MICROGRAVITY 

RESEARCH 

DIVISION

Bioreactor expands health research

NASA device gives new dimension to cell science

Good neighborhoods help you grow. It is true of humans,

and of the cells of which we and all complex organisms

are made. A challenge which has faced cell science throughout its short history is that most cell cultures produce flat,

one-cell-thick specimens that offer limited insight into how

cells work together. Such knowledge is crucial if we are to

understand the chemistry and mechanics of healthy organs

and of cancers, infectious diseases, immune system failures, and other public health problems.

The National Aeronautics and Space Administration, in

concert with the biomedical community, has initiated work

that offers significant advances in cell culturing technology on Earth that enables further unique research progress

aboard Space Shuttle, Mir, and — soon — International

Space Station. The NASA Biotechnology Cell Science

Program, directed by Dr. Neal Pellis at NASA’s Johnson

Space Center, involves more than 100 scientists, engineers,

and support personnel around the nation.

A new spin on cell growth

They are meeting the challenge with a unique new technology, the rotating wall vessel bioreactor. It spins a fluid

medium filled with cells to neutralize most of gravity’s

effects and encourage cells to grow in a natural manner.

The rotating bioreactor was invented by NASA as a model

of microgravity effects on cells.

Ground tests of the bioreactor yielded three-dimensional

tissue specimens approximating natural growth, a striking

change from the pancake shapes of traditional cultures.

As cells replicate, they “self associate” to form a complex matrix of collagens, proteins, fibers, and other chemicals. This highly evolved microenvironment tells the cells

who is next door or nearby, how they should grow and into

what shapes, and how to respond to stimuli such as bacteria and wounds. Thus, we have the opportunity to study

the complex order of tissue in a culture system that can be

manipulated by drugs, hormones, and genetic engineering.

Staying in touch

As with a city, the lives of a cell are governed by its

neighborhood connections. Those connections that don’t

work have been implicated in muscular dystrophy, osteoporosis, glaucoma, and the spread (metastasis) of cancer cells throughout the body.

Studying these mechanisms outside the body on Earth is

limited by gravity’s effects because the cells do not easily

self-associate to grow naturally. The bioreactor promotes

self-association in a container about the size of a soup can.

Targeted health issues

Infectious disease: Human immunodeficiency virus (HIV), Ebola virus, Lyme disease.

Cancer models: Prostate, breast, ovary, lung, and colon cancers.

Diabetes: Work on a pancreatic tissue for transplant.

Therapies: Musculoskeletal tissue disorders (in space and on earth).

Drug efficacy: Non-animal/nonclinical testing of drug effects and toxicity.

The heart of the Bioreactor is the rotating wall vessel, shown

here without its support equipment.

The clear shell allows scientists to check growth, and the

center holds a cylindrical filter that passes oxygen and

nutrients in and carbon dioxide and wastes out. This ensures that the fluid rotates without shear forces that would

destroy the cells. The rotating vessel does not really cancel gravity, but ideally maintains cells in continual freefall similar to that experienced by astronauts in the

microgravity of space.

Earth-based bioreactor

cultures typically maintain cell growth for at

least 60 days. On Earth,

a sample then becomes

so large (about 1 cm)

that it is no longer suspended. In long space

missions, large-sample

growth in bioreactors

can be studied in order

to understand the extended growth and differentiation of cells,

such as engineering

complex tissue or modeling slow-growing tumors.

With long-term stays

aboard the Mir space station and, soon, the International

Space Station, NASA and the biomedical community have

this opportunity to explore this new frontier in cell science and contribute to public health.

Native

cartilage

Bioreactor

cartilage

Stationary

culture

Lacunae

Matrix

Lacunae

Matrix

No lacunae

No matrix

The Bioreactor allows cells to reach

a level of maturity rarely achieved

by other methods.
Tools

The Bioreactor rotating wall vessel sits at the front of the incubator module (window of large module in left photo) behind a window so

the crew may monitor cell progress. The bioreactor is controlled by the Experiment Control Computer (ECC; flat module in the left

photo), and the crew can reprogram the system through the payload general support computer atop the ECC. The BSTC (closed, top

right, and open, bottom right) is an integral part of the bioreactor development program aboard Mir.

at 11.5 rpm to produce a gentle flow that

ensures removal of waste products as

fresh media are infused. Periodically,

some spent media are pumped into a waste

bag and replaced by fresh media. When

the waste bag is filled, an astronaut drains

the waste bag and refills the supply bag

through ports on the face of the incubator. “Pinch” valves and a perfusion pump

ensure that no media are exposed to moving parts. An Experiment Control Computer controls the Bioreactor, records conditions, and alerts the crew when problems occur. The crew operates the system

through a laptop computer displaying

graphics designed for easy crew training

and operation.

NASA’s cell science program uses experimental hardware fitted to the requirements of the science community and the

resources of the Space Shuttle orbiter

middeck, and taking advantage of the endurance of the Mir space station. These

are forerunners of systems for long-term

investigations aboard the International

Space Station.

Bioreactor Demonstration System

(BDS) comprises an electronics module,

a gas supply module, and the incubator

module housing the rotating wall vessel

and its support systems. Nutrient media

are pumped through an oxygenator and

the culture vessel. The shell rotates at 0.5

rpm while the inner filter typically rotates

Biotechnology Specimen Temperature Controller (BSTC) will cultivate

cells until their turn in the bioreactor; it

can also be used in culturing experiments

that do not require the bioreactor. The

BSTC comprises four incubation/refrigeration chambers individually set at 4 to

50  ̊C (near-freezing to above body temperature). Each chamber holds three rugged tissue chamber modules (12 total),

clear Teflon bags holding 30 ml of growth

media, all positioned by a metal frame.

Every 7 to 21 days (depending on growth

rates), an astronaut uses a shrouded syringe and the bags’ needleless injection

ports to transfer a few cells to a fresh

media bag, and to introduce a fixative so

that the cells may be studied after flight.

The design also lets the crew sample the

media to measure glucose, gas, and pH

levels, and to inspect cells with a microscope. The controller is monitored by the

flight crew through a 23-cm (9-inch)

color computer display on the face of the

BSTC.

Biotechnology Refrigerator (BTR)

holds fixed tissue culture bags at 4  ̊C to

preserve them for return to Earth and postflight analysis.

Gas Supply Module contains four rechargeable bottles of an air/carbon dioxide mixture to support optimal cell growth

over long periods of time aboard a space

station.

Bioreactors are entering the medical research community via the Technology

Transfer Act. In 1990, NASA granted

Synthecon, Inc., of Houston exclusive commercial

license to NASA patents

for the bioreactor system.

This ensures that the research potential of the

bioreactor will be available to a wide

range of institutes and companies. More

than $2,000,000 in Rotary Cell Culture

SystemsTM had been sold by mid-1998.

Synthecon has sponsored research agreements with the University of North Texas

Science Center, Fort Worth, to commercialize replacement skin

products, and Baylor College of Medicine, Houston,

to develop a novel diabetic

therapy. Synthecon is negotiating other agreements

with major medical institutes to commercialize additional biological products. In

addition, Synthecon develops new designs to advance their growth potential.

Into the commercial sector
Since its invention in 1992, the bioreactor

has been used in terrestrial laboratories for

significant research and for work leading

to clinical studies in a range of diseases. In

May 1997, the Society for In Vitro Biology

devoted an entire issue of In Vitro Cellular

& Developmental Biology—Animal (33:5,

325-405) to the results of a special NASA

workshop on bioreactor results. Many of the

authors are investigators in NASA’s

bioreactor flight research program. Topics

covered by In Vitro or reported through

other media include:

Cancer research. Bioreactors have cultured single cells of cancers of the skin

(melanoma), prostate, ovary, breast, bone

(osteosarcoma), and colon into viable cell

cultures. Tissue structures can be grown for

at least 60 days before they become too large

to remain suspended in the bioreactor

growth medium.

Infectious disease. The U.S. Army Medical Research Institute of Infectious Diseases

at Fort Detrick, Md., uses the bioreactor to

grow cultures—rather than using live animals—in the study of how the Ebola virus

is transmitted. At the NASA/NIH Center for

Three Dimensional Tissue Culture (see box

above), scientists use the bioreactor in a

wide range of infectious disease

studies.

Kidney failure. In addition to filtering waste from the bloodstream,

the kidneys secrete crucial hormones, including erythropoietin and

1-25-diOH-D3. Replacing these hormones now costs $2.5 billion. Further, the technology for supplying

them is (as with insulin for diabetics) inadequate to maintain a healthy

body. Culturing of kidney cells offers the potential to produce these

key hormones so that the health and

Ground-based research results

In 1994, NASA and the National Institutes of Health signed an interagency

agreement to provide NASA bioreactor

technology to NIH and to establish a joint

Center for Three Dimensional Tissue Culture at the National Institute of Child

Health and Human Development. Since

the original agreement, the bioreactor has

been incorporated into more than a dozen

laboratories within NIH.

The bioreactor is an excellent example

of how the skills and resources of two distinctly different agencies can complement

each other for the public good. Where

NASA is chartered to explore and exploit

space, NIH is chartered to develop tools

to defeat disease.

This new center is conducting 16

bioreactor research projects. Foremost

among these is the first in vitro tissue system permitting the study of the HIV pathway through the human lymphatic tissue.

The other 15 projects address a range of

human health issues. NASA bioreactor

technology is used within several NIH institutes and other agencies covering virtually all of human health: allergies, dentistry, the human genome, digestive and

kidney disease, neurology, and heart, lung,

and circulatory health.

At the NASA/NIH Center for Three Dimensional Tissue Culture, scientists use

tonsil tissue to grow live Human Immunodeficiency Virus (HIV-1) and thus observe more closely the transmission of the

virus. The cultures have demonstrated the

same progressive loss of CD4 T-cells as

seen in AIDS patients. The NASA/NIH

Center also is studying an intestinal parasite found on some imported fruit.

National Institutes of Health

Understanding that a key hormone is

needed to move sugar from the bloodstream into cells allowed doctors to develop a treatment, daily insulin injections

for life, for diabetes. While staving off

death, this therapy is crude compared to

the fine balancing act performed by the

beta cells of a healthy pancreas. About half

of the blindness, kidney failure, and limb

loss in the United States are due to longterm complications of diabetes, for a public cost of $1 out of $7 spent in health care.

Bioreactor cell science opens new possibilities for more natural treatments. In 1997,

NASA and the Juvenile Diabetes Foundation signed a Space Act Agreement for collaborative work including bioreactor research to understand the best route for cultivating and transplanting beta cells into

Type I diabetics, and to develop a non-invasive blood sugar monitor which could

enhance bioreactor operations.

Under a Technology Transfer Act Agreement, VivoRx of Santa Monica, Calif., is

developing a method of encapsulating beta

cells in treated seaweed membranes for implant in the abdomen. The seaweed allows

insulin and glucose to diffuse back and

forth so the transplanted cells work as an

artificial pancreas. Microencapsulation results in human volunteers are highly promising. The bioreactor is being used to develop methods to expand the small numbers of cells available from donors to supply the large numbers of cells needed by

diabetics.

Attacking diabetes

quality of life of kidney patients can be

improved.

Immune system repression. One of the

marvels of the human immune system is

how lymphocytes, like escape artists,

squeeze through the tight spaces between

cells in search of invading disease.

Bioreactor studies using collagen reveal that

lymphocytes exposed to simulated

low-g for 72 hours do not move. This

has been verified with experiments

aboard Shuttle missions, and demonstrates that the bioreactor is a good

simulation of low-g growth conditions.

Drug efficacy. Kidney and heart

tissues cultured in the bioreactor

show the appropriate drug receptor

sites that allow testing of drugs to determine their safety without using

animals. This also reduces the need to

use human volunteers in final testing.

Cells from kidneys lose some of their special features in conventional culture (left) but form spheres replete with specialized cell microvilli (“hair”) and synthesize hormones that

may be clinically useful.
These results demonstrate the feasibility of

microgravity tissue engineering and may

have implications for long human space

voyages and for treating musculoskeletal

disorders on earth. A separate commentary

(PNAS, pp. 13380-2) notes that bioreactor

research in space

“offers an unprecedented opportunity for studying

complex fluid assemblies....”

By mid-1998, the

bioreactor investigators were deep

into analysis of

specimens from Mir Increments 6 and 7.

Increment 6 (Sept. 25, 1997-Jan. 31-1998)

will provide a stop-action movie of bone

marrow, neuroendocrine, and kidney tubule

cells as they are cultivated and then fixed

every 7 to 10 days for up to 105 days in

space (a portion of each batch is injected

into new media to continue the cell line).

Increment 7 (Jan. 22-June 12, 1998), was

the first opportunity to grow human breast

cancer cells in this most lifelike artificial

setting as we work to understand how this

killer grows and moves through the body.

While bioreactor research in space is relatively new, it shows great promise. For 5

days on the STS-70 mission, a bioreactor

cultivated human colon cancer cells, which

grew to 30 times the volume of control

specimens grown on Earth. This significant

result was reproduced on STS-85

which grew mature

structures that more

closely match what

are found in tumors

in humans.

Mir Increment 3

(Sept. 16, 1996 -

Jan. 22, 1997) grew

specimens of tissue engineered cartilage

that surprised investigators. In the December 1997 issue of the Proceedings of the National Academy of Sciences (94: 13885-90,

1997), Dr. Lisa E. Freed of the Massachusetts Institute of Technology and her colleagues reported that initially disclike specimens tend to become spherical in space,

demonstrating that tissues can grow and

differentiate into distinct structures in

microgravity. The Mir samples were

smaller, more spherical, and mechanically

weaker than Earth-grown control samples.

Flight results

Bioreactor research

aboard the Shuttle

(above) is valuable but limited in

duration. Completing work on

Mir will let the

bioreactor science

team make full use

of the Biotechnology Facility (right)

proposed for International Space

Station.

REPLACE REPLACE

Experience aboard Mir has turned

microgravity bioreactor research into a

mature science. The first long-duration

Bioreactor experiment

(Increment 3) saw the

Bioreactor grow large

cultures of bovine cartilage cells. A supporting

technology mission (Bio3D on Increment 6) demonstrated the BSTC as a

repository for cells awaiting their turns in the

bioreactor. The last

NASA stay aboard Mir

(Increment 7) was crucial

as it brought everything

together in an effort to culture human tissue in the Bioreactor. This was a full-scale

rehearsal for operations aboard the International Space Station (center).

In addition to important science results, this work provided

basic training on crew

procedures and equipment design and operation. At the end of NASA

activities aboard Mir in

June 1998, the NASA

bioreactor team will synthesize these lessons into

an advanced program that

will let them take maximum advantage of International Space Station at

the earliest opportunity,

and produce groundbreaking scientific results that will advance medical science.

International Space Station

Research experiment

1991-94

Ground-based research

Shuttle flights (STS-XX): Technology demonstration

Developing technology Maturing science

44, 54, 56, 57, 62 70 79, 81 85

Mir increment ISS

86, 89, 91 99

1995 1996 1997 1998 1999 2000 21st century

Bioreactor schedule

3 67

3

Photos: Proceedings of the National Academy of

Sciences (page 4, top right). Synthecon (page 2,

bottom). All others, NASA.

Rotary Cell Culture is a trademark of Synthecon Inc.

Engineered cartilage samples.

BIOTECHNOLOGY

RESEARCH PROGRAM

For additional information, contact:

Dr. Stephen Davison

Biotechnology Enterprise Scientist

Microgravity Research Division—Code UG

National Aeronautics and Space Administration

Washington, DC 20546

202-358-0647

steve.davison@hq.nasa.gov

Ron Porter, Manager

Microgravity Biotechnology Project Office—MG20

Marshall Space Flight Center

Huntsville, AL 35812

205-544-1323

microgravity@msfc.nasa.gov

Dr. Neal Pellis, Director

Biotechnology Cell Science Program

Johnson Space Center

Houston, TX 77058

283-483-7171

neal.r.pellis1@jsc.nasa.gov

http://microgravity.msfc.nasa.gov

FS-1998-09-65-MSFC; 9/4/98

Mir Ground

1 mm