotechnology research focuses on how

organisms and their components function. Large

organisms are composed of systems of organs. If

you look in the mirror, you can see the largest organ

in the human body — the skin. The skin and other

organs consist of tissues specialized to perform spe-

cific functions in the body. These tissues in turn are

made up of a smaller structure — the cell. How the

cell functions in a particular tissue is determined by

its molecular components. Cells contain billions of

biological macromolecules, which are much larger

and more complex than nonbiological molecules.

The unique chemical traits of these molecules deter-

mine how a cell differentiates to become part of a

particular type of tissue and, ultimately, how an

organism grows, lives, and dies.

The microgravity environment of space

provides special advantages to biotechnology

researchers studying cell growth and biological mol-

ecules. NASA’s microgravity biotechnology pro-

gram, therefore, supports research in two main

areas: macromolecular biotechnology, overseen by

Marshall Space Flight Center (MSFC) in Huntsville,

Alabama, and cell science, overseen by Johnson

Space Center in Houston, Texas. The program’s

contributions to understanding the foundations of

life at the molecular and cellular levels may enable

the development of new drugs and other therapies

for disease and dysfunction, as well as measures

to safely send humans into space for extended

time periods.

OVERVIEW3 BIOTECHNOLOG

Cheese is a product ofbiotechnology. Bacteriaproduce lactic acid to aidcurd formation and influ-ence the cheese’s flavorand quality during ripen-ing. Genetic engineeringenables yeast to producecalf chymosin, the enzymeused to accelerate cheesecurd formation.

here are tens of thousands of biological

macromolecules at work in the human body. These

molecules, mostly proteins and nucleic acids, per-

form or regulate all functions that maintain life.

Proteins, for example, transport oxygen and chemi-

cals in the blood, form major components of muscle

and skin, and, in the form of antibodies, aid in fight-

ing infection. Enzymes, which are a class of pro-

teins, catalyze specific chemical reactions in cells

and control metabolic pathways, which are a series

of chemical reactions that together perform one or

more important functions, like the conversion of

sugar to energy.

Nucleic acids are another type of biological

macromolecule. The best-known examples of nucleic

acids are ribonucleic acid (RNA) and deoxyribonu-

cleic acid (DNA). Nucleotides, which are subunits

of nucleic acids, exist in a particular order along the

DNA molecule. Each unit of three nucleotides along

a strand of DNA forms a “letter” of the genetic

code, with the letters specifying particular amino

acids, the building blocks of proteins. So each sec-

tion of the genetic code actually specifies the pro-

duction of a specific protein, which in turn supports

the maintenance of life at both the cellular and

whole-organism levels. Small differences in genetic

codes can result in major differences within and

between organisms.

To unlock some of the mysteries about how

a biological molecule carries out its role, scientists

need knowledge of the molecule’s structure. A bio-

logical molecule’s shape and chemical components

determine the types of other molecules with which it

can interact. Proteins have active sites that allow

them to fit with other molecules to perform a specif-

ic function. Active sites on proteins, when inappro-

priately triggered, can cause disease or unwanted

functions. Drug designers seek knowledge of these

sites so they can develop drugs to block the sites or

otherwise render them inactive.

OVERVIEW


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MACROMOLECULAR BIOTECHNOLOGY 3

Proteins are the building blocks of our bodies and the living worldaround us. If the structure of a protein is known, then companiescan develop new or improved drugs to fight the disease of whichthe protein is a part. On Earth, convection currents, sedimenta-tion, and other gravity-induced phenomena hamper crystalgrowth efforts, and the result is crystals with flaws, as shown onthe left. In microgravity, researchers can grow high-quality crys-tals in an environment free of these effects to obtain better quali-ty crystals that yield more structural data, as shown on the right.Research on crystals of human insulin, like these, could lead toimproved treatments for diabetes.

cred

it: N

ASA


Information about molecular structure is

important to scientists in other fields as well.

Genetic engineers use this information to chemically

alter genetic codes to make bacteria, plants, or fungi

with desirable properties, such as yeast that has been

altered to produce insulin. Knowledge of molecular

structure is also the key to understanding how some

species survive and even thrive in extreme condi-

tions like the arctic or in volcanic vents. And

because some biological molecules, such as

enzymes, catalyze processes, understanding their

structure may enable their use as miniature manu-

facturing plants to process materials — the ultimate

in nanotechnology.

X-ray crystallography is the most common

method by which scientists study the structure of

biological molecules. Crystals of the molecule of

interest are formed, and X-rays are passed through a

single crystal at various angles. The resulting dif-

fraction patterns are analyzed using computers to

estimate the size, shape, and structure of the mole-

cule. A flawed crystal will yield a blurry and/or

weak diffraction pattern, whereas a well-ordered

crystal will yield a sharp and/or strong diffraction

pattern and thus useful information about the struc-

ture of the crystal.

A microgravity environment reduces the

effects of fluid flows and sedimentation within the

crystallization solution that can interfere with the

crystal growth process and the quality of the crystal.

OVERVIEW3MACROMOLECULAR BIOTECHNOLO3

This unusually large cubic crystal of satellite tobacco mosaic virusgrown under microgravity conditions is more than 30 times the size ofsimilar crystals grown on Earth.

credit: NASA

When a crystal begins to form in a solution, mole-

cules diffuse from the solution around the crystal to

join the growing crystal lattice. As a result, the solu-

tion in the immediate vicinity of the crystal has a

lower concentration of the crystal-forming material

than the remainder of the solution, and therefore has

a lower density. Under the influence of Earth’s grav-

ity, this difference in density creates currents next to

the growing crystal. Such fluid flows can alter the

orientation and position of the biological molecules

added to the crystal lattice, thereby creating disorder

in the crystal. Molecules are added to the crystal lat-

tice in the same way on Earth and in microgravity,

but in microgravity the lower concentration at the

crystal surface can slow crystal growth enough to

enable misplaced crystals to disassociate and then

reattach in a better orientation.

Likewise, sedimentation, another effect of

gravity, can result in poor-quality crystals. When

crystals grow to a size that cannot be supported by

suspension in the drop of solution in which the crys-

tals are forming, then the crystals will drift to the

bottom of the drop. There they may settle on top of

other crystals and grow into those adjacent crystals.

X-ray crystallography requires single crystals for

analysis, and thus sedimentation can render poten-

tially high-quality crystals unusable. In the micro-

gravity environment of low Earth orbit, the effects

of sedimentation and fluid flow are nearly eliminated,

and the conditions for growing diffraction-quality

crystals are improved.

Ground-based research in molecular science

includes crystallization of biological macromole-

cules (including analysis of crystals and methods to

control crystal quality); the development of bioma-

terials, which are substances that are synthetic or

natural in origin that can be used to treat, augment,

or replace a tissue, organ, or function of the body;

research on separation technology; and biologically

oriented nanotechnology.

OVERVIEW



Enzymes catalyze specific chemical reactions incells and control metabolic pathways. Studying thestructure of enzymes will help researchers to betterunderstand how the enzymes function. Creatinekinase, pictured here, converts the major storageform of high energy phosphate into a usable energyform. Creatine kinase is a major muscle enzyme andis implicated in some muscle diseases. Understandinghow the enzyme works could lead to therapies forthose diseases.

credit: NASA


3 MACROMOLECULAR BIOTECHNOLOGY

Program Summary

Following the release in 2000 of the NationalResearch Council’s* (NRC) report, titled FutureBiotechnology Research on the International SpaceStation, the biotechnology program implemented theStructural Biology Initiative to enact the recommendationsof the NRC panel. The goals of the initiative are to accel-erate the process by which investigators get their researchprojects to flight, to decrease the time interval betweendeveloping a research idea and obtaining data, and to match the speed of the ground-based researchprocess.

To achieve these goals, the biotechnology pro-gram will integrate new hardware for use on theInternational Space Station (ISS) that will allow increasedsample throughput and provide video microscopy of crys-tallization experiments. Also in response to the NRCpanel, the biotechnology program began developing andtesting an external review process that will accommodateboth large-scale research projects funded by NASA’speer-reviewed grant process and small-scale ad-hocinvestigations. An external, nonadvocate panel will beused to peer-review and prioritize experiments and tomake decisions in a timely manner to better match thepace of ground-based biotechnology research. This pro-gram will provide the scientific community with one,well-advertised point of contact for access to spaceflightexperiments. To obtain information on the associateinvestigator program, visit http://crystal.nasa.gov/technical/assoc_invest_prg.html or http://www.nisb.org.

The NASA Research Announcement (NRA) formacromolecular and cellular biotechnology that wasreleased in August 2000 directly addressed the recom-mendations of the NRC report. Research proposals in anumber of areas, detailed in the following paragraphs,were solicited from scientists.

Proposals were sought for structural biologyresearch to produce crystals of macromolecular assem-blies with important implications for cutting-edge biolo-gy problems, as recommended by the NRC. Systems thatmeet the criteria set forth in the NRA include membraneproteins, molecular motors, and biopolymer syntheticmachinery. The NRC report described all of these sys-tems as elaborate and fragile, which makes them difficultto crystallize except under optimal conditions. In thesecases, microgravity conditions might improve the qualityof the crystals enough to allow determination of key

structures. Also included were macromolecular systemsfor which research efforts have already been undertakenbut which have presented challenges for crystallization.In the area of crystallization studies and technologies,proposals were invited to support the aforementionedresearch with emphasis on providing a framework forunderstanding microgravity crystallization results, opti-mizing crystal growth conditions, characterizing crystaldefect formation and the relationship between defectformation and crystal growth, and providing a morerational approach to the growth of macromolecularcrystals.

NASA also invited proposals for developingtechnologies that seek to improve macromolecular crys-tallization throughput for structural biology and pro-teomics research on the ISS. Proteomics is the identifica-tion and study of proteins in the body, genes that codefor particular proteins, protein-protein interactions, andthe role of proteins in such activities as transmitting dis-ease. Research for improving throughput includes auto-mated crystal growth technologies, screening methods,and cryopreservation techniques.

In the area of biological nanotechnology, NASAsought research proposals for the development of molecular-sized sensors, signalers, and receptors; nanometer-scalebiomaterials; and technologies to manipulate biomole-cules to form useful devices or nanometer-scale struc-tures. Nanotechnology research is important because itcan be used to reduce experiments’ weight, volume, andneed for electrical power, all limiting factors duringspace missions.

Research solicited in the area of biomolecularself-assembling materials includes polymer biosynthesis,self-assembled monolayers and multilayers, decoratedmembranes, mesoscopic organized structures, and bio-mineralization. The area of biomolecular self-assemblingmaterials combines molecular biology, physical sciences,and materials engineering. Biomolecular materials haveability to assemble themselves without external interven-tion, and understanding the mechanisms involved in suchself-assembly could lead to the development of newprocesses and materials with significant technologicalimpact, including applications in life support to enablehumans to live and work permanently in space, as wellas other Office of Biological and Physical Research(OBPR) goals.

Finally, in the area of structural protein-basedmaterials, NASA solicited proposals for the production

* The NRC was organized by the National Academy of Sciences to associate the science and technology communities and to be the principaloperating agency that provides services to the government, the public, and the scientific and engineering communities. These services includeinvestigating, examining, experimenting, and reporting on any subject of science or art.



of protein-based materials or the isolation, in useableform, of such materials from cells. Collagen, keratin,and silk are examples of structural proteins. Researchersmay be able to incorporate novel properties in suchmaterials by genetically engineering the sequences orincorporating modular components from other proteins.Because these materials could be produced usingrecombinant DNA technology, it is possible to create auniform and controllable architecture of the resultingmaterial. Such biomaterials could also support OBPRgoals.

Notices of intent for this NRA were due onSeptember 6, 2000, and 225 proposals were received bythe October 27, 2000, due date. Selections were made inJune 2001; 20 of the selected proposals were to conductresearch in macromolecular biotechnology, includingprojects on challenging problems in structural biology,artificial biomembranes, and membrane proteins. Foradditional information on the NRA and selections, visithttp://research.hq.nasa.gov/code_u/nra/current/NRA-00-HEDS-03/winners.html on the WWW.

NASA macromolecular biotechnology principalinvestigators (PIs), co-investigators, guest investigators,and associate investigators published 69 peer-reviewedarticles in scientific journals in fiscal year (FY) 2001 and62 in FY 2002.

In early FY 2001, the Spallation Neutron Source(SNS) project and NASA cosponsored a workshop inKnoxville, Tennessee. Representatives from the biologi-cal neutron diffraction and microgravity crystal growthcommunities met to discuss the future use of the SNS formacromolecular single-crystal neutron diffraction.Academic, industry, and government advisers represent-ing the countries of England, France, Japan, and theUnited States participated in the workshop and devel-oped a set of recommendations regarding biological neu-tron diffraction crystallography.

Using neutrons to produce a diffraction patternof protein crystals has advantages over X-ray diffraction.About one-half of the atoms that make up a protein arehydrogen atoms. When protein crystals are bombardedwith X-rays, the X-rays are diffracted from the electronclouds of the individual atoms within the protein crystalto form a pattern from which the structure of the proteincan be determined. But it is an incomplete picture of thestructure, because hydrogen atoms have very little elec-tron density and so go undetected by X-ray diffraction.In contrast, when a protein crystal is bombarded withneutrons, the neutrons interact with the nuclei of the pro-tein crystal atoms. The diffraction pattern of the neutronsthen allows the position of hydrogen atoms to be identi-fied, and thus a more complete structure of the proteincan be determined.

However, neutron diffraction techniques pose aparticular challenge. Although neutron diffraction canprovide a complete structural analysis using a singlecrystal, that crystal must be much larger than crystalsthat are suitable for X-ray analysis. The colloquium for-mally recognized that because of research supported byboth NASA and the European Space Agency (ESA), theproduction of crystals sufficiently large for neutron dif-fraction studies is now an attainable goal. Growth ofcrystals 2 mm x 1.5 mm x 1 mm or larger is now com-mon for an increasing number of proteins. Based on pre-vious microgravity crystal growth experiments and theavailability of a controlled environment for extended-duration missions afforded by the ISS, it was estimatedthat approximately 90 percent of proteins crystallized inorbit will have the potential to reach 1 mm x 1 mm x 1 mm— the size range needed for analysis by current andfuture neutron sources.

NASA was well-represented at the AmericanCrystallographic Association’s 2002 Annual Meeting,held in San Antonio, Texas, May 25–30, 2002. One-thirdof the microgravity macromolecular biotechnology pro-gram’s principal investigators were key presenters at themeeting, which is the nation’s largest gathering of struc-tural biologists, drawing 800 attendees in 2002. At awell-attended session, “Biomacromolecular CrystalGrowth and Perfection,” which was sponsored by themacromolecular biotechnology program, NASA investi-gators made six presentations that covered hardwaredevelopment, crystal quality analysis method develop-ment, and a new technique for judging the quality of thecrystal cryocooling process.

Among the program presentations was a talkgiven by NASA Project Scientist Mark van der Woerdtitled “About Small Streams and Shiny Rocks:Macromolecular Crystal Growth in Microfluidics.” Vander Woerd provided an overview of work being conductedat MSFC using microfluidic technology for protein crys-tallization and reported on preliminary results from hard-ware incorporating that technology for crystal growth.

Aniruddha Achari, of MSFC, and his researchteam presented a poster titled “Equilibrium KineticsStudies and Crystallization Aboard the InternationalSpace Station Using the Protein CrystallizationApparatus for Microgravity (PCAM).” The PCAM hasbeen used to grow macromolecular crystals in a micro-gravity environment using a “sitting drop” method ofvapor diffusion. The experiments were set up to gatherdata for a series of days of activation with differentdroplet volumes and precipitants. The results of theseexperiments will help future PCAM users to choose pre-cipitants that will optimize crystallization conditions fortheir target macromolecules for a particular mission witha known duration.


3 MACROMOLECULAR BIOTECHNOLOG

In April 2002, a patent was awarded to macro-molecular biotechnology PIs and project scientistsWilliam Witherow, of MSFC, R. L. Kurtz, of Pace andWaite Inc., Huntsville, Alabama; and R. R. Holmes, ofMSFC, for their Laser Image Contrast EnhancementSystem (LICES). LICES allows objects that are hotenough to emit blackbody radiation to be illuminated andimaged. (A blackbody is a theoretically ideal radiator andabsorber of energy at all electromagnetic wavelengths.)For example, in a furnace, an object is heated until itemits blackbody radiation. It is then illuminated fromoutside with laser light and viewed with a camera with aspecial optical system.

In FYs 2001 and 2002 the macromolecularbiotechnology program made progress toward optimizingthe analysis of microgravity-grown crystals by advancingtechniques for cryocooling. Rapid cooling, or cryopreser-vation, is a technique routinely used to preserve crystalsof biological molecules for structural analysis by X-raydiffraction. It is important that crystals are carefully pre-served and stored not only to remain intact for lateranalysis but also to withstand radiation damage from theintense X-rays used. Flash cooling of crystals to near 100kelvins (cryocooling) extends a crystal’slifetime and makes it less susceptible tothe secondary radiation damage thatoccurs during X-ray analysis. Cryocoolingalso reduces thermal motions of the mole-cules and allows for data collection fromvery thin or small crystals.

Edward Snell, Russell Judge, andMark van der Woerd, of MSFC, have pro-vided a method by which scientists, forthe first time, can actually see images ofthe temperature gradients as crystals ofcertain molecules are rapidly cooled.Using a camera sensitive to infrared radi-ation, the MSFC scientists determined thelength of time it took to complete the cryo-cooling process. The experiment alsodemonstrated that it is possible to observedefects created by improper cooling orhandling of the crystals. Being able toactually assess temperature distributionacross a crystal and to observe defectscaused by improper handling will helpscientists to improve crystal preservationmethods and ultimately obtain more com-plete and accurate data.

The researchers presented theirwork on cryocooling at several venues in FY 2002, including the AmericanCrystallographic Association meeting in San Antonio. While investigating

cryocooling of crystals, Snell and van der Woerd alsostudied water in the macromolecular structure to under-stand how cryoprotectants interact with the crystal at themolecular level. Cryoprotectants replace water in thestructure and slow the formation of ice so that flash cool-ing the crystal vitrifies it — turns it into a glassy sub-stance — rather than freezes it. The cryoprotectants donot react with the crystal and are simply present to pro-tect the crystal from the effects of freezing. This workresulted in an invited talk in 2001, which was publishedin 2002 (“Neutrons and Microgravity,” by E. H. Snell, inProceedings of the 3rd International Symposium onDevelopment of New Structural Biology IncludingHydrogen and Hydration in Organized ResearchCombination System, 33–41). Complete neutron datasets have been collected to complement X-ray studies;these results will be published in 2003.

Flight Experiments

Three different pieces of macromolecular experi-ment hardware flew on the ISS in FYs 2001 and 2002,accommodating hundreds of macromolecular samplesthat successfully crystallized in the microgravity

The structural model of thaumatin shown here was developed from informationgleaned from thaumatin crystals grown in microgravity. The crystals grown in theEnhanced Gaseous Nitrogen Dewar on the International Space Station were of higherquality than any of those grown on Earth. Synchrotron studies of these crystals pro-duced 50 percent more data than had been obtained from the best ground-grown crys-tal. Thaumatin is a protein from the African Serendipity berry and is highly prized for itssweet taste.

credit: NASA



environment. Several technologies were also deployed toadvance crystal studies and analysis, including real-timeimaging of crystals during in-flight growth.

The first biological crystal growth experimentsconducted aboard the ISS took place in the EnhancedGaseous Nitrogen (EGN) Dewar in FY 2000 and werereturned to Earth in early FY 2001. The dewar, whichwas developed by PI Alexander McPherson of theUniversity of California, Irvine, flew on three ISS mis-sions in 2001 (each lasting approximately 40 days) andcarried a total of 881 samples of macromolecules to orbitfor crystallization.

In the dewar, crystals were grown using the liq-uid-liquid diffusion method. In liquid-liquid diffusionsamples, the material to be crystallized and the precipi-tant solutions are frozen separately, and then are thawedonce in orbit, diffusing with each other and resulting incrystal formation. Under microgravity conditions, crys-tals of the biomolecular materials form without interfer-ence from the container, other crystals, or turbulentflows, which often results in a crystal with a more nearperfect structure than those grown on Earth.

This was the case for the dewar-grown crystalsof thaumatin, which were of higher quality than any ofthis molecule grown on the ground. Thaumatin is a pro-tein from the African Serendipity berry (Thaumatococcusdanielli) and is valued for its intensely sweet taste.NASA PI Craig Kundrot, of MSFC, grew crystals ofthaumatin using the liquid-liquid diffusion method in theEGN. Synchrotron diffraction data collected from thebest crystal extended to 1.28 angstroms and produced 50percent more data than the best ground-grown crystaland 100 percent more data than earlier reports on thau-matin crystals in scientific literature. Results of the thau-matin crystal growth investigation were published in2002 in a paper titled “Thaumatin CrystallizationAboard the International Space Station Using Liquid-Liquid Diffusion in the Enhanced Gaseous NitrogenDewar,” by C. L. Barnes, E. H. Snell, and C. E. Kundrot,in Acta Crystallographica Section D: BiologicalCrystallography(58), 751–60.

FY 2001 also saw the transport of the ProteinCrystallization Apparatus for Microgravity aboard STS-100, on April 19, 2001. PCAM had flown on 11 previousshuttle flights. PCAM, developed by Daniel Carter, ofNew Century Pharmaceuticals, and his colleagues atMSFC, is a self-contained crystal growth apparatus thatuses multiple seven-chamber trays as a disposable inter-face. The sample chambers, which each hold a drop ofprotein solution, are surrounded by a “moat” ofabsorbent material that controls the crystal growthprocess after activation. The wells are filled prior tolaunch and sealed with rubber to prevent evaporation

and subsequent crystal formation before launch. Nine plas-tic trays can be loaded in one PCAM cylinder, and sixcylinders can be carried in a temperature-controlledlocker.

For its first ISS mission, in April 2001, PCAMtrays containing 756 samples of 11 different proteinswere housed in two Single-Locker Thermal EnclosureSystems. The scientific objectives of these experimentsranged from producing crystals of superior size and qual-ity for X-ray structure determination to experimentsaimed at improving understanding of the underlyingphysical processes involved in biological macromolecu-lar crystal growth in microgravity. PCAM equilibrationstudies conducted during the ISS increment produceddata that will help future users of the PCAM equipmentto optimize growth conditions for the macromolecules inwhich they are interested.

One of the PCAM experiments, led by Co-Investigator Jean-Paul Declercq of the University ofLouvain, in Belgium, resulted in crystals of peroxire-doxin 5. Peroxiredoxin 5 is a protein thought to play animportant antioxidant protective role in various tissuesunder both healthy and disease states. Peroxiredoxin mayalso be important to signal transduction, or communica-tion, between cells. Crystals of the oxidized form of thisprotein grown on the ISS showed an improvement in res-olution from 7 angstroms to 3.8 angstroms.

In FY 2002, PCAM flew on two space shuttlemissions headed to the ISS, STS-108 in December 2001and STS-111 in June 2002. On the STS-108 mission,several proteins produced significantly larger crystalsand, in some cases, crystals that diffracted to the highestresolution to date for Earth- or space-grown crystals. Onthis flight, Carter and New Century Pharmaceuticalscrystallized human serum albumin, the major protein ofthe human circulatory system. It contributes 80 percentto osmotic blood pressure and is chiefly responsible formaintaining blood pH. Additionally, albumin is involvedwith the binding and transportation of a variety of smallmolecules throughout the circulatory system, includingthe majority of currently known pharmaceuticals.Structural details of albumin and albumin-drug com-plexes can be used to explore the potential for improvingthe safety and efficacy of a broad base of therapeuticsand for developing novel engineered albumins for a vari-ety of applications. The highest resolution and qualitynative data to date on human serum albumin crystalswere collected from one of the crystals grown on theISS. Data were collected at a resolution of 1.9 angstroms,and these data indicated that even higher resolution datashould be obtainable.

Co-Investigator Mark Wardell, of New CenturyPharmaceuticals, crystallized human antithrombin III,


3 MACROMOLECULAR BIOTECHNOLO

which controls blood coagulation in human plasma andis an important target for understanding strokes andthrombolytic diseases, which include deep vein thrombo-sis, pulmonary embolism, and cerebral infarction. Thefirst few ISS crystals analyzed have shown diffraction toat least 1.8 angstroms, with an overall completeness ofmore than 95 percent. As with the human serum albumincrystals, even higher resolution data may be obtainablefrom the antithrombin crystals. A detailed analysis of theimproved structure is currently under way and will bepublished in the future. Another goal that was achievedduring PCAM experiments flown to the ISS on STS-108was the exploratory growth of crystals with a definedinternal symmetry, called a space group, and morphologysuitable for neutron diffraction. Carter’s PCAM experi-ment was geared toward proof of concept for this pro-tein/space group combination as a prelude for the morecostly-to-prepare samples that are currently aboard theISS. Neutron diffraction experiments are performedusing specialized nuclear reactors and require unusuallylarge crystals, which can be difficult to grow. The re-searcher’s efforts, if successful, can be rewarded withan exceptional view into the hydrogen arrangementwithin the protein molecule — a key to understandingmany of the chemical processes that underlie a protein’sfunction.

The Dynamically Controlled Protein CrystalGrowth (DCPCG) experiment flew on the ISS in FY2001. The DCPCG hardware was developed by theCenter for Biophysical Sciences and Engineering at theUniversity of Alabama, Birmingham. The hardware isthe first of its kind to allow the study of the physics of

the biological crystal growth process. The DCPCG designincludes a laser light scattering system that will be used toattempt to automatically detect the onset of nucleation,when the crystal begins to form. Microscopic high-resolution video cameras provide constant monitoring ofcrystal growth.

Carried to the station on STS-104 in July 2001,the DCPCG allowed, for the first time, dynamic controlover crystal growth. This was accomplished through theability to vary the rate of evaporation of the crystalliza-tion experiments using computers from the ground. Thiswas also the first microgravity payload that allowedautomated imaging of the crystal experiments inprogress. Video images were collected every four hoursand transmitted to Earth at a minimum of once each day.During operation of the DCPCG, half the chambers wereactivated and the experiments were monitored.Experiment conditions for the second half of the experi-ments were changed from the ground on the basis of theinformation obtained from the first set of chambers. Theremote control and imaging capability of the DCPCGpermitted scientists to observe two important phenomenaregarding crystallization in microgravity: the effect ofevaporation rates on crystal formation and the occur-rence of significant movement of the crystals in solution.

Although differences in diffraction resultsbetween ground- and microgravity-grown crystals of thetwo model proteins flown in DCPCG were not statisti-cally significant, the microgravity samples, having aslower evaporation rate, grew fewer and larger proteincrystals. The ability to see the samples every four hours

This series of images taken by Delta-L of one of 10 glucose isomerase crystals imaged automatically at 1.5-hourintervals can help give researchers insight into how growth rate dispersion can affect crystal growth and quality.

credit: NASA



gave some very intriguing results regarding crystalmovement, which was much more pronounced in themicrogravity environment of the ISS than had beenanticipated. It is not yet clear to what extent this move-ment was due to Marangoni effects, caused by convec-tion that occurs as the result of surface tension differ-ences, and what may have been the result of accelera-tions from various ISS activities. It is also not knownwhether the movement is a disadvantage to crystalgrowth — although it may be detrimental to ultimatecrystal quality, it may also help to grow larger crystalvolumes by moving the growing crystals into areas offresh nutrient.

Engineers and scientists at MSFC have teamedto produce award-winning flight hardware namedDelta-L. This equipment, which will fly on the ISS inlate 2003, is expected to provide data that will test thehypothesis that growth rate dispersion plays a role in

crystal quality improvement in microgravity. Growthrate dispersion is an occurrence in which individual crys-tals grow at slightly different growth rates under thesame solution conditions. MSFC scientists participatingin the study believe that microgravity may act to improvecrystal quality by reducing growth rate dispersion. Areduction in dispersion has been shown to be an indicatorof quality crystals on the ground.

The Delta-L experiment comprises a fluid assem-bly that allows crystallization fluid in the growth cell tobe exchanged, thereby providing fresh growth solution to enable continued crystal growth; a data acquisitionand control system; and an imaging system that allowsimages of crystals to be collected by using a videomicroscope camera.

MSFC scientists and engineers involved in thedevelopment of Delta-L are Dyana Beabout, Robert

Delta-L flight hardware, shown being tested in the ISS Microgravity Science Glovebox ground testunit, is expected to provide information to help researchers improve the quality of microgravity-grown crystals.

credit: NASA



Cooper, Eric Corder, Willie Dawson, Tim Dowling,Russell Judge, Paul Julino, Sharon Manley, Jim Meehan,Teresa Miller, Edward Snell, Mark van der Woerd, andJason Waggoner.

Highlights

Understanding How Antioxidants Protect the Body

Few people look forward to aging, and history isfull of stories of searches for a “fountain of youth.”Theories abound as to why and how aging happens. Oneof the more popular of these theories states that aging isdue to DNA and other cellular structures being damagedby a class of molecules known as free radicals. The bodyhas its own defenders against free radicals, and NASAPrincipal Investigator Gloria Borgstahl, of the Universityof Toledo, is using space-grown crystals to discover howone of these “antioxidants” works.

Free radicals are produced in the body duringoxidation, the reaction of oxygen with other molecules,which is a necessary chemical reaction that provides theenergy to maintain life. A free radical has an unpairedelectron in its outer orbital shell that is highly reactiveand wants to pair with another electron to gain a morestable state. This electron makes free radicals very unsta-ble. By reacting rapidly with nearby molecules, theunpaired electron is able to pair off with another elec-tron, but the result is yet another unpaired electron,which leads to a kind of chain reaction of free radicals.The role of antioxidants is to react with free radicals,thereby stopping their chain reaction and pre-venting damage to molecules that are importantto biochemical processes in the body.

The aging body somehow loses itsability to provide the necessary antioxidants toprotect vital biochemical processes from oxi-dation and the production of free radicals andbecomes subject to various aging-related prob-lems such as heart disease, diabetes, cancer,and Parkinson’s disease. Free radicals can alsoreact with fatty acids in the body to make themmore saturated and can cause cross-linking ofprotein molecules. One of the best-knownresults of this type of cross-linking is theappearance of wrinkles and the loss of elastic-ity in skin as we age.

Borgstahl is studying the antioxidantcalled superoxide dismutases (SOD), whichprotects the body from the oxidative damagethat is associated with aging. SODs are impor-tant enzymes that protect all living cells byreacting with the toxic superoxide radical, anoxygen molecule with a negative charge

because of an extra electron, which is a normal by-prod-uct of respiration, the oxidation or burning of fuel withincells.

The ultimate goal of Borgstahl’s research is tostudy the chemistry of SOD at the atomic level as it performs its job of detoxifying superoxides. She and her team hope that high-quality crystals of this enzymegrown in microgravity will help advance the understand-ing of how SOD works and will enable several types oftechnically challenging structure determinations.Although a naturally occurring manganese-containingform of superoxide dismutase (MnSOD) — the specificenzyme Borgstahl is working with — has been exten-sively studied biochemically, the crystal structure of this

This microgravity-grown MnSODcrystal is pink due to the manganesemetal ion in the active site. Earth-grown crystals typically grow as thinplates and are never thick enough toallow the viewer to see this vibrantpink color.

credit: Gloria Borgstahl

According to one popular theory, aging is due to DNA and other cellular struc-tures’ being damaged by a class of molecules known as free radicals. The bodyhas it own defenders against free radicals, including superoxide dismutases(SODs). PI Gloria Borgstahl hopes that high-quality crystals of MnSOD grownin microgravity, pictured here, will help advance the understanding of howSODs work and will enable several types of technically challenging structuredeterminations.

credit: Gloria Borgstahl



enzyme has not been solved. MnSOD and other metalSODs are part of the cell’s defense against free radi-cal–mediated damage. MnSOD also protects against theproduction of free radicals during inflammatory pro-cesses in the body.

Borgstahl’s first microgravity crystallizationexperiments on the manganese-containing SODs weretransported to the ISS by STS-108 in December 2001and were returned to Earth in April 2002. At AdvancedPhoton Source in Chicago, Illinois, Borgstahl and hercolleagues were able to collect the best X-ray diffractiondata available to date from the crystals grown on the ISS.Crystals grown in Earth laboratories are typically longrectangular rods that are thin in cross section (120microns x 50 microns x 1,000 microns). To the researchteam’s great satisfaction, all 35 crystallization experi-ments on the ISS produced crystals, and more than halfof them were of a dramatically larger volume in crosssection (400 microns x 400 microns x 3,000 microns)than similar crystals grown on Earth. Several of theMnSOD crystals grown on the ISS were 80 timesgreater in crystal volume than Earth-grown crystals,and diffraction spots to 1.26-angstrom resolution wereobserved, providing significantly improved data com-pared with that obtained from crystals grown in Earthlaboratories. Borgstahl has said that the difference incrystal size was “like comparing toothpicks to 4-inch x 4-inch planks of wood.”

To answer fundamental biochemical questionsconcerning this enzyme, Borgstahl and her team neededto obtain these large, high-quality crystals of SOD forneutron studies and for time-resolved Laue studies. Bothneutron and Laue methods require large (greater than1 cubic millimeter), perfect crystals. With the neutronexperiment, the researchers hope to be able to obtain thenever-before-seen, three-dimensional structure of thehydrogens on each amino acid of the protein and therebyanswer the unsolved questions concerning the source ofthese hydrogens in the enzyme reaction mechanism.With the time-resolved Laue experiments, the team will be able to generate the superoxide substrate within the crystals with a laser pulse and then make a“movie” of the enzyme converting it to peroxide andwater.

The role of manganese-containing SODs in thebody is important, and in-depth study of their structure isnot only vital to understanding their function, but alsomay lead to new therapeutic treatments for variousdegenerative processes.

Stamp-Sized Laboratories for Space

Just as the world of electronics was reshaped bythe philosophy that “smaller is better,” biotechnology

systems are being transformed by the drive to miniaturize.The result is the production of tiny biological laborato-ries on the scale of microns and millimeters. The func-tion of several pieces of standard laboratory equipmentand a lab technician can now be replaced with a postagestamp–sized “lab-on-a-chip.” The science of microflu-idics is making this new technology possible as itrequires the ability to manipulate processes that involvefluid volumes measured in nanoliters (10-9 L) and pico-liters (10-12 L). In the life sciences, microfluidic systemsmay be used for biochemical assays, genetic analysis,drug screening, electrochromatography (separating the components of a substance by applying a voltage),and blood-cell separation/analysis (to determine bloodcell counts and the presence of disease), reducing thetime and cost of performing complex biochemicalprocesses.

NASA has also recognized the potential of thesechips to process samples of macromolecules for crystalgrowth experiments in space. The tiny chips couldgreatly minimize the volume of valuable biological sam-ples required to obtain results. With this objective, in late2001, a collaboration began between NASA’s IterativeBiological Crystallization (IBC) project and CaliperTechnologies Corporation of Mountain View, California,the renowned mass producer of LabChip® devices. Theresult is chip NS374.

In future space travel, miniaturized systemswill be essential for reducing spacecraftsystem mass and volume. The functions ofseveral laboratory instruments can now beplaced on a chip that is not much largerthan a dime. The size of the chips greatlyminimizes the volume of valuable biologicalsamples that must be used to obtainresults, and the automated equipment thatmanages the chips allows scientists onEarth to use the Internet to set up and trackcrystallization experiments on the ISS.

credit: NASA

ACTUALCHIP


In any experiment, for crystal growth to occur,a sample is prepared that contains the macromoleculein a solution and a precipitant that initiates evaporationand thus crystal formation. The new chip is capable ofmixing prescribed recipes from up to five solutioncomponents and of selectively delivering each of therecipe mixtures to separate growth wells that reside onthe same chip. Testing of this chip, which has beenongoing for several months in the IBC laboratories,has proven the efficacy of the approach. The IBC lab-on-a-chip system will provide a statistically significantsample, much higher throughput, and greater repeata-bility of macromolecular experiments. Automatedequipment that manages the chips allows scientists onthe ground to use the Internet to iteratively set up andvisually evaluate hundreds of crystallization experi-ments throughout the duration of an ISS flight incre-ment.

The lab-on-a-chip technology developed byIBC, with its capacity to meter and mix biological flu-ids with picoliter accuracy, is well suited for commer-cial or academic structural biology research on Earthas well as in space. The unit will also be adaptable forother areas of research that employ microfluidics.

In future space travel, miniaturized systemswill be essential in reducing the mass and volume ofspacecraft systems. Microfluidics has the potential tofacilitate and automate scientific research across multi-ple disciplines. As NASA seeks to develop tools thatwill diminish the negative effects of long-term spacetravel on humans, lab-on-a-chip technology is apotential springboard for medical diagnostic and ther-apeutic devices that will ultimately make spaceflightsafer for humans.

A New NASA Institute

In November 2001, NASA awarded theHauptman-Woodward Medical Research Institute(HWMI) in Buffalo, New York, a grant to establish theNASA Institute for Structural Biology (NISB).Hauptman-Woodward is an independent, nonprofit facili-ty specializing in basic research using structural biologyand is known worldwide for its expertise in crystalgrowth. The new NASA institute will be devoted to fos-tering research in the field of macromolecular biologyand in facilitating the use of low-gravity research oppor-tunities. Principal Investigator George DeTitta and hisco-investigator, HWMI Research Scientist Joseph Luft,and HWMI Executive Vice President and PrincipleResearch Scientist Walter Pangborn were named to headthe institute.

NISB was formed in part to help structural biolo-gists access flight hardware through NASA’s unified

Associate Investigator Program, which is focused onopening up spaceflight opportunities to a larger commu-nity of scientific researchers. Shortly after its inception,NISB began to promote awareness of the new program.The NISB contacted groups and individual members ofthe structural biology community, providing informationabout how the institute can assist investigators who wishto fly macromolecular crystallization samples in ISSexperiment hardware sponsored by NASA’s PhysicalSciences Research Division. Also provided was a list ofupcoming flights available with instructions on how tomake an application. An independent peer-review panelwas set up to evaluate flight proposals, and the groupbegan reviewing applications for early 2003.

Another important task for the NISB is to helpselected researchers through the process of flying experi-ments on the ISS. To that end, the NISB will provide thesupport necessary to do adequate ground crystallizationexperiments and diffraction analyses to assess the effectsof microgravity, including making synchrotron radiationavailable. Timely access to synchrotron beam time fol-lowing retrieval of flight experiments is a high priorityfor researchers, as using electromagnetic radiation hasbecome an indispensable tool in the field of X-raycrystallography for molecular structure determination.NISB will help investigators secure access to theStanford Synchrotron Research Laboratory at StanfordUniversity.


In June 2002 New York State Senator Hillary Rodham Clinton(second from left) announced the award of a $2.6 million grantover three years from NASA to the Hauptman-Woodward MedicalResearch Institute (HWMI) to establish the NASA Institute forStructural Biology at HWMI’s Buffalo Niagara Medical Campus.From left to right: George DeTitta, HWMI executive director andCEO; Senator Hillary Rodham Clinton; Ron Porter, manager,Science Planning and Program Management Group, MSFC;Herbert Hauptman, HWMI president; and Christopher Greene,HWMI chairman of the board.

credit: Hauptman-Woodward Medical Research Institute


ore than 70 years ago, cellular biologist

E. B. Wilson wrote in his book The Cell in

Development and Heredity that “the key to every

biological problem must finally be sought in the

cell.” All living creatures are made of cells — small,

membrane-bound compartments filled with a con-

centrated water-based solution of chemicals. The

simplest forms of life are solitary cells that propagate

by dividing in two. More complex organisms such as

humans are like cellular cities in which groups of

cells perform specialized functions and are linked by

intricate communication systems. Cells occupy a

halfway point on the scale of biological complexity.

Scientists study cells to try to understand their

molecular makeup and to learn about how they

cooperate to enable a complex organism to function.

More than 200 types of cells make up the

human body. They are assembled into a variety of

tissues, such as skin, bone, and muscle. Most tissues

contain a mixture of cell types. Cells are small and

complex — a typical animal cell is about five times

smaller than the smallest visible particle, and it con-

tains all the molecules necessary to enable an organ-

ism to survive and reproduce itself. A cell’s small

size makes it difficult for scientists to see its struc-

ture, to discover its molecular composition, and

especially to find out how its various components

function. Differentiated cells perform specialized

functions. For example, a heart muscle cell looks

different from and performs different functions than

a nerve cell. Specialized cells interact and communi-

cate with one another, setting up signals to govern

the character of each cell according to its place in the

structure as a whole.

What can be learned about cells depends on

the available tools. Culturing (growing) cells is one

of the most basic techniques used by medical

researchers. The growth of human cells outside the

body enables the investigation of the basic biological

and physiological phenomena that govern the normal

life cycle and many of the mechanisms of disease. In

traditional research methods, mammalian cells are

cultured using vessels in which cells settle to the bot-

tom surface of the vessel under the influence of

gravity. This gravitational influence results in a thin

sheet of cells, with the depth of a single cell, called a

monolayer. Cells in human tissues, however, are

arranged in complex, three-dimensional structures.

MM

OVERVIEWCELLULAR BIOTECHNOLOGY 3 NASA’s ground-based

rotating bioreactor isan analog of micro-gravity cell culturethat has made it pos-sible for cells toaggregate, differenti-ate, and grow in threedimensions in cultureson Earth.

credit:: NASA


When cells are grown in a monolayer, they do not

perform all the functions that the original tissue does.

Although much valuable information can be

gained from monolayer cell cultures, further under-

standing of the processes that govern gene expres-

sion and cellular differentiation is limited because

the cells are not arranged as they are in the human

body. When the influence of gravity is decreased,

the cells are able to grow in more tissue-like, three-

dimensional aggregates, or clusters. Until the cellular

biotechnology program developed a unique technol-

ogy called the NASA Bioreactor, experiments to

form three-dimensional cell formations were con-

fined to the microgravity environment of space.

The NASA-designed bioreactor allows cells

to be cultured in a continuous freefall state, simulat-

ing microgravity and providing a unique cell culture

environment on the ground. The growth medium–

filled cylindrical vessel rotates about a horizontal

axis, suspending the cells in a low-shear* culturing

environment. This allows for cell aggregation, differ-

entiation, and growth. The bioreactor affords

researchers exciting opportunities to create three-

dimensional cell cultures that are similar to the

tissues found in the human body.

Using both space- and ground-based bioreac-

tors, scientists are investigating the prospect of

developing tissues that can be used in medical trans-

plantation to replace failed organs and tissues.

Additionally, investigators are striving to produce

models of human disease to be used in the develop-

ment of novel drugs and vaccines for the treatment

and prevention of disease, to devise strategies to

reengineer defective tissues, and to develop new

hypotheses for the progression of diseases such as

cancer. Finally, cells exposed to simulated and true

microgravity respond by making adaptations that

give new insights into cellular processes, establish a

cellular basis for the human response to microgravity

and the space environment, and pave the way for cell

biology research in space regarding the transition of

terrestrial life to low-gravity environments.

OVERVIEWBIOTECHNOLOG3 CELLULAR BIOTECHNOLO

The NASA rotating wall vessel bioreactor provides a low-turbulenceculture environment that promotes the formation of large, three-dimensional cell clusters. Cell constructs grown in the bioreactormore closely resemble tumors or tissues found in the body. Cellconstructs grown in a rotating bioreactor on Earth (left) eventuallybecome too large to stay suspended in the nutrient medium. In themicrogravity of orbit, the cells stay suspended (right). Rotationprovides gentle stirring to replenish the medium around the cells.

Rotating Wall Vessel Bioreactor

EARTH (1g) ORBIT (µµg)

credit:NASA

* Shear is the force caused by the cells sliding against one another.


Program Summary

A recommendation was made by the NationalResearch Council in 2000 for the Cellular BiotechnologyProgram to work with the Fundamental Biology Programof NASA’s Life Sciences Division to take advantage ofoverlapping interests. Therefore NASA Cell Science con-ferences, which had for many years been sponsored bythe Cellular Biotechnology Program (CBP) at JohnsonSpace Center in Houston, Texas, were jointly sponsoredby the CBP and the Fundamental Biology Program atAmes Research Center in Moffett Field, California, in2001 and in 2002. This successful collaboration betweenthe two centers will continue.

The 2001 NASA Cell Science Conference andAnnual Investigators Working Group Meeting was heldMarch 6–8, 2001, in Houston, Texas; this was the firstagencywide cell science conference. Approximately 190scientists from universities, NASA centers, the NationalInstitutes of Health, the National Space BiomedicalResearch Institute, and commercial cell culture enterpris-es attended the three-day conference. Sixty-three invitedspeakers gave talks in the following areas: cell move-ment/cytoskeleton, tissue modeling, biological responsesto physical forces, models in lower organisms, immunol-ogy, cell culture technology, proliferation and differentia-tion, and gene expression. Eight industry exhibitors alsoattended to showcase their products.

The 2002 conference was held February 26–28,2002, in Palo Alto, California, and was attended byapproximately 160 scientists and 10 exhibitors.Currently, planning is under way for the 2003 NASACell Science Conference, scheduled for February 20–22,2003. In addition to the research areas covered in pastconferences, the 2003 conference will be expanded toinclude presentations on neoplastic disease (cancer), sen-sors and analytical equipment, and gravity and mechani-cal sensing. The area of sensors and analytical equipmentencompasses work in advancing the state of the art inautomated cell culture technology. In space, the culturingof cells must be highly automated because it may be per-formed by crewmembers who are not proficient in thisvery time-consuming and skill-intensive procedure.Thus, sensing systems that can detect cell culture condi-tions and control them autonomously to ensure theyremain viable are necessary to ensure successful science.Gravity and mechanical sensing covers investigationsinto the molecular and cellular mechanisms behind themany varied responses seen in microgravity. Understandingthese is critical to understanding why we see many decreas-es in quality of physiological functions such as muscleatrophy and bone loss associated with spaceflight.

Under the Cellular Biotechnology Program in fiscal years (FYs) 2001 and 2002, 47 principal

investigators conducted scientific investigations in bothground- and flight-based environments, resulting in morethan 50 publications in peer-reviewed scientific journalsand proceedings. Additionally, a NASA ResearchAnnouncement for cellular biotechnology (NRA 01-OBPR-08-B) was issued in June 2002.

Research solicited under this announcementsought to establish the scientific foundations for futureexperiments on the International Space Station (ISS) andto support the development of biotechnology applica-tions for long-duration spaceflight. The solicitation alsosought coordinated research efforts involving both space-and ground-based research that would lead to potentialflight experiments or development of new technologiesfor future NASA missions. NASA not only invitedresearch in the areas that it has previously supported,such as tissue engineering, bioreactor design, andchanges in gene expression, but also expanded the scopeto include other research areas that have been identifiedas having potential to contribute to human exploration ofspace. These new areas of supported research includeseparation, purification, and remediation methods;microbiosensor monitoring devices; and selective pres-sures on cell populations, among others.

Separation, purification, and remediation meth-ods are needed to clean and recycle water on spacecraftduring future long-duration missions. Purification meth-ods must be specific for toxic molecules, reliable, andinexpensive and must make minimal demands on space-craft resources. The cellular biotechnology program cancontribute in this area by researching the use of cellu-lar organisms to convert or catalyze fluid waste tousable products such as drinking water, oxygen, ormethane.

Likewise, the cellular biotechnology programcan assist in the development of microbiosensor monitor-ing devices. The sensors will be microtechnology- andnanotechnology-based, will be extremely stable andsmall, and will be used for monitoring biological systemsand experiments to aid in the advancement of biotechno-logical processes and their use in support of long-duration space missions.

Assessment of selective pressures on mammalianand microbial cell populations is critical to long-termoccupation of space. Changes in cells that are both geno-typic (changes in the makeup of the genes themselves)and phenotypic (changes in how the genes express them-selves externally) occurring over numerous generationsof cells exposed only to a space environment must bestudied in order to determine risks to our biologicalintegrity and to our life-based support systems wroughtby extended (and even permanent) stays in space.

CELLULAR BIOTECHNOLOGY 3


Proposals in response to the NRA were dueSeptember 6, 2002, and selections are expected to bemade in May 2003. For additional information, visithttp://research.hq.nasa.gov/code_u/nra/current/NRA-01-OBPR-08-B/index.html on the World Wide Web.

Most of NASA’s previous work in cell sciencehas taken place on shuttle flights and on the Russianspace station, Mir. These experiments have demonstratedthat microgravity and the space environment affect cellshape, signal transduction (the transfer of signals fromoutside the cell to inside the cell), replication and prolif-eration, gene expression, apoptosis (cell disintegration),and synthesis and orientation of intracellular and extra-cellular macromolecules. With the increased availabilityof research opportunities on the ISS and the new hard-ware developed specifically for this platform, more fre-quent and longer term investigations will undoubtedlyaccelerate the advancement of our understanding of howthe microgravity environment affects cell structure,processes, and functions.

Flight Experiments

FY 2001 and FY 2002 were again years ofadvances for the cellular biotechnology flight program.Several researchers flew experiments on space shuttleflights and on the ISS. Flight experiments included stud-ies of colon cancer metastasis, kidney cell gene expres-sion, and erythroleukemia. Additionally, in FY 2002 theCellular Biotechnology Programextended its efforts to expand biotech-nology commercial ventures by enlarg-ing its agreement with StelSys Inc. ofBaltimore, Maryland, to include flightexperiments aboard the ISS. Progresswas also made on the development ofthe Biotechnology Facility for the ISS.

Principal Investigator (PI) J.Milburn Jessup’s study of colon carci-noma metastasis using the NASAbioreactor flew to the ISS aboard STS-105 in August 2001. Jessup, of theGeorgetown University MedicalCenter, is a veteran of two spaceflightexperiments on shuttle missions STS-70 (July 1995) and STS-85 (August1997). STS-70 provided the proof thatNASA’s rotating wall vessel bioreactor(RWV) could be used to grow three-dimensional cellular aggregates. Thecarcinoma cells provided by Jessup forthe experiment formed masses 10millimeters in diameter — 30 timesthe volume of those grown in thecontrol experiment on the ground.

The experiment was repeated on STS-85, again resultingin mature differentiated tissue samples and confirmingthat microgravity is an environment beneficial to cellculture and tissue growth.

Experimental results from these two space shut-tle flights indicated that programmed cell death, or apop-tosis, occurred in the RWV on the ground but wasreduced in the actual microgravity cultures. The rate ofapoptosis in the MIP-101 (human colorectal carcinoma)cells approached that of the same cells growing as nonro-tated masses in three dimensions on a surface to whichthe cells did not attach. This finding was importantbecause it suggested that rotation at the speeds necessaryto suspend cells on Earth in the RWV may actually hurtthe cells. Other researchers have reported that RWVsoperated on Earth may also change the cytoskeleton, orbackbone, of cells in such a way that rotation may leadto cell death. In microgravity, the RWV does not need tospin as fast to keep the cells suspended, so the cells morenearly approach the nonrotated three-dimensional cul-tures on the ground. Jessup has recently found thatrotation on the ground also increases nitric oxide andreactive oxygen species production by as much as six toeight times. These substances can be quite toxic to cellsand cause the apoptosis seen in the RWV.

Jessup’s work from these flights resulted in twopeer-reviewed publications in national journals regardingmetastatic characteristics of colon carcinoma.

3 CELLULAR BIOTECHNOLO

Timothy Hammond is examining how microgravity alters the gene expression in renal cells thatultimately enables kidneys to develop and function normally. He has found that the geneticexpression of human renal cells can be manipulated in microgravity to produce hormones thatare valuable in the treatment of disease.

credit: NASA


Preliminary ground research indicated that the metabo-lism of the MIP-101 line of colon cancer cells is signifi-cantly increased in microgravity; the cells essentiallyoutgrew the capacity of the rotating wall vessel bioreac-tor under simulated microgravity conditions.

Additional flight experiments were conductedduring the STS-108 mission in December 2001; analysisof those experimental samples is in progress. Jessup usedthe MIP-101 cells for the additional experiments becausethey differentiate, producing certain proteins, in theRWV. He conducted the experiment to assess howaggressively such cells will consume nutrients in micro-gravity when a three-dimensional culture is attempted, aswell as to test the metabolic requirements of space-basedbioreactors. Ultimately, Jessup hopes his research willaid in gaining additional needed information regardingthe mechanisms involved in colon cancer metastasis.

Jessup is looking forward to testing the hypothe-sis that the apoptosis seen in the RWV on the ground andin some spaceflights is due to oxidative stress. In addi-tion to the results gathered from his studies of cell deathin MIP-101 cells, much evidence exists that reactiveoxygen and nitrogen species are generated both in cellsin culture and in muscles and organs in crewmembersand animals in space. This work may ultimately lead to abetter understanding of the effects of reduced gravity on

subcellular organelle distribution and oxidative stress.This may also help provide a means to assess new andbetter countermeasures for the deleterious aspects ofweightlessness.

PI Timothy Hammond, of Tulane UniversityMedical Center and the Veterans Affairs Medical Centerin New Orleans, Louisiana, has conducted a series ofspaceflight cell culture experiments using renal (kidney)cells. He is examining how microgravity alters the geneexpression in renal cells that ultimately enables kidneysto develop and function normally. During shuttle missionSTS-106 (September 2000), Hammond cultured three-dimensional constructs of normal human renal cells, andin early FY 2001, he analyzed the results regarding thegenetic expression of human cells in microgravity andtheir ability to be manipulated to produce renal hor-mones that are valuable in the treatment of disease.

Continued studies of renal cells inmicrogravity aboard STS-105 in August 2001revealed additional information about themechanisms involved in these genetic manipu-lations and responses. Hammond was able toassess the cultured tissue’s production of ery-thropoietin, a hormone produced mainly by thekidneys that stimulates the production of redblood cells by stem cells in bone marrow, andvitamin D3, a substance converted by the kid-neys that plays an important role in the absorp-tion of calcium from the intestine, helping tomaintain strong bones. As expected, the pro-duction of both of these substances increaseddramatically in space. Hammond hopes toadapt the three-dimensional tissue model forcommercial production of these hormones.

In FY 2002, Hammond flew renal cellson the ISS during Expedition 4 (December2001–June 2002). In this study, Hammondexamined the responses of normal human renalcells to a peptide sequence known to inhibitthe vitamin D receptor under microgravity. Inpatients with kidney disease characterized byheavy protein excretion, it is believed that theelevated levels of protein in the kidney cause

tissue damage and scarring. This toxicity is thought to bedue to the binding of low–molecular weight proteins toscavenger receptors on the surface of the renal proximaltubules. Molecules that can disrupt the scavenger path-way by binding to the scavenger receptors include cer-tain classes of antibiotics as well as artificial blood andhormone precursors like vitamin D. Hammond evaluatedcellular structure and assessed the distribution of thevitamin D receptor and other biological molecules thatcontrol gene expression to understand the molecular


Microgravity is valuable for peeling away the interfering effects of gravity and lay-ing bare functions of an organism that might not be apparent on Earth. Thesehuman liver cells were flown on the International Space Station and then culturedfor 24 hours on the ground. Scientists studying how space changes life-formshope that a comparison between cells grown in microgravity and those grown onEarth will provide insight into the effects of microgravity on liver cell functions andresult in a better understanding of liver functions both in space and on Earth.

credit: StelSys Inc.


BIOTECHNOLOG

mechanisms mediating gene expression changes in space.Long-term goals of Hammond’s research include identi-fication of genes that respond to microgravity, modelingof renal injury mechanisms, and production of renal hor-mones of pharmacological importance. Changes in geneexpression in cells grown in space have demonstrated themetabolic pathways important in the response to micro-gravity, which is allowing the researchers to mimic thebiologically and pharmacologically useful elements ofthis response on the ground.

PI Arthur Sytkowski, of Harvard MedicalSchool, Cambridge, Massachusetts, conducted a flightexperiment during ISS Expedition 4 to study ery-throleukemia (EMS-3) cells. Erythroleukemia is a cancerof the blood-forming tissues in which large numbers ofimmature, abnormal red blood cells are found in theblood and bone marrow. The EMS-3 cells respond toerythropoietin, the natural inducer of the formation of redblood cells, and to other chemical inducers such asdimethyl sulfoxide (DMSO). Sytkowski and his teamcultured EMS-3 cells in orbit to test their responsivenesswhen exposed to these inducers in microgravity andcompare results to data from previous ground-basedrotating wall vessel bioreactor experiments.

Because it has been known for some time thatred blood cells do not evolve well in microgravity, thisresearch has a direct bearing on the future of long-termhuman spaceflight as well as on human diseases experi-enced on Earth. Data from these experiments willimprove our knowledge of the effects of microgravity onthe hematopoietic (blood-forming) system and will sug-gest possible in-flight countermeasures and treatmentsfor negative effects of microgravity on astronauts andprovide insight into developing therapies for patients on Earth with diseases affecting blood cell formation.

To carry on NASA’s commitment to developingreal-world applications of NASA’s bioreactor technologyand to substantiate NASA’s interest in the commercial-ization of microgravity research in areas related to bio-logical systems, NASA signed an agreement with StelSysInc. in 2002. This follows on the heels of the ground-breaking agreement NASA signed with StelSys inSeptember 2000, which began a new biotechnology com-mercial venture. The first agreement fostered theexploration of a new frontier in biotechnology, infec-tious disease research and the development of a liver-assist device for patients in need of transplant surgery.The agreement signed in 2002 augmented the initialventure by providing for the flight of experiments onthe ISS.

The main objective of the StelSys series ofexperiments is to test the hypothesis that a microgravity

environment will facilitate three-dimensional propagationof cultured liver cells into differentiated, functional tissueequivalents. As with other tissues grown in microgravity,obtaining three-dimensional constructs that function likethe liver in vivo would help researchers to better under-stand liver functions and develop drug therapies and testtheir efficacy before administering the drugs to patients.One of the specialized functions of the liver is to breakdown drugs or toxins into less harmful and more water-soluble substances that can be excreted from the body.ISS-based research will examine how human liver cellsprocess drugs in space, using the microgravity environ-ment to isolate individual cell functions.

Onboard the ISS, the StelSys experiments willtest the function of human liver cells in microgravity ver-sus the function of duplicate cells on Earth. Sponsors ofthis experiment hope that this work will elucidate theeffects of microgravity on the proper functioning of livercells and lead to earlier and more reliable screening ofnew drugs for patients in need of liver and kidney treat-ments prior to transplant. It could also acceleratedevelopment of new lifesaving drugs by pharmaceuti-cal companies because drug developers would be able totest their drug candidates in tissue constructs that main-tain their liver-specific functions for up to a week.Researchers could then choose only the best therapeuticcandidates for further testing, which may take place inhumans.

Albert Li, of StelSys, grew liver cells in theCellular Biotechnology Operations Support System,managed by Neal Pellis at Johnson Space Center. Cellswere transported on STS-111 in June 2002 to the ISS,where they were nurtured and grown. When cell growthwas complete, the samples were frozen and then trans-ported back to Earth for study by STS-112 in October2002. Li and his colleagues will assess the liver con-structs for true functionality to assess their usefulness fordrug screening and to determine their utility for produc-ing compounds that could improve human health.

Great progress was made during FY 2002 ondesign and development of the ISS BiotechnologyFacility (BTF). The BTF is a complement of hardwareand science experiments designed to use the uniquemicrogravity environment of low Earth orbit as a tool inbasic and applied cell biology. Researchers will be ableto use BTF hardware that is based on an extensive her-itage of spaceflight-proven designs, including incubators,refrigerators, analytical instruments, and gas- and water-supply devices. This hardware will be contained insidetwo refrigerator-sized enclosures known as researchracks. For more about the BTF and milestones achievedin FYs 2001 and 2002, see the ISS chapter of this annualreport.



The cellular biotechnology program has alsomade significant progress in the development ofadvanced sensors to support tissue culture. Growth oflong-duration mammalian cell and tissue cultures inspaceflight bioreactor systems requires automated moni-toring of culture parameters such as pH, glucose, andoxygen concentration. Four invention disclosures weremade to NASA during 2002 for a pH control process, aglucose control process, a glucose sensor, and an oxygensensor. The glucose sensor can continuously measureglucose present in cell culture medium in a perfusedbioreactor system, in which cells are grown in an excessof medium that continuously flows through the bioreac-tor. The oxygen sensor is an optical sensor based ondynamic fluorescent quenching of a pulsed blue lightthat is emitted by a light-emitting diode. The sensor isdesigned for long-term continuous measurement of dis-solved oxygen concentration in the cell culture mediumin perfused bioreactors. In 2002, two papers describingthe pH control process and the glucose sensor were pub-lished: “Continuous pH Monitoring in a PerfusedBioreactor System Using an Optical pH Sensor,” by A.S. Jeevarajan, V. Sundeep, T. D. Taylor, and M. M.Anderson (in Biotechnology and Bioengineering,78(4),467–72), and “On-Line Measurement of Glucose in aRotating Wall Perfused Vessel Bioreactor Using anAmperometric Glucose Sensor,” by X. Yuanhang, A. S.Jeevarajan, J. M. Fay, T. D. Taylor, and M. M. Anderson (inJournal of the Electrochemical Society, 149(4), H103–106).

Highlights

Bringing Cancer Cells to Their Knees

While much progress has been made in identify-ing the processes that give rise to cancer, new therapiesfor its treatment have not kept pace. Chemotherapy,which involves using drugs, including chemicals thatdamage DNA, remains the primary cancer treatmentoption for physicians. Unfortunately, cancer cells canexhibit resistance to chemotherapeutic agents. In somecases, this resistance develops during or very shortlyafter chemotherapy treatment, and often the resistancecan happen with several therapy agents, even when onlyone was administered. This is called acquired resistance.In other cases, tumor cells appear to be completely unre-sponsive to treatment with therapeutic agents, even ifthey are agents to which the cancer cells have never beenexposed. This is known as intrinsic resistance. In bothscenarios, the result is the same: the chemotherapy doesnot destroy the cancer cells. The mechanisms underlyingthis rapid onset of drug resistance in human cancer arenot clear. One problem in studying and combating thisresistance is the lack of cancer models that reproduceconditions occurring in vivo, or in the body. This is alsoa problem in studying the effects of various therapies oncancer cells.

NASA investigator Jeanne Becker and her teamof researchers at the University of South Florida, inTampa, have successfully used the NASA-developedHigh Aspect Ratio Rotating-Wall Vessel (HARV) to cul-ture three-dimensional constructs of human ovariantumor cells, which, as are breast tumor cells, are notori-ously difficult to grow outside the body. Becker beganworking with the rotating wall vessel bioreactor in 1992and continued with the HARV in her attempt to growthree-dimensional cancer cell aggregates that wouldfunction more like human tumors than the two-dimen-sional tissues obtained by traditional culture methods.The earlier rotating wall vessel bioreactor and the HARVboth provide a growing environment for cell cultures thatis similar to the one available in the microgravity condi-tions of low Earth orbit. The continuous rotation of thebioreactor keeps the growing cells in a state similar tothe freefall experienced by the space shuttle and the ISSas they orbit Earth, thereby mitigating the effects ofgravity that normally prevent the cells from growing inmore than a single layer. The constructs grown in therotating wall vessel bioreactor provide a model that ismore biologically representative of conditions that occurin vivo than models afforded by traditional culture sys-tems, and Becker plans to use them to study chemothera-peutic drug resistance.

Becker also prepared a spaceflight experiment tocompare ovarian tumor growth in a true microgravityenvironment to cells cultured in concurrent experimentson the ground and in the HARV. In August 2001, herexperiment was transported aboard STS-105 to the ISS,where it remained until December 2001. Human ovariantumor cells were cultured in microgravity for a 14-daygrowth period. The cells were preserved at three timepoints during culture so that they could be analyzed for


In August 2001, PI Jeanne Becker sent human ovarian tumor cells to theISS aboard STS-105. The tumor cells were cultured in microgravity for a14-day growth period and were analyzed for changes in the rate of cellgrowth and for synthesis of associated proteins, as well as evaluated forthe expression of several proteins that are the products of oncogenes,which cause the transformation of normal cells into cancer cells. Thisphoto, which was taken by astronaut Frank Culbertson while he was per-forming the experiment for Becker, shows two cell culture bags containingLN1 ovarian carcinoma cell cultures.

credit: NASA


changes in the rate of cell growth and for synthesis ofassociated proteins, as well as evaluated for the expres-sion of several proteins that are the products of onco-genes, which cause the transformation of normal cellsinto cancer cells.

The experiment results will be used to definepotential points of tumor cellular development that maybe targeted by chemotherapeutic drugs. Finding new tar-gets for chemotherapeutic drugs is especially importantin the case of ovarian cancer, which is usually not detect-ed until it is already in an advanced, incurable stage.

Ultimately, Becker hopes that her research willprovide oncologists with a better chance of predictingwhich drug treatments will work against ovarian cancer.With a three-dimensional model that behaves the waycancerous ovarian tissue in the body does, researcherswill have a more reliable means of predicting drug andhormone treatment efficacy before administering thosetreatments to patients. Becker’s study of three-dimen-sional cell development offers great potential for improv-ing therapies for ovarian and other cancers.

In another example of using the rotating wallvessel bioreactor to culture three-dimensional constructsof cancer cells, Peter Lelkes, of Drexel University, inPhiladelphia, Pennsylvania, is attempting to grow vascu-larized tissue, which contains blood vessels, in vitro.Cancerous tumors are able to grow only because the for-mation of new blood vessels within the tumor providesthe oxygen and nutrients that are necessary to sustaingrowth. One of the strategies in combating such tumor-ous cancers, therefore, has been to look for ways to inter-fere with this blood vessel growth. If Lelkes can grow avascularized tissue in vitro, he will have created a toolwith which to investigate the efficacy of drug therapiesthat can interfere with or prevent the blood vessel growththat sustains tumors as they grow in the body, therebyslowing or stopping tumor growth. Lelkes is currentlyattempting to co-culture microvascular endothelial cellswith prostate cancer cells in rotating wall bioreactors.

Becker and Lelkes are just two researchers out ofmany who are using rotating wall vessel bioreactors andthe microgravity environment of low Earth orbit to try todevelop a better understanding of the mechanisms ofcancer development and better means of fighting cancerin humans.

Neutralizing Virulent Microbes

Spacefarers can remain in a closed system forweeks, sometimes months — and for proposed long-duration flights, maybe even years — breathing recycledair and drinking recycled water. Given that some virulentmicrobes appear to thrive in microgravity, that’s not a

promising scenario for health, according to CherylNickerson, assistant professor of microbiology andimmunology at Tulane University Health SciencesCenter’s program in molecular pathogenesis and immunity. Nickerson says that spacegoers alreadyappear to have a higher risk of falling ill.

In ground-based studies simulating microgravity,Nickerson and her research team have found that a com-mon strain of bacteria known as Salmonella typhimuriumcan alter its genetic profile, upping the production of cer-tain self-protecting proteins that may enhance virulence.That could be unwelcome news for future astronauts.Microgravity may also reduce antibiotic effectiveness,and absent any new pharmacological approach, the dif-ficult task of in-space treatment is made even morechallenging.

In the course of their investigation, Nickersonand her colleagues found that more than 100 Salmonellagenes, or about 3 percent of the salmonella genome,altered genetic expression. The changes made the bacte-ria far more lethal: mice injected with the strains grownin modeled microgravity died, on average, three daysearlier than expected from shock and from large-organfailure.

Nickerson’s original studies in simulated micro-gravity involved the use of the rotating wall vessel biore-actor, which mimics reduced gravity. Cells of S.typhimurium were placed in a culture within the bioreac-tor chamber. When the bioreactor spun, it maintained thecells in close approximation of freefall, which astronautsexperience as up to one-millionth of Earth’s normal gravity.


Cheryl Nickerson’s research focuses on the well-known pathogen,Salmonella typhimurium, whose genetic response to gravity’s nearabsence could provide clues to infection protection. Here, Nickerson (faright) works with her laboratory staff: from left to right, Carly LeBlanc,Rajee Ramamurthy, Kerstin Honer zu Bentrup, and Jim Wilson.

credit: Tulane Univers


The researchers also cultured S. typhimurium under nor-mal-gravity conditions.

In addition, to study how S. typhimurium causesinfection in people, Nickerson and her colleagues usedthe bioreactor to culture three-dimensional human intes-tinal epithelial cells, which more accurately model thephysiology of human intestinal tissue than does conven-tional tissue culture. In response to the microbial inva-sion, the cells produced higher levels of substancescalled anti-inflammatory cytokines, which may help limitdamage to the intestinal tissue following salmonellainfection. The three-dimensional intestinal cells alsoshowed less damage and cell death following salmonellainfection when compared with other types of cellsknown as monolayers. These observations are consistentwith the self-limiting nature of salmonella infection,according to Nickerson, which can damage or kill epithe-lial cells in otherwise healthy individuals before beingdestroyed by immune reaction.

According to the Centers for Disease Control,salmonella-related maladies are among the most com-mon intestinal infections in the United States, with40,000 cases reported yearly. However, scientists esti-mate that because only 3 to 5 percent of salmonella casesare actually reported nationwide, and many milder casesare never diagnosed, the true incidence is much higher,likely in the millions. As many as 1,000 Americans dieannually from salmonella infections.

Bacteria are not premeditated killers. Theirgoals, like all organisms, are to survive, thrive, andreproduce. To do so, they release certain proteins. In nat-ural environments, these proteins neutralize substancesharmful to the bacteria. When ingested into a humandigestive tract, the same mechanisms are engaged.Although the strong acids found in the stomach kill up to99 percent of the would-be bacterial colonizers, the 1percent that do survive are able to “express,” or release,the protective proteins that cause so much upset to theirhuman hosts. The immunologic battle between host andpathogen can be fierce. Most of the time, the immunesystem wins, containing the infection, but sometimesthe bacteria can overcome all defenses, and death canresult.

Although most S. typhimurium–caused infectionsin the United States don’t require hospitalization or seri-ous medical intervention, at least in healthy people, theyare potentially fatal if untreated in people with weakenedimmune systems. Deciphering the bacteria’s molecularresponses could lead — with new drugs and vaccines —to a means to treat or even neutralize salmonella infec-tions, quickly lessening or eliminating the characteristicnausea, vomiting, intestinal inflammation, and diarrheathat they cause.

As humans work for longer periods in space,they may bring with them preexisting infections.Moreover, despite precautions, foods brought on boardcould conceivably harbor salmonella bacteria. Dependingon severity, a salmonella-induced illness could pose seri-ous dangers. And those dangers could be even worse ona space mission, where astronaut immune systems mayalready be stressed. Nickerson explains, “Something likefood poisoning could put a mission at risk, or in theworst case, threaten crew survival.”

Nickerson plans to send S. typhimurium intoorbit to see if the results she obtains there are similar tothose she obtained on the ground. Her hope is to build adetailed roadmap of how salmonella bacteria sense andrespond to microgravity. Once that roadmap is complete,she hopes it will be a guide for developing effectiveremediation strategies.

How the Body Fights Back

One concern with space travel is the fact thatexposure to the microgravity environment apparentlycauses impairment of the immune system. NASA’s high-est priority is to ensure the health and safety of astro-nauts in space, and consequently, NASA supports manyresearch investigations related to the immune system.NASA investigator Joshua Zimmerberg, with a team ofresearchers at the NASA/National Institutes of HealthCenter for Three-Dimensional Tissue Culture, Bethesda,Maryland, is contributing to unraveling the mystery ofhow the immune system changes in microgravity. Theteam is looking specifically at the effects on lymph tissueand lymphocytes.

The immune system is complex and composedof many elements, all of which work in concert to pro-tect the body from foreign invaders like bacteria andviruses. Among the components of the immune systemare the lymph system, a passive system of lymph fluid,or blood plasma, which provides nutrients obtained fromthe blood to cells and carries waste away; the thymus;the spleen; bone marrow; white blood cells; antibodies,also known as immunoglobulins; and hormones. Eachcomponent has a specific role in the body’s immuneresponse.

The best-known defenders within the body arethe white blood cells, which differ from other cells in thebody in that they behave more like independent, single-celled organisms that are incapable of reproducing.Lymphocytes are a type of white blood cell. Some lym-phocytes, known as B cells, produce specific antibodiesfor specific germs. When a B cell recognizes a markeron a germ called an antigen, it will clone itself and pro-duce millions of antibodies against that germ. In con-trast, T cells, the other type of lymphocyte, must actually



come into contact with cells that contain viruses or bac-teria in order to be able to kill them. Both B cells and Tcells can be found in the bloodstream, but they tend toconcentrate in the lymph tissue.

Zimmerberg and his team conducted experimentsthat involved growing human lymphoid tissue cells in theNASA-developed rotating wall vessel bioreactor (RWV),which simulates some aspects of microgravity by gentlyrotating growing cells to maintain them in an environ-ment similar to freefall. The sample cells were isolatedfrom the tonsils of five human donors for use during thisexperiment. Tonsils are granular tissue similar to lymphnodes that are found at the back of the throat. They workas part of the immune system by sampling and filteringgerms that enter the body through the mouth. The resultsdemonstrated that some immune functions becameimpaired with exposure to the simulated microgravityconditions provided by the RWV.

When the tissues cultured in the RWV were chal-lenged with recall antigens, markers to which the lym-phoid tissues had previously been exposed, they did notrespond by producing specific antibodies, as they shouldhave. The previous exposure should have caused thelymphoid tissue to recognize those markers and mountan immune response. The tissues grown in the RWVwere also challenged with polyclonal antigens, whichwere descended from more than one group of cells, to tryto obtain a general immune response, but unlike culturesgrown by traditional, nonrotating methods, the tissuesdid not respond with increased immunoglobulin produc-tion. These results indicate that lymphocytes lose theirability to be activated when cultured in the RWV.However, when the lymphocytes were activated by expo-sure to antigens prior to being cultured in the RWV, they

remained activated during the culture. This showsthat the timing of the activation period is critical tothe cells’ immunogenic capability.

Subsequent studies were conducted on the ISS inorder to determine if results obtained from theRWV cultures could be replicated in a true micro-gravity environment. The RWV creates an envi-ronment that mimics some, though not all, aspectsof microgravity, and cells cultured in ground stud-ies do not experience other factors associated withspaceflight that may affect immune function.Zimmerberg’s flight experiment flew to the ISS onshuttle mission STS-108 in December 2001. TheISS samples were returned to investigators foranalysis in April 2002.

On the ISS, the tonsil cell cultures were grown inTeflon bags and challenged with antigens.Preliminary results indicate that differences existbetween the flight and ground samples and demon-

strate an impaired immune response in the microgravitysamples. Further analysis will determine whether thesedifferences are similar to those seen between RWV andground samples. Future experiments will examine pat-terns of membrane reorganization and changes in thecytoskeletons of cells cultured in the RWV; thesechanges could impair the cells’ ability to recognize andrespond to viruses and bacteria.

Preliminary results suggest that responses in truemicrogravity are similar to those seen in simulatedmicrogravity. A second experiment to continue gatheringinformation regarding T cell and B cell interactions lead-ing to lymphocyte activation has been scheduled. If theresults are indeed similar in both simulated and truemicrogravity, then it could be much easier to begin iden-tifying the cause of immune impairments, becauseresearchers could rely on the RWV for their experimentsinstead of having to wait for infrequent spaceflightopportunities. Conducting experiments in the RWVwould also allow researchers to replicate their experi-ments, which is currently difficult due to limitations onpayload capacity and time for conducting researchaboard the ISS. The study of these immune impairmentscould have important impact on the future of space traveland on human health on Earth. The human immuneresponse in space is blunted, and thus the potential forpathological diseases associated with reduced immuno-logical capability on long-duration spaceflights, such as amission to Mars, becomes a significant risk. Understandingwhy the immune response is adversely affected is a neces-sary first step in developing countermeasures that can miti-gate this risk. On Earth, understanding the mechanisms ofimpaired immune response has potential applications in thestudy and treatment of autoimmune diseases and immuno-deficiencies, such as AIDS.

3 CELLULAR BIOTECHNOLOG

PI Joshua Zimmerberg is studying how microgravity affects the human immunesystem. In ground-based studies, Zimmerberg exposed human tonsil tissue toantigen markers and then grew that tissue under simulated microgravity in arotating wall vessel. He then re-exposed the tissue to antigen markers to see ifthe cells would respond by producing antibodies.

credit: National Institutes of Health

BIOTECHNOLOGY 3 OVERVIEW - Mains Associates · 2002-09-06 · Biotechnology is the application of...

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