A long-term 3D organoid culture system was established for mouse and human primary hepatocytes by Matrigel, which is a hydrogel based on solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in ECM proteins (Kleinman et al., 1982). In this system, single hepatocytes can create organoids that can be cultivated for several months while keeping important morphological, functional, and gene expression characteristics (Hu et al., 2018). Furthermore, PEG hydrogels were employed to culture and expand a range of neuronal and glial cell types (Lampe et al., 2010) and hepatocytes (Christoffersson et al., 2019) by simply changing the material properties of the hydrogel. In addition, RGD-modified alginate hydrogels accelerated the development of retinal pigment epithelium and neuroretina in 3D-cultured human embryonic stem cells (hESCs)/human induced pluripotent stem cells (hiPSCs) (Hunt et al., 2017).

Since collagen is the most abundant natural type of fibrin hydrogel in vivo (Faraj et al., 2007), research has shown that collagen type II scaffolds may significantly improve the chondrogenic development of human mesenchymal stem cells (hMSCs) when compared to collagen type I hydrogel scaffolds (Tamaddon et al., 2017). Although type I and type II collagens support chondrogenic phenotypes in various ways, collagen hydrogel scaffolds can construct cartilage tissue (Tamaddon et al., 2017). For example, in a cartilage deficiency rat model, the collagen hydrogel scaffolds covered with rat mesenchymal stem cells (rMSCs) promoted rMSC chondrogenic development and had a statistically greater cartilage healing capability (Rouwkema and Khademhosseini, 2016). In addition, HA-based hydrogel scaffolds might stimulate the neural development of human-induced pluripotent stem cell-derived neural progenitors (hiPSC-NPCS) and the proliferation of neuroblastoma cells (Seidlits et al., 2010; Lei et al., 2011b; Yang et al., 2016). Another work encapsulated hiPSCs in HA-rich core-shell hydrogel microcapsules via microencapsulation to increase cell bulk and promote effective cardiac differentiation (Xu et al., 2021). In addition, growth factor-containing PVA hydrogels may accelerate the differentiation of mouse spermatogonial stem cells (mSCCs) into meiotic and postmeiotic cells (Kashani et al., 2020).

In a hydrogel-based 3D cell culture, cells can naturally form 3D structures rather than being restricted to a 2D surface. As shown in Figure 1, hepatocytes and hESCs form spheroids in PIC-based hydrogel (Bar = 200 μm). Similarly, the 3D culture experiments of chondrocytes showed that the collagen type I hydrogel scaffold could retain the chondrogenic phenotype of rat chondrocytes in a 3D growth pattern (Smeriglio et al., 2015; Jin and Kim, 2017; Kashani et al., 2020). Customizable HA hydrogels were also created with variable hardness for 3D rMSC growth (Wu et al., 2017). HA hydrogel scaffold can maintain the survival of bone marrow stromal cells (BMSCS) and promote direct tubular chondrogenic development (Ren et al., 2021). Furthermore, in 3D cultures of PVA hydrogel-coated cell plates, various human glioma cell lines (LN299, U87MG, and Gli36) may form tumor spheres like their morphology in vivo (Molyneaux et al., 2021). The alginate-collagen hydrogels improve cell adhesion of hiPSCs-derived neurons and stimulate the creation of complex neural networks in 3D culture models (Moxon et al., 2019).

The digested 2D cultured cells develop into a 3D cell mass after adding hydrogel into the cell suspension as a cell scaffold, as opposed to 2D culture with a flat morphology (Figure 1, left panel). Two types of cells, hepatocytes and hESCs, are depicted to show the difference between 2D patterned cells and 3D hydrogel-based cells (Figure 1, right panel). The hydrogel used here is polyisocyanopeptide (PIC) -based synthetic fiber hydrogels. hESCs, human embryonic stem cells; Bar = 200 μm.

It is known that a variety of growth factors may bind to ECM proteins. These secluded growth factors can produce gradients in concentration that direct the differentiation and morphogenesis of stem cells during in vivo development (Muncie and Weaver, 2018). Traditional 2D cell culture employs a liquid medium that does not allow for the storage and release of growth agents or the creation of concentration gradients. In contrast, 3D culture allows cells to construct 3D structures instead of being limited to a single layer in 2D (Jose et al., 2020). While more technically difficult than traditional 2D cell culture, hydrogel-based 3D cell culture facilitates normal cell-cell and cell-matrix interactions and tissue-specific activity. Materigel-based 3D cell culture, but not 2D models, increased tauopathy in human stem cell-derived neurons with familial Alzheimer’s disease by increasing the accumulation of β-amyloid aggregates in the ECM (Choi et al., 2014). PIC hydrogels enable the generation of mammary gland organoids from mammary fragments or pure single mammary epithelial cells by decorating with the adhesive peptide RGD for cell adhesion. Furthermore, the cell-gel interactions via the cell binding peptide density regulate the ratio of the major cell types in the mammary gland organoids (Zhang et al., 2020).

Given that alterations in cell-to-cell interactions and interactions with the ECM are the main effects of microgravity on cells and may alter cell fate through these effects (Andreeva et al., 2022), hydrogel-based 3D cell models should be employed for microgravity studies to study the response of human tissues and organs to microgravity.

Most studies show that microgravity affects cell differentiation by promoting reagent-induced differentiation of stem cells or specific cell types into specific tissue types. Short-term exposure (3 days) of cardiac progenitors to space microgravity upregulated genes involved in cardiac differentiation (Rampoldi et al., 2022). hBMSCs were affected by RMG and responded to RMG stresses, reverting to quiescence after a moderate osteogenic differentiation aboard ISS for 3 weeks (Bradamante et al., 2018). It was demonstrated that SMG facilitates hESCs to differentiate into hematopoietic stem cells (HSCs) and progenitor cells with more efficient induction of CD34⁺ CD31⁺ hemogenic endothelium progenitors (Ma et al., 2021). ADSCs displayed enhanced neural differentiation in neurons and increased neurotrophin expression and their specific Trk receptors, especially BDNF and TrkB, when cultured on fast rotating clinostat (Zarrinpour et al., 2017). When Postnatal stem cells from the apical papilla of teeth were cultured with NGF, EGF, and bFGF on high-aspect rotating vessels for 7 days, they formed nerve tissue (Kim et al., 2017). Limbal fibroblasts (LFs) cells could differentiate into adipocytes, osteocytes, and chondrocytes on RCCS/HARV for 3 days, compared to 1 g controls (Pao et al., 2017). Neonatal cardiac progenitor cells showed an increased expression of early developmental markers on ISS, 3 weeks (Baio et al., 2018). This less visible differentiation could be attributed to additional environmental factors or the poor cultural conditions of space travel experiments. However, some evidence suggests that microgravity can preserve pluripotency. Mouse ESCs can be maintained without leukemia inhibitory factor (LIF) and retain pluripotency under a simulated microgravity environment (Kawahara et al., 2009). Similar results were obtained for cancer stem cells (CSCs), which have similar differentiation potential to ESCs (Arun et al., 2017). SMG also increased stemness in human colorectal cancer cell HCT116 using RCCS, indicating CD133/CD44 dual-positive cells (Arun et al., 2019).

Another important aspect of the effect of microgravity on cells is the influence on cell adhesion, morphology, and cytoskeleton. When subjected to microgravity, some cells grew into a monolayer altering their growth behavior, while the remaining continued to develop naturally (Infanger et al., 2006; Ulbrich et al., 2010; Pietsch et al., 2011b). Although these cells detect the absence of gravity within seconds (Ulbrich et al., 2011; Grosse et al., 2012), it takes at least 12 h to see spheroids (Infanger et al., 2006; Pietsch et al., 2011b) and up to 7 days to see constructions like tubes resembling an intima floating in a culture flask (Grimm et al., 2009; Grimm et al., 2010). Our previous study found that SMG promotes hESCs to differentiate between HSCs and hematopoietic progenitor cells (HSPC). Interestingly, HSPC prefers floating growth, and transcriptome sequencing results showed that cell adhesion and ECM-related genes were downregulated (Ma et al., 2021). When retinal pigment epithelial cells, ARPE19 cells, were cultured on a 3D clinostat, actin cytoskeleton regulators were modulated, and the cells showed multilayered growth with increased expression of epithelial-mesenchymal transition (EMT) markers (Son et al., 2022).

Exposure of cells from epithelial tissue to microgravity creates 3D structures. For example, human microvascular endothelial cells (HMVEC) were exposed to RMG for 5 days and 12 days and SMG for 7 days, showing tubular structures (Pietsch et al., 2017). The yes-associated protein (YAP) and HIPPO signaling changes, known to be correlated with organ growth, cytoskeleton, and stress sensing, are also present in specific cells with enlarged morphology (Ma et al., 2019). For example, the upregulation of Hippo signaling with downstream genes, YAP1, was detected in cardiac progenitor cells (CPCs) when cultured on ISS and 2D clinostat (Camberos et al., 2019). Colorectal cancer cells, HCT116, are giant cancer cells formed with complete nuclear localization of YAP in the culture on RCCS (Arun et al., 2019). Changes in the cell adhesion, morphology, and cytoskeleton, which can impact various cellular functions, maybe a possible substrate for a cell’s response to microgravity. Since the 2D cell model differs from the in vivo stress environment, eliminating the effects of microgravity on cells from the experimental model should ensure that they are in a 3D development state akin to the in vivo environment.

For tissue engineering, various hydrogels were combined with a microgravity bioreactor. A multicellular 3D organotypic model used collagen I matrix of the human intestinal mucosa was composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells, and fibroblasts cultured under microgravity provided by the RWV bioreactor (Salerno-Goncalves et al., 2016). When chondrocytes were seeded onto poly (DL-lactic-co-glycolic acid) (PLGA) sponges and cultured in a chondrogenic induction medium containing TGF-β 1 for 3 weeks, the engineered cartilage then emerged in a microgravity bioreactor for another 3 weeks. The results showed that it had a similar structure and composition to native rat cartilage (Emin et al., 2008). Human periodontal ligament fibroblasts (hPDLFs) were 3D cultivated on a mineralized PLGA scaffold to mimic microgravity in the NASA-approved bioreactor. The outcomes demonstrated a successful strategy for producing hPDLF-PLGA structures with improved osteogenic potential using a 3D system and microgravity settings (Inanc et al., 2006). hPDLFs encapsulated in C/HA microspheres exhibited significantly higher osteogenic differentiation potential when compared to those not encapsulated. The 3D-osteogenic culture environment can potentially improve the osteogenic differentiation of hPDLFs (Inanc et al., 2007). The collagen-HA is a type of implant material used to create a permanent implant. The collagen-HA material allowed the embedded MLO-Y4 cells to survive and grow for 6 months. This technology creates permanent implants for patients with spinal cord injuries (Fournier and Harrison, 2021). However, microgravity raises the likelihood of irreversible changes that weaken skeletal integrity and the gradual start of fracture injuries in space travelers (Nelson et al., 2009; Genah et al., 2021). According to the findings of the previous studies, the 3D -matrix bone differentiation model can be used to test potential drugs against bone loss or to promote bone regeneration.

Hydrogel 3D culture models were used to investigate the influence of microgravity on stem cell development, an important research area in regenerative medicine and tissue engineering. An efficient and integrated 3D bioprocess has developed based on the encapsulation of undifferentiated mouse embryonic stem cells (mESCs) within alginate hydrogels. The osteogenic lineage’s morphological, phenotypic, and molecular characteristics were represented in 3D mineralized constructions with mechanical strength and mineralized calcium/phosphate deposition. This bioprocess represents a significant advance in bone differentiation from mESCs (Hwang et al., 2009). Similar findings have been discovered in improved odontogenic differentiation abilities of Human dental pulp stem cells (hDPSCs) on PLGA scaffolds in the 3D SMG culture system (Li et al., 2017), indicating that these models have the potential to be used to explore tissue regeneration processes in regenerative medicine and microgravity conditions.

It may be preferable to create a physiological and pathological research model similar to that used in vivo. Human pancreatic cancer NOR-P1 cells, fibroblasts, or minced pancreatic carcinoma tissue were grown in solid collagen gels for seven days in a microgravity environment. Compared to NOR-P1 3D cultures treated to the static 1 g condition, cultures subjected to the SMG condition had more mitotic, cycling (Ki67-positive), nuclear factor-kappa B-activating cells and fewer apoptotic cells. Additionally, compared to static culture conditions, human pancreatic cancer specimens better preserved the original carcinoma tissue’s heterogeneous makeup and cellular activity (measured by the cycling cell ratio and mitotic index) (Nakamura et al., 2002). When fibroblast differentiation was studied in SMG using collagen-based 3D matrices to approximate interstitial tissue, SMG exposure decreased alpha-smooth muscle actin (SMA) expression and Smad2/3 translocation into the cell nucleus compared to the 1 g control (Sapudom et al., 2021). Compared to 2D cell culture, 3D cell culture attenuates the effects of SMG on the T cells transcriptome and nuclear abnormalities, which were closer to the in vivo findings (ElGindi et al., 2022).

Compared to 2D culture, 3D cell culture is more effective for enhancing cell differentiation and organ-like tissue formation from a variety of cells to study the bio-effects of microgravity (Figure 2, lower panel). Exposing 3D cells to SMG circumstances increased the knowledge of biological response mechanisms to SMG and RMG in several tissues, including the liver, bone, vasculature, skin, and capillary tissue. Microgravity affects physiology, pathology, and medical research (Figure 2, right panel). MSCs, mesenchymal stem cells; ESCs, embryonic stem cells (the Figure was created with BioRender.com).