Use of an adaptable cell culture kit for performing lymphocyte and monocyte cell cultures in microgravity

Abstract

The results of experiments performed in recent years on board facilities such as the Space Shuttle/Spacelab have demonstrated that many cell systems, ranging from simple bacteria to mammalian cells, are sensitive to the microgravity environment, suggesting gravity affects fundamental cellular processes. However, performing well-controlled experiments aboard spacecraft offers unique challenges to the cell biologist. Although systems such as the European 'Biorack' provide generic experiment facilities including an incubator, on-board 1-g reference centrifuge, and contained area for manipulations, the experimenter must still establish a system for performing cell culture experiments that is compatible with the constraints of spaceflight. Two different cell culture kits developed by the French Space Agency, CNES, were recently used to perform a series of experiments during four flights of the 'Biorack' facility aboard the Space Shuttle. The first unit, Generic Cell Activation Kit 1 (GCAK-1), contains six separate culture units per cassette, each consisting of a culture chamber, activator chamber, filtration system (permitting separation of cells from supernatant in-flight), injection port, and supernatant collection chamber. The second unit (GCAK-2) also contains six separate culture units, including a culture, activator, and fixation chambers. Both hardware units permit relatively complex cell culture manipulations without extensive use of spacecraft resources (crew time, volume, mass, power), or the need for excessive safety measures. Possible operations include stimulation of cultures with activators, separation of cells from supernatant, fixation/lysis, manipulation of radiolabelled reagents, and medium exchange. Investigations performed aboard the Space Shuttle in six different experiments used Jurkat, purified T-cells or U937 cells, the results of which are reported separately. We report here the behaviour of Jurkat and U937 cells in the GCAK hardware in ground-based investigations simulating the conditions expected in the flight experiment. Several parameters including cell concentration, time between cell loading and activation, and storage temperature on cell survival were examined to characterise cell response and optimise the experiments to be flown aboard the Space Shuttle. Results indicate that the objectives of the experiments could be met with delays up to 5 days between cell loading into the hardware and initial in flight experiment activation, without the need for medium exchange. Experiment hardware of this kind, which is adaptable to a wide range of cell types and can be easily interfaced to different spacecraft facilities, offers the possibility for a wide range of experimenters successfully and easily to utilise future flight opportunities.

The kinetics of translocation and cellular quantity of protein kinase C in human leukocytes are modified during spaceflight

“…In both 37°C syringe and flask cultures over 90% of the d-glucose in the medium was used by the 3rd day of culture, whereas over half of the initial concentration remained in 25°C cultures at the 4th day of culture. These data are in agreement with experiments performed in the cell culture cassettes used for the space shuttle experiment reported separately (18), indicating that syringe culture is a good analog of the conditions within the cassette system.…”

“…Sterile hypodermic 2-ml syringes were used to simulate culture in the GCAK-2 cassettes. Comparison of cell viability data between these two culture systems confirmed that syringes are good analogs of the cassette culture environment (18). A rapid decrease in cell viability after the 3rd day of culture was noted in both 37°C syringe and flask cultures, with the rate of decrease being highest in 37°C syringes.…”

“…A specially designed cell culture/activation cassette known as the General Cell Activation Kit-2 (GCAK-2) was used to perform the experiment within the technical and safety constraints of space shuttle flight. This cassette is described in detail elsewhere (18). Briefly, each cassette consisted of a machined polycarbonate block enclosing six independent culture activation units.…”

“…The results of these optimization experiments are described in detail elsewhere (18). To further understand the effect of environmental conditions on PKC distribution and quantity we assessed PKC isoforms in U937 cells under different culture conditions simulating those encountered in the space shuttle experiment during spaceflight.…”

Monitoring functional immune responses with a cytokine release assay: ISS flight hardware design and experimental protocol for whole blood cultures executed under microgravity conditions

“…The test procedure was carried out at the Biotechnology Space Support Center (BIOTESC), University of Luzerne (USLU), Switzerland using the accessible ground model of the Kubik incubator (FM2, ESA). A previously used ( Hatton et al, 1998 ) Kayser Italia Container-Spider (KIC-Spider, Kayser Italia, Italy) was combined with a 3D printed insert (Kayser Italia, Italy) and evaluated for insertion of RTs in Kubik. This KIC insert (length 40.30 mm, width 20.30 mm, height 85.10 mm, and mass 10.90 g) was manufactured in Acrilonitrile-Butadiene-Stirene (ABS) + P340 through 3D printing (Fortus 250mc, Fortus 3D Production Systems) and contains 4 bays for harboring 4 RTs at a time.…”