IntroductionThe development of immunocompetent in vitro tissue models is an important step in the study of physiological or disease-related processes and in the testing and evaluation of therapeutics and harmful substances1,2,3,4,5,6. What is already a challenge on Earth becomes even more complex in gravitational biology and space medicine. On Earth, from both the National Aeronautics and Space Administration (NASA) and European Space Agency (ESA) acknowledged, “ground-based facilities” are often used to simulate microgravity conditions for experimental approaches7,8. In most cases, these systems use a rotation pattern or magnetic levitation to keep a cultivated cell system in suspension. For instance, the Random Positioning Machine (RPM) works by constantly changing the orientation of the gravity vector, effectively averaging it to zero over time. The rotation of the fluid environment promotes efficient mixing of nutrients and gases within the medium, while also allowing suspension cells to experience a state analogous to free fall, thereby reducing sedimentation over prolonged periods9. The “simulated microgravity” conditions generated in this way are far from perfect weightlessness and have numerous side effects, which have a greater or lesser impact on the results depending on the setup and the model organism used9,10,11,12,13. Nevertheless, rotational bioreactors such as the RPM are still the means of choice for preparatory experiments and important tools in biomedical space research10,14. Unlike spaceflight experiments, which are logistically complex, expensive, and limited in availability, the RPM enables broader and more frequent experimentation, particularly important for preliminary studies, mechanistic insights, and hypothesis generation. Moreover, for cell types like monocytes, whose short lifespan complicates the use in real microgravity, RPM-based models provide an essential platform for studying immune responses under altered gravitational conditions. Although the shear stresses generated on the RPM (10–25 mPa) are much lower compared to the typical physiological stresses generated by blood flow15, which are typically between 0.1 and 9.5 Pa16, the RPM is gaining recognition in new research areas of mechanobiology. Mechanical loading has already been described to modulate differentiation and plasticity of human monocytes17. However, adequate controls are required. Therefore, the RPM is a valuable tool to characterize exactly how rotating bioreactors affect immune cell function. This will allow to understand data generated in RPM experiments and to transfer results from RPM experiments to both space microgravity and physiological systems on Earth.Monocytes are central players in the immune system, serving crucial roles as professional antigen-presenting cells, immune sentinels, and precursors to macrophages and dendritic cells. Understanding how they respond to mechanical influences like shear stress, which is significant for circulatory homeostasis, is essential for comprehending immune processes18. The decisive advantage of their use in RPM experiments is that monocytes are suspension cells that are automatically kept floating by random positioning. This probably comes closest to the conditions in microgravity9. The human leukemia monocytic cell line THP-1 is widely used to study both monocytes and macrophages19,20. THP-1 cells have several technical advantages over human primary monocytes or macrophages, as they enable a very homogeneous and long-lasting stable cell culture. This makes them particularly interesting for automated long-term experiments in harsh environments (e.g. in spaceflights). THP-1 monocytes provide several further advantages over tissue-resident or peripheral blood mononuclear cells (PBMCs), being ease of access, long-term storage, faster proliferation rates, relative homogeneity with the same genetic background and the absence of “contaminating” cells21. However, the use of THP-1 cells also has some disadvantages. Several studies have shown that leukemic cell lines such as THP-1 cells respond differently to inflammatory stimuli than blood-derived monocytes22,23. Furthermore, THP-1 cells express only small amounts of CD14 and are, therefore, a poor model for blood-derived monocytes with regard to lipopolysaccharide (LPS) reactions, for example24. THP-1-derived macrophages can thus be considered a simplified model of primary macrophages when it comes to studying relatively simple biological processes, but they fail as an alternative source for more comprehensive immunopharmacology and drug screening programs25.From a space research perspective, we focused on the feasibility and usefulness of monocyte cultures exposed to the RPM environment. Therefore, we examined and compared cultures of the model cell line THP-1 and CD14+ PBMCs from adult blood after random positioning for morphology, typical differentiation markers, and functionality. Dynamic culture systems for studying immune cell function are however also being used outside of gravitational biology to increase the physiological relevance of immune cell culture systems, as these systems more closely resemble the in vivo environment of suspension cells in the blood system. This way, we considered the utility of the RPM for developing better physiological cell culture systems in the study of immune cell function.ResultsMorphological and biological effects of random positioning on THP-1 cellsTo assess the effects of random positioning on human immune cells, we first cultured monocyte-like THP-1 cells on the RPM for up to 7 days. Note, that cell culture flasks were completely filled with medium for both static and RPM conditions, while conventional cell culture is done in a partly filled culture flask with medium/air contact (Fig. 1a). Compared to a static cell culture, the RPM-exposed cells showed a changed morphology after 5 to 7 days: qualitatively they seem to grow less in aggregates, and individual larger cells with a progressively smoother shape were visible (Fig. 1a, arrows; Fig. 1b). F-actin staining in large THP-1 cells from RPM cultures revealed the typical marginal localization, as also observed in statically cultured cells. In addition, these cells displayed actin accumulation within outward protrusions (Fig. 1c, arrow). In our estimation, such protrusions were predominantly observed in RPM cultures, infrequently under static conditions, but not in conventional cell cultures (Supplementary Fig. S1).Fig. 1Effects of the random positioning machine (RPM) on human monocyte-like THP-1 cells. (a) Typical appearance of a static and a dynamic THP-1 cell culture. Random positioning of the THP-1 cells leads to an altered appearance of the cells within 5–7 d. The small section shows the presumed movement of the cells inside the culture flask due to RPM rotation. Scale bars: 100 μm. (b) Close-up images of individual larger cells with altered morphology on the RPM. Scale bars: 100 μm. (c) Immunofluorescence of F-actin after 3 d shown as cross-section (small pictures, scale bars: 5 μm) and 3D model of a hyper stack (big pictures). (d) Total cell numbers during a 7 d experiment (n = 5) (below) Observed cell culture effects on the RPM. (e) Effects on cell size analyzed by flow cytometry (top, n = 5) and cell viability analysis (middle, n = 6). Number of large cells per microscopic image (20×). (f) Effects on cell granularity analyzed by flow cytometry (n = 5). (g) Effects on autofluorescence (AF) in the FITC and the PE channels. Non-parametric Mann–Whitney U test ** p ≤ 0.01, *** p ≤ 0.001, ns non-significant. FSC-A: forward scatter area; SSC-A: sideward scatter area. Parts of the figure were drawn by using pictures from Biorender.com and Servier Medical Art.Full size imageIn addition, the rotated cell culture contained a larger number of cells after 7 days (+ 30%), indicating a higher cell proliferation on the RPM, which starts only after 4 days of rotation (+ 10%) (Fig. 1d). Although observations after 5 to 7 days showed individual larger cells on the RPM (Fig. 1b and e, lower panel), flow cytometry results of the entire cell population did not indicate a significant change in cell size between the two culture conditions. However, a small population of cells with larger forward scatter was observed in the RPM group (Fig. 1e, upper panel), that confirms presence of individual larger cells. Furthermore, independent automated cell counting analysis confirmed our visual observations and showed a slightly increased average diameter of the THP-1 cells on the RPM after 7 days, but not after 3 days (Fig. 1e, middle panel). Flow cytometry findings showed a short-term increase in granularity of THP-1 cells (1–3 days) on the RPM compared to static conditions, followed by a decrease after 7 days (Fig. 1f). It is often reported that monocyte differentiation is accompanied by an increased autofluorescence of the cells21,26. In our experiments, the autofluorescence intensities in the FITC and PE channels of THP-1 cells showed no significant change between the two groups (Fig. 1g) and were therefore complemented by an additional analysis of differentiation markers at a later time point to rule out a false-negative result as conclusions about differentiation should not be based on autofluorescence alone.Effects of random positioning on the differentiation capability of THP-1 cellsVarious biophysical properties of a cell (such as cell size, cell shape and distribution of intracellular structures) represent the emergent properties of cell phenotype and function27. Since changes in these properties can indicate monocyte differentiation21, we next looked at the differentiation capability of THP-1 cells. In preliminary experiments, we chemically differentiated THP-1 cells into M0 macrophages using PMA (phorbol 12-myristate 13-acetate), which led to an optical change in cell morphology under the microscope (Supplementary Fig. S2a). Differentiation success was further determined by the cellular expression of activation antigens (CD14, CD71) as evidence of the maturation of THP-1 cells28,29. Both activation markers CD14 and CD71 increased in PMA-treated THP-1 cells (Supplementary Fig. S2b).Next, we pre-cultured the THP-1 cells on the RPM for 4 and 7 days prior to PMA-differentiation. Control cells were cultured under conventional (conv.; medium/air contact) or static (static; medium filled flask) conditions for the same amount of time before PMA differentiation (Fig. 2a). Differentiation success was confirmed by optical inspection. Differentiated cells attached to the bottom of the well plate and showed either an elongated or ‘egg-shaped’ morphology. Interestingly, static cultured cells showed a more elongated morphology (Fig. 2b, blue arrows) while cells on the RPM became more ‘egg-shaped’ (Fig. 2b, red arrows). PMA-treated THP-1 cells, after 4 days on the RPM, increased more in size and granularity compared with static cell culture (Fig. 2b, top). However, this difference was not observed anymore after 7 days (Fig. 2b, bottom). Changes in granularity could thus be a remaining culture effect independent of PMA treatment, as this was already observed in cells not treated with PMA (Fig. 1f). Interestingly, conventional cell culture with medium/air contact resulted in better differentiated M0 macrophages as seen by the largest increase in cell size and granularity with a mixed morphology in the response to PMA (Fig. 2b).Fig. 2Effects of cell culture conditions on the response of THP-1 cells to PMA stimulation. (a) Schematic overview of experimental timeline. (b) Effects of pre-culture conditions on cell size and granularity of M0 macrophages (top n = 4, bottom n = 6). Scale bars: 50 μm (20×). (c) Marker expression on PMA-differentiated M0 macrophages after 4 d under different culture conditions (n = 4). (d) Mean Fluorescence Intensity (MFI) of CD14, CD16, CD71, CD80, CD206 and CD209 as determined by flow cytometry after 4 d (n = 5). (e) and after 7 d (n = 6) under different culture conditions. Independent sample t-test (n = 4) or non-parametric Mann–Whitney U test (n ≥ 5) * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001. Parts of the figure were drawn using pictures from Biorender.com and Servier Medical Art.Full size imageMacrophages and their distinct functional phenotypes depend on environmental stimuli and are characterized by plasticity30. Thus, we additionally analyzed the expression of various surface markers for macrophage differentiation. The selected marker panel consisted of activation markers CD14, CD71 and phenotypical antigens such as CD80, CD209, CD206 and CD1631,32,33. Under all analyzed culture conditions, 4-day PMA-differentiated cells revealed an increased surface expression of both activation markers and CD16, CD80 and CD206, but not CD209 (Fig. 2c). Interestingly, cells pre-cultured on the RPM for 4 days showed the highest expression of these antigens, while cells cultured under conventional and static conditions express comparable amounts of analyzed surface markers (Fig. 2d). This suggests an increased plasticity in response to PMA of cells cultured on the RPM. After 7 days, these effects could not be observed anymore. Instead, RPM-cultured cells showed a slightly reduced activation status as marked by reduced CD14 and CD71 expression. Phenotypical antigens were either unchanged (CD80, CD16) or slightly increased (CD206, CD209) compared to the conventional culture group. Moreover, static cultured cells showed the highest expression levels for CD14, CD16 and CD80 (Fig. 2e). These findings suggest a strong influence of the type and duration of culture systems used on monocyte differentiation and macrophage plasticity. While conventional cell culture with medium/air contact seemed to be superior to the completely filled flask conditions in terms of morphology, the RPM induced an increased expression of important phenotypical and functional surface markers, after four days of culture. This suggests that the cells potentially benefit from transiently rotated culture conditions, as evidenced by an increased responsiveness and activation to PMA.Effects of random positioning on the phenotype of THP-1 cellsThe characterization of the cell phenotype could also provide further insights into the previously observed PMA-independent changes by RPM. Thus, the panel consisting of the surface markers CD14, CD16, CD71, CD80, CD206 and CD209 was used to analyze activation of THP-1 cells independent of PMA. Only 30% of the THP-1 cells expressed the monocyte-typical marker CD14 under conventional cell culture. Additionally, THP-1 cells cultured under conventional conditions did not express CD16 and CD206, and only partially expressed CD71 (34%), CD80 (22%) and CD209 (35%) (Fig. 3a).Fig. 3Effects of the RPM on cluster-of-differentiation (CD) markers expressed by THP-1 cells. (a) Marker panel and basal expression on THP-1 cells. The percentage of CD+ THP-1 cells in normal cell culture is shown in pie charts. Pie charts correspond to 3b grey bar plots (conv.). (b) Percentage of cells expressing certain surface markers under different culture conditions. (c) Mean Fluorescence Intensity (MFI) of CD14, CD16, CD71, CD80 and CD209 as determined by flow cytometry (n = 5); conventional cell culture (dotted line) corresponds to 1. Non-parametric Mann–Whitney U test * p ≤ 0.05, ** p ≤ 0.01. Parts of the figure were drawn using pictures from Biorender.com and Servier Medical Art.Full size imageNext, the functional phenotype was compared between static and dynamic culture conditions after 1, 3 and 7 days. The percentage of CD14+ and CD16+ THP-1 cells strongly increased after 3 days of rotated or static cell culture (~ 80% and 60%, respectively) and decreased again after 7 days (~ 30%; Fig. 3b). This observation was partly reflected in the expression levels of CD14 on individual cells, which was higher in RPM-cultured cells than in static cultured cells after 1 and 3 days but lower after 7 days. RPM-exposure also increased the surface expression levels of CD209 and CD16 compared to static culture after 1 and 3 days, respectively (Fig. 3c). The transient increase in CD14 and CD16 expression after 3 days on the RPM indicates a transient more physiological state than under conventional or static conditions. It is important to note that the largest differences in marker positive cells (Fig. 3b) were observed between completely medium filled culture conditions (static/RPM) and conventional cell culture, especially after 3 days. For instance, the frequency of CD71+ cells increased to approximately 95–100% in both conditions when cells were cultured in completely medium filled flasks. Similar trends were observed for the other markers, which increased in cells cultured in completely medium filled flask conditions after 3 days, which leads us to believe that other factors besides rotation, such as the physico-chemical environment, have a large influence on THP-1 phenotype that should not be ignored.After 7 days, the percentage of CD14+ and CD209+ cells was comparable between all culture conditions. Notably, the percentage of CD209+ cells decreased more in RPM-cultured THP-1 monocytes than in static cultured cells after 7 days. The percentage of CD71+ and CD80+ cells remained elevated upon cells cultured with both completely medium filled flask conditions (Fig. 3b).Interestingly, static-cultured THP-1 monocytes after 7 days expressed higher levels of surface antigens CD14, CD71, CD80 and CD209 compared to RPM-cultured THP-1 cells, and higher levels of surface antigens CD14, C71 and CD209 compared to conventional cell culture (Fig. 3c). This again highlights that length and type of THP-1 culture system is critical for optimal outcomes of differentiation.Effects of random positioning on the viability of THP-1 cellsThe extent to which the rotated cell culture influences the viability of THP-1 cells after 7 days was tested with an annexin V/propidium iodide (PI) staining (Fig. 4a). A significant difference between static and dynamic cell culture was observed with a higher frequency of viable cells and a lower frequency of necrotic/dead cells on the RPM (Fig. 4b).Fig. 4Effects of an RPM cell culture on the viability and functionality of THP-1 cells. (a) Annexin V and PI staining in THP-1 cells cultured for 7 d under static or RPM conditions. The fourth quadrant represents living cells (annexin V, PI negative), the third quadrant early apoptotic cells (annexin V positive, PI negative), the second quadrant late apoptotic (annexin and PI positive) and the first quadrant necrotic or dead cells (annexin V negative and PI positive). (b) Percentages of viable, apoptotic and necrotic cells of different individual experiments (n = 3). (c) Effects of lipopolysaccharide (LPS) stimulation on THP-1 cells. Despite low basal expression of CD14, CD14+ THP-1 cells should upregulate CD14 in response to LPS stimulation. (d) Mean Fluorescence Intensity (MFI) of CD14 in response to LPS after different culture conditions (n = 2–5). The hatched bars show CD14 expression without LPS, the filled bars show the effect of LPS on CD14 expression. (e) Micromere particle uptake by THP-1 derived M0 macrophages. Phagocytosis was confirmed by an increase in cell size and autofluorescence in the PE-A channel. (f) Phagocytosis after different culture conditions (3–7 days) reflected by changes in autofluorescence (PE) and cell size (FSC-A). Independent sample t-test (n