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Microgravity cell culture in the space and on earth

Microgravity cell culture in the space and on earth.

One hundred years ago the hanging drop method was usual in microbiology, and with this method Robert Koch was able for the first time to see under the microscope cultured anthrax bacilli and live Vibrio cholera in Egypt’s contaminated waters. The method is simple: just put a drop of water, containing the microorganisms, on the surface of a microscope slide and invert it with a rapid hand turn, leaving the drop of water hanging from the slide, sustained by the surface tension. Inside the water drop, the microbial moves freely and can be observed with a light microscope. Microbial inside the water drop are confined to the water drop, unable to cross the water-air interface and therefore unable to touch a surface to adhere. Identical principle of that used today to create microscopic 3D cell aggregates.

About the same time, around 1880, Jules Verne published a satiric novel, “From the Earth to the Moon”, regarding the possibility of humans reaching the Earth’s satellite in a space flight, inside a cannon shell. But, only about a hundred years later humans walked on the Moon, transported by a complicated combination of rockets and propulsion devices.

As early as 1961 US scientist showed interest on studying space flight influence on cell biology and carried out organ and cell culture experiments in the Discoverer satellites XXIX and XXX (1) (2). Two basic physic factors were the target in this research: The influence of gravity and electromagnetic radiations on living organisms, from single cells, to embryo development and to mature complex organisms.

From 1966 to 1969 NASA launched “Biosatellites” as part of a research program to assess the effects of spaceflight, especially microgravity, on life processes, studying basic cellular biochemistry, growth structure of cells and tissues, growth and form of plants and animals. Similarly in another biological space research program, carried out by an international group of scientists, with the Cosmos 1129, brought important data about the behavior of stem cells and organism development, by confirming with carrot embryos that cultured stem cells in microgravity can give rise to embryos and somatic embryos and that the space hypo gravity environment can support the normal growth of already-organized somatic embryos giving rise to fully developed plantlets (3).
At least two basic conclusions were obtained from all these early experiments: A) Cell life and general structure are not gravity-dependent. B) Embryogenesis is also gravity independent

During a shuttle missions in the 1980s, NASA Life Sciences Division carried out studies on the effects of microgravity on cell behavior. The main goal of this research was to analyze the effect on living organisms of the weightless status, since in orbit this is the normal ambient. Unfortunately results were inconclusive due to the fact that between the time the cargo’s pre-launch loadings and the orbital flight phase, the cells were not under microgravity conditions. After these experiments, scientists realized that this kind of research carried out in space vehicles was limited, but it could be simulated on earth with special technologies. Soon gravity reduction/disturbing instruments such as Clinostats, Random Positioning Machines (RPM), Free Fall Machine (FFM) and parabolic flight aircrafts were developed (http://www.descsite.nl/RPM_Frames.htm).

NASA engineers developed the Rotating Wall Vessel (RWV) bioreactor which provides a “simulated microgravity” on Earth. The rotary motion of the device counteracted gravitational force to keep cells in a "simulated microgravity" environment. In these conditions, cells growing inside the rotating wall vessel aggregate together forming 3D multi-cellular structures or clumps comparable to 3D tissue structures, even more if different cell types are cultured together (7). This device was produce industrially in 1993 and since many people has conducted important research on 3D cultures obtained with RWV.
Soon the research was not concentrated exclusively in microgravity or g force reduction, it was also important the gravity vector direction and the hyper-gravity as increment of the gravity force.

Most of these culture experiments were developed in semisolid (gel) media; however, the bioreactors with liquid water based media showed additional problems in microgravity, either the media spread on any internal walls surface, living a central bubble of air, or the media removes away from the walls forming a big central droplet, leaving the cells attached to the internal surfaces of the flask without media. Moreover, gas diffusion between the gas and the liquid phases in the flask is not stable in microgravity conditions.

In 1997 Dr. E. Barbera-Guillem developed the first cell culture device without internal gas phase, the OptiCell, based on the use of respiratory membranes (controlled gas diffusion membranes) (4) These devices avoid the mentioned space cell culture problems, and were rapidly adopted by NASA and are still in use for many space bio-experiments (5) (6).

From 1993 to 1996 scientist studied in space the role of gravity on the early development of the Xenopus laevis, in cooperation with the Swedish Space Agency, showing that a short period of microgravity during fertilization and the first few minutes of development results in abnormal axis formation. Adding a special centrifuge inside a parabolic flight rocket was possible to discriminate between the influences of flight perturbations and actual microgravity, showing that the eggs fertilized in microgravity produced morphology changes of the blastocoels, but these embryos recovered and resumed normal development in earth (8). These results showed the need for longer periods of microgravity to really disclose the effects on the development processes.

In the mid 1990s the Columbia shuttle carried multiple biological experiments including cell cultures, most of them prepared on earth but developed in the space vehicle under the crew control. Because of the specialized skills required for these experiments and the crew’s tasks overload, instruments were developed to automatically perform most tasks and be controlled from earth, like plunger boxes for egg fertilization (9), the Generic Cell Activation Kits 1 &2 designed for immune cells research in microgravity environment (10), and bone marrow cells maturation (11).

In November 1998, NASA in cooperation with Russia’s space program initiates the assembly of the International Space Station (ISS) and only five years later the orbital laboratory started real scientific operations, including cell biology research, opening a promising future for research in orbital environments (12).

In early 2008 the European Columbus laboratory was assembled to the ISS carrying the Biolab module designed to support biological experiments including, what role weightlessness plays in cells and tissue cultures from a single cell to complex cellular structures. Based on these opportunities specific hardware is being developed for small eggs incubation, plant cultivation or for support of insect species.

Lately, in order to expand research opportunities and capabilities a Cell Culture Unit has been integrated, as well as an Advanced Animal Habitat for rodents, an Aquatic Facility to support small fish and aquatic specimens, a Plant Research Unit for plant cultivation and a specialized Egg Incubator for developmental biology studies. Host systems such as a 2.5 m Centrifuge Rotor, for direct comparisons between g and selectable g levels, the Life Sciences Glovebox for contained manipulations, and Habitat Holding Racks which will provide electrical power, communication links and cooling to the habitats. Habitats will provide food, water, light, air and waste management as well as humidity and temperature control for a variety of research organisms. Common laboratory equipment such as microscopes, cryo freezers, radiation dosimeters, and mass measurement devices are also available to be operated either by the crew on the ISS or from the Earth, scientist will be able to send commands to the laboratory equipment and monitor the environmental and experimental parameters inside specific habitats (12,13,14,15)
petaka microgravity
Since these laboratories had been in place many “earth” instruments have been developed and validated to simulate space conditions in earth, expanding the research scenario and the possibilities for these special conditions to be applied in medicine and industry. The main attribute on space experimental setting is g force being close to zero; therefore using centrifuges allows increasing and redirecting the g vector, exposing the cell culture to different g forces in intensity and direction. Studies in these fields have brought important data about the immune system cells behavior in microgravity ambient combined with oriented vector forces (12) using a remote control cell culture bioreactor (Techshot, multi-specimen variable gravity platform). Important discoveries in possible applications of simulated microgravity in tissue engineering have also been made (17). Free Fall Technology has been implemented PetakaG3 hypoxia minichamber in order to produce microgravity like 3D cell agregates on earth.

Experiments conducted in space microgravity environment by cancer biologist are not frequent. However, microgravity provides physical conditions that can be exploited to study mechanisms and pathways that control cell growth and functions (18), altering biological processes relevant to cancer research (19) including endothelial cell alterations (20) and immune deregulation (21).
Simulated microgravity provides 3D cultures, closing a technology circle that started with the simple, but effective, hanging drop method, now in fashion again with in different modalities and solutions, like the cell attachment impairment methods and the microgravity technology.
Space research introduced data on cell biology impossible to imagine before the orbital flights and the ISS, even with the imagination and creativity of inspired novelists as Jules Verne. But not only had that, like in many other fields space flights have stimulated new technologies and the development of high-tech instruments that used on earth, in any laboratory, reproduce the ambient of the orbital spacecrafts, therefore experiments can be executed before validating them in the space.

More than a hundred years ago experimental embryology showed that one egg becomes a blastula by cell division. This blastula is made of a number of cells (blastomeres) that as long as they remain together and healthy will develop a complicated and perfect organism. However, if one blastomere dies, the neighbor will be unable to reproduce the normal body as long as the dead cell remains attached to the healthy one. Nevertheless, Hans Driesch in 1892 was able to demonstrate that each separated blastomere has the ability to develop a complete animal. The development steps that each separate blastomere follows are identical, always growing the number of cells in contiguity of each other, coaching the neighbor’s fate. So the differentiation is a centrifugal and stochastic process and it does not parallel with today’s 3D cultures, which are simply convenient aggregates of cells without an inherited organization program.


1.            PRINCE JE, MABRY JR. Biologic systems of Discoverer satellites XXIX and XXX. Organ and tissue cultures. 2. Ciliary activity of embryonic chick choroid plexus. Tech Doc Rep SAM-TDR USAF Sch Aerosp Med. 1962 Apr;62-62:29–32.

2.            KATZBERG AA, MORI LH. Biologic systems of Discoverer satellites XXIX and XXX. Organ and tissue cultures. 1. Embryonic chick heart and human cell cultures. Tech Doc Rep SAM-TDR USAF Sch Aerosp Med. 1962 Apr;62-62:25–7.

3.            Krikorian AD, Dutcher FR, Quinn CE, Steward FC. Growth and development of cultured carrot cells and embryos under spaceflight conditions. Adv Space Res Off J Comm Space Res COSPAR. 1981;1(14):117–27.

4.            GEN | Magazine Articles: Novel Cell Culture Optimization Strategy [Internet]. GEN. [cited 2014 Feb 5]. Available from: http://www.genengnews.com/gen-articles/novel-cell-culture-optimization-strategy/2623/

5.            Johnson TE, Nelson GA. Caenorhabditis elegans: a model system for space biology studies. Exp Gerontol. 1991;26(2-3):299–309.

6.            Talbot NC, Caperna TJ, Blomberg L, Graninger PG, Stodieck LS. The effects of space flight and microgravity on the growth and differentiation of PICM-19 pig liver stem cells. In Vitro Cell Dev Biol Anim. 2010 Jun;46(6):502–15.

7.            Schwarz RP, Goodwin TJ, Wolf DA. Cell culture for three-dimensional modeling in rotating-wall vessels: An application of simulated microgravity. J Tissue Cult Methods. 1992 Jun 1;14(2):51–7.

8.            De Mazière A, Gonzalez-Jurado J, Reijnen M, Narraway J, Ubbels GA. Transient effects of microgravity on early embryos of Xenopus laevis. Adv Space Res Off J Comm Space Res COSPAR. 1996;17(6-7):219–23.

9.            Ubbels GA, Reijnen M, Meijerink J, Narraway J. Xenopus laevis embryos can establish their spatial bilateral symmetrical body pattern without gravity. Adv Space Res Off J Comm Space Res COSPAR. 1994;14(8):257–69.

10.          Hatton JP, Lewis ML, Roquefeuil SB, Chaput D, Cazenave JP, Schmitt DA. Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity. J Cell Biochem. 1998 Aug 1;70(2):252–67.

11.          Ortega MT, Pecaut MJ, Gridley DS, Stodieck LS, Ferguson V, Chapes SK. Shifts in bone marrow cell phenotypes caused by spaceflight. J Appl Physiol. 2009 Feb;106(2):548–55.

12.          Katovich MJ. Gravitational biology opportunities planned for the International Space Station. J Gravitational Physiol J Int Soc Gravitational Physiol. 1998 Jul;5(1):P189–192.

13.          Kern VD, Bhattacharya S, Bowman RN, Donovan FM, Elland C, Fahlen TF, et al. Life sciences flight hardware development for the International Space Station. Adv Space Res Off J Comm Space Res COSPAR. 2001;27(5):1023–30.

14.          Vandendriesche D, Parrish J, Kirven-Brooks M, Fahlen T, Larenas P, Havens C, et al. Space Station Biological Research Project (SSBRP) Cell Culture Unit (CCU) and incubator for International Space Station (ISS) cell culture experiments. J Gravitational Physiol J Int Soc Gravitational Physiol. 2004 Mar;11(1):93–103.

15.          Uchida S. Experiment facilities for life science experiments in space. Uchū Seibutsu Kagaku. 2004 Nov;18(3):140–1.

16.          Goodwin T, Lundquist C, Tuxhorn J, Hurlbert K. The Biotechnology Facility for International Space Station. J Gravitational Physiol J Int Soc Gravitational Physiol. 2004 Mar;11(1):75–80.

17.          Stamenković V, Keller G, Nesic D, Cogoli A, Grogan SP. Neocartilage formation in 1 g, simulated, and microgravity environments: implications for tissue engineering. Tissue Eng Part A. 2010;(16(5)):1729–36.

18.          Sambandam Y, Townsend MT, Pierce JJ, Lipman CM, Haque A, Bateman TA, et al. Microgravity control of autophagy modulates osteoclastogenesis. Bone. 2014 Jan 23;

19.          Becker JL, Souza GR. Using space-based investigations to inform cancer research on Earth. Nat Rev Cancer. 2013 May;13(5):315–27.

20.          Versari S, Longinotti G, Barenghi L, Maier JAM, Bradamante S. The challenging environment on board the International Space Station affects endothelial cell function by triggering oxidative stress through thioredoxin interacting protein overexpression: the ESA-SPHINX experiment. FASEB J. 2013 Nov 1;27(11):4466–75.

21.          Crucian B, Simpson RJ, Mehta S, Stowe R, Chouker A, Hwang S-A, et al. Terrestrial stress analogs for spaceflight associated immune system dysregulation. Brain Behav Immun [Internet]. [cited 2014 Feb 9]; Available from: http://www.sciencedirect.com/science/article/pii/S0889159114000129

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