A White Paper by Ronawk®
Tiffany Edwards1, Heather Decker1, Savannah Lowe2, Anna Smith2
1Ronawk, Overland Park, KS; 2Amnion Foundation, Winston-Salem, NC
May 21, 2026
Executive Summary
Scientific progress in placental biology is increasingly constrained not by a lack of knowledge, but by inadequate model systems. This limitation is particularly evident in the study of human placental cytotrophoblasts (CTBs), which are central to placental development as progenitors of both multinucleated syncytiotrophoblasts (STBs), responsible for nutrient and gas exchange, and extravillous trophoblasts (EVTs), which invade the maternal decidua and remodel uterine vasculature (1, 2). When cultured using conventional two-dimensional (2D) culture systems under atmospheric oxygen, CTBs rapidly and spontaneously differentiate, and the mechanisms governing lineage specification remain incompletely understood (3, 4). 2D culture systems fail to address this challenge, requiring non-atmospheric conditions to maintain CTB morphology while lacking important spatial and microenvironmental cues that help regulate differentiation.
In this white paper, we evaluate the Ronawk Bio-OS platform as a three-dimensional (3D) culture system for primary human CTBs. Compared to standard approaches, Bio-OS enables enhanced structural organization, improved retention of CTB-associated markers, and preservation of differentiation capacity under ambient conditions. These findings demonstrate that biomimetic 3D environment provides a physiological model to study trophoblast biology and placental function.
Introduction
The development of the human placenta is essential for successful pregnancy and fetal health, yet many of the molecular and cellular mechanisms governing placental formation remain poorly understood (1-5). Abnormal placental development is associated with severe pregnancy complications including miscarriage, stillbirth, preterm birth, intrauterine growth restriction, preeclampsia, and placental insufficiency (5). Because the placenta serves as the critical interface between mother and fetus, understanding the cellular systems that regulate placental growth and function is fundamental to reproductive biology and maternal–fetal medicine.
Trophoblasts are specialized placental cells that play central roles throughout pregnancy, including establishment of histiotrophic nutrition, remodeling of maternal vasculature, immune regulation, and attachment of the placenta to the uterine wall (1). Among these trophoblast populations, placental cytotrophoblasts (CTBs) represent a critical progenitor lineage. CTBs are proliferative epithelial cells derived from the trophectoderm shortly after implantation and serve as the source of the major trophoblast subtypes that establish and help maintain the placenta throughout gestation (1,3).
CTBs differentiate into two major trophoblast lineages with distinct biological functions (1-5):
- Syncytiotrophoblasts (STBs): Multinucleated cells formed through CTB fusion that mediate nutrient and gas exchange, hormone production, and immune regulation at the maternal–fetal interface.
- Extravillous trophoblasts (EVTs): Invasive trophoblasts that migrate into the maternal decidua and remodel uterine spiral arteries to establish low-resistance blood flow to the developing fetus.
CTB differentiation must be tightly regulated to ensure proper placental development and maternal–fetal exchange throughout pregnancy. Disruption of this process can lead to severe pregnancy complications, including preeclampsia, fetal growth restriction, miscarriage, placental insufficiency, and abnormal placental invasion disorders such as placenta accreta spectrum disease (1, 6). Inadequate differentiation toward the extravillous trophoblast (EVT) lineage impairs uterine spiral artery remodeling and reduces placental perfusion, while abnormal syncytiotrophoblast (STB) formation compromises nutrient exchange, hormone production, and immune regulation at the maternal–fetal interface (1, 7). CTB lineage specification is controlled by a complex combination of biochemical and biomechanical signals, including oxygen tension, extracellular matrix composition, tissue stiffness, cell–cell interactions, and developmental pathways such as Wnt, Notch, AP-2α, TGF-β and PPAR-γ signaling (1, 4, 8-11). However, many of these regulatory mechanisms remain poorly understood because conventional two-dimensional (2D) culture systems fail to replicate the structural organization and microenvironmental cues present in native placental tissue. CTBs cultured in vitro rapidly lose progenitor-associated phenotypes and undergo spontaneous differentiation, limiting the physiological relevance and reproducibility of existing placental models (11, 12). Improved biomimetic three-dimensional (3D) culture systems are therefore required to better preserve CTB phenotype, enable controlled differentiation, and more accurately model human placental biology and disease.
Limitations of Current CTB Culture Methodologies
Conventional cell culture systems were developed primarily for ease of expansion and experimental accessibility and remain poorly suited for maintaining the physiological behavior of primary mammalian cells in vitro (13-18). Cells cultured in two-dimensional (2D) environments exhibit substantial differences in gene expression, morphology, physiology, and cellular behavior compared to cells grown in three-dimensional (3D) systems or native tissues (19-20). One major driver of these differences is the altered tissue microenvironment and signaling landscape experienced in traditional 2D culture.
Native tissues provide highly regulated biochemical and biomechanical cues that govern cell differentiation, viability, phenotype, morphology, and gene expression (21-23). Among the most influential of these cues is substrate stiffness. While native tissues typically range from approximately 0.1–100 kPa in stiffness, standard tissue culture plastic is orders of magnitude stiffer (~1000 kPa) (20, 24). This mechanical mismatch alters cellular behavior and contributes to loss of multipotency, increased senescence, and reduced bioactivity in vitro, particularly in stem and progenitor cell populations (25-29). Increasing evidence demonstrates that faithful replication of native microenvironmental signaling is essential for physiologically relevant cell culture; however, conventional two-dimensional (2D) systems fail to recreate the structural, biochemical, and biomechanical complexity of native tissue environments (20, 30-33).
The limitations of 2D culture are further compounded with the difficulty of culturing primary cytotrophoblasts (CTBs). To culture CTBs scientists face several other significant constraints:
- Reliance on collagen IV coated plastic cultureware increases cost and technical complexity.
- Atmospheric oxygen conditions accelerate senescence and spontaneous differentiation.
- Hypoxic culture conditions (2% O₂), while partially protective, require specialized infrastructure that limits accessibility and reproducibility.
- CTBs rapidly lose progenitor-associated phenotypes during serial passaging.
- Freshly isolated CTBs from term placentas have significant donor-to-donor variability and some cell populations are nearly senescent when compared to early term placentas.
- Standard 2D systems fail to reproduce the three-dimensional microenvironment and biomechanical cues that regulate trophoblast organization and lineage specification (Fig. 1).
3D culture systems have emerged as a promising alternative because they better recapitulate native tissue architecture, cell–cell interactions, and tissue-like signaling. However, many existing 3D systems remain limited by challenges related to nutrient and oxygen diffusion into the core of the matrix, scalability, usability, reproducibility, and compatibility with translational or regulatory workflows. Together, these limitations create a major bottleneck in placental biology research and highlight the need for physiologically relevant, scalable 3D CTB culture systems.
Figure 1: In vivo placental structure versus conventional 2D culture limitations. Comparison of the complex placental microenvironment vs 2D culture. Image on the left adapted and simplified from Knofler et al., 2019. AC amniotic cavity, ChC chorionic cavity, CTB cytotrophoblast, EC ectoderm, EVT extra-villous trophoblast, pSC placental stromal cell, STB syncytiotrophoblasts.
Bio-OS™ Platform
Ronawk’s Bio-OS platform is a modular 3D cell culture and biologics manufacturing system designed to standardize and scale mammalian cell production from research through manufacturing (Fig. 2). The platform integrates three tightly coupled components:
- Bio-Blocks® hardware: Tissue-mimetic hydrogel substrates.
- Standardized process logic and SOPs.
- The living biology cultured within the system.
Together, these layers create a reproducible biological operating system that reduces variability, enables coordinated workflows across sites, and supports scalable cell manufacturing.
Figure 2. Bio-OS: Ronawk’s Biological Operating System, a novel platform-based technology for the culture of cells in a physiologically relevant tissue-mimetic environment.
At the core of Bio-OS are Bio-Blocks, customizable bioprinted hydrogels engineered to recreate native tissue microenvironments through defined mechanical properties and porous microchannel architecture (Fig. 3). Bio-Blocks are fully customizable. Its standard “puzzle piece” modular macrostructure allows customizable, building block-like configurations that can be adapted to different tissue types and easily expanded without disrupting ongoing cultures, eliminating the requirement for sub-passaging. Nutrients and oxygen are easily diffusible throughout the scaffold which supports long-term viability and stable phenotypes. At the microscale, an interconnected network of polymer pores and microchannels creates a dynamic microenvironment that maximizes surface area for cell attachment while supporting robust cell growth and tissue formation. The material and mechanical properties of Bio-Blocks are precisely tunable to match the elastic modulus of specific human tissues, enabling physiologically relevant mechanotransduction and tissue-specific function. eliminating the need for repeated passaging.
The porous microarchitecture of Bio-Blocks functions as a compact bioreactor, enabling:
- Efficient nutrient and oxygen diffusion.
- Continuous cell migration and expansion.
- Eliminates need for repeated subculturing.
- Enhanced retention of stem-like phenotypes.
- Retention of differentiation potential.
- Easy collection and enhanced secretion of biologics including extracellular vesicles, proteins, and antibodies
- Together, these features reduce cellular stress, minimize phenotype drift and contamination risk, and deliver reproducible, high-fidelity tissue models that scale seamlessly from discovery to production within a compact footprint.
By supporting tissue-like cellular organization and behavior, Bio-OS promotes long-term cellular stability, functional retention, and enhanced biological productivity within a compact footprint.
Figure 3. Schematic of Bio-Block architecture and porous microchannel structure. (A) Classic Bio-Block in the puzzle piece configuration. Bio-Blocks are ~1-cm3 and have the equivalent culturable surface area as a T150 flask. Center microchannel shown with red circle. (B) Illustrative depiction of interconnected microchannels.
Methods
Figure 4 displays a graphical overview of the methods used for the study. A brief description of the methods is provided below.
Primary Cytotrophoblast Isolation and Expansion
Primary human placental cytotrophoblasts (CTBPRO2®) were isolated from term human placentas following uncomplicated birth. Cells were enriched using density gradient centrifugation followed by CD49f-positive selection and expanded to passage 2 under hypoxic conditions (2% O₂). Expanded CTBs were cryopreserved by Amnion Foundation and transferred to Ronawk for downstream studies.
2D and 3D Cell Culture
Cryopreserved CTBs were thawed and seeded into either conventional two-dimensional (2D) culture systems or Bio-OS Bio-Blocks (3D).
| 2D | Bio-Block |
Seeding Density | 750,000 | 500,000* |
| Culture Surface Area | 75cm2 | ~150cm2 |
CTB passage at time start of experiment | 3 | 3 |
Number of subpassages | 5 | 0 |
Total days in culture | 20 | 20 |
Temperature | 37°C | 37°C |
Oxygen | Atmospheric | Atmospheric |
CO2 | 5% | 5% |
Vessel coating | 1% Gelatin | Proprietary hydrogel polymer |
Number of Bio-Blocks per well | N/A | 4 (12 total) |
*Seeding density adjusted for total number of cells available.
All cultures were maintained under ambient oxygen conditions using CTBPRO2GRO cytotrophoblast media. Media exchanges were performed every 24–48 hours. Conditioned media was collected during each media exchange and analyzed for hCG expression.
Morphological and Phenotypic Analysis
Cell morphology and spatial organization were monitored throughout culture using brightfield microscopy.
At experimental endpoints, cultures were fixed in 2% paraformaldehyde (PFA) and analyzed by morphological staining and immunolabeling. 2D cultures were stained on 24-well glass plates seeded at 20,000 cells/well and fixed 24 hours post seeding. Bio-Block cultures were analyzed either in situ within the hydrogel platform or cryo-embedded, sectioned transversely and labeled. Structural stains included:
- DAPI/Hoechst 33342 (DNA)
- Wheat Germ Agglutinin (Lectins)
- Phalloidin (F-actin)
- MemGlow (Lipids)
Immunolabeling was performed for the following trophoblast-associated markers:
- CD49f: Cytotrophoblast progenitor marker.
- CK7: Epithelial trophoblast marker.
- CD138: Syncytiotrophoblast-associated marker.
- HLA-G: Sxtravillous trophoblast marker.
Figure 4. Experimental workflow schematic. Image created using Bio-Render.
Results
CTBs were cultured in Bio-Blocks with either 300 µM or 500 µM microchannel sizes for up to 20 days. CTBs cultured in the Bio-Block exhibited pronounced 3D organization and preferential localization within 500 µm microchannels (Fig. 5). Cells formed tightly packed clusters of cells in the microchannels and on the surface of the Bio-Blocks, eventually forming a dense layered sheet of cells surrounding the Bio-Blocks (Fig. 5E) and cells bridged the gaps between each Bio-Block forming unique structures. At increased magnification, cells cultured in the Bio-Blocks were found to have large vacuoles and significant granulation (Fig. 5F). In contrast, 2D cultures formed uniform monolayers (Fig. 5A). Over serial passage, 2D cultures showed a bias toward STB differentiation (Fig 5D) and a small percentage of cells differentiated into the extravillious trophoblast (Fig. 5D).
Figure 5: Brightfield Morphological Assessment of 2D and 3D Cultures. (A, D) show representative images of CTBs cultured on gelatin coated T75 flasks and serial passaged over 20 days. B, C, E, F) show CTBs cultured in Ronawk 500V Bio-Blocks for 20 days without subculturing. dps, days post seed.
After 20 days in culture, the cells were fixed in 2% PFA and stained with morphological markers including DAPI to stain DNA, Wheat Germ Agglutinin (WGA) to stain lectins, MemGlow to stain lipids, and Phalloidin to stain F-actin. Cells cultured in the Bio-Blocks were found to have an increase in lipid staining within the cellular membrane (Fig. 6A), while the lectin staining was less pronounced (Fig. 6B). In comparison, 2D cultures had less punctate lectin staining (Fig. 6D) and more diffuse lipid staining (Fig. 6E).
Figure 6: Morphological assessment panels. (A, B, C) CTBs cultured in Ronawk Bio-Block for 20 days followed by PFA fixation and morphological staining with Memglow (A), WGA (B), DAPI, and Phalloidin. 3D overlay (C). (D, E, F) Passage 7 CTBs cultured on 24-well gelatin coated glass bottom plate, fixed in 2% PFA and stained with morphological markers Memglow (D), WGA (E), DAPI and Phalloidin. 2D overlay (F).
Immunolabeling for canonical trophoblast was performed. The selected markers were CD49f (cytotrophoblast progenitor marker), cytokeratin 7 (CK7; epithelial trophoblast marker), CD138 (syncytiotrophoblast-associated marker), and HLA-G (extra-villous trophoblast marker). Bio-Block cultures retained stronger expression of CTB-associated markers (CD49f) alongside CD138, indicating enhanced maintenance of progenitor phenotype with concurrent differentiation capacity (Fig 7). In contrast, 2D cultures showed strong STB differentiation bias.
Figure 7. Immunofluorescence staining panels: Evaluation of CTB lineage markers CTBs cultured in Bio-OS or 2D. Blue, Hoechst, Green WGA, Red CD138 (A, B, D, E) or CD49f (G, H, J, K), Magenta CK7 (A, C,D, F) or HLAG (G, I, J, L). 3D max-project of 50 µM Bio-Block section overlay (A,G). P8 2D culture overlay (D, J). Fluorescent threshold of the 2D panels for CD138 and CD49f was increased for easier visualization.
Figure 8: Longitudinal hCG expression. 3D Bio-Blocks shown in Blue, 2D shown in Black. *subculturing of 2D cells.
Human chorionic gonadotropin (hCG) is produced by trophoblasts after implantation of the developing embryo and expression/secretion continues throughout pregnancy primarily in differentiated STBs. Quantification of hCG secretion in spent cell culture media has been used to support differentiation of primary cytotrophoblasts into the STB phenotype; primary CTBs secrete a modest amount of hCG but STBs have increased hCG secretion (data unpublished, 34-36). In the Bio-Block 3-day lag period was observed and then consistent levels of hCG were produced over the duration of the culture (Fig. 8). In comparison, 2D cultures showed a larger lag period from date of seed to culture confluency of ≥75% in earlier passages. During later passages, a higher concentration of hCG was detected within 24 hours post subculture indicating that higher proportion of the cells had undergone differentiation into STB, which was supported by brightfield microscope examinations.
Collectively, these results demonstrate that the Bio-Block platform provides a more biomimetic environment that supports CTB phenotype retention and spatial organization, offering a valuable system for studying human trophoblast biology and placental function in vitro.
Comparative Summary
Feature | 2D Culture | Bio-OS Platform |
Structure | Monolayer | 3D organization |
Phenotype retention | Poor over passage | Enhanced |
Differentiation bias | STB skewed | Balanced potential |
Culture duration | Requires passaging | Long-term without passaging |
Discussion and Future Work Applications
The results presented here demonstrate that microenvironmental context is an important determinant of cytotrophoblast (CTB) behavior and highlights the limitations of conventional 2D culture systems in modeling placental biology. While traditional 2D culture systems remain valuable for some research and development experimental applications due to their simplicity and accessibility, these findings underscore the importance of incorporating physiologically relevant 3D culture systems whenever possible to better recapitulate native tissue architecture and function. By enabling three-dimensional organization and reducing dependence on non-physiological culture conditions, the Bio-OS platform supports sustained CTB phenotype retention while preserving differentiation capacity, and future studies may be performed to investigate the signals required to force differentiation into either the STB or EVT lineage. This balance between stability and plasticity is critical for accurately studying trophoblast lineage dynamics and function. The observed structural organization and CTB marker expression suggest that biomimetic architecture of the Bio-Block may support maintenance of physiologically relevant CTB cell states. Collectively, these findings position Bio-OS as a foundational platform for advancing in vitro placental models, with the potential to improve experimental reproducibility, biological insight, and translational relevance.
Bio-OS enables studies of CTB lineage regulation, modeling of placental diseases (e.g., preeclampsia, IUGR), and evaluation of drug effects, including placental transfer and toxicity. The platform also supports the development of advanced placental models and integration into multi-organ systems. The possibilities are extensive (Fig. 9); for more information about Ronawk’s Bio-OS platform, reach out to us directly.
Figure 9. Potential applications of Bio-OS in placental biology
Conclusion
The Bio-OS platform provides a biomimetic 3D culture environment that improves cytotrophoblast phenotype retention and organization compared to conventional 2D systems. This approach offers a powerful new tool for advancing the study of human placental biology and developing translational applications.
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About Ronawk
Ronawk has created Bio-OS™ is a modular platform that replicates native human biomechanics and tissue signaling, bridging In Vitro and In Vivo biology. Bio-OS scales from discovery to biomanufacturing for mammalian cells and biologics while reducing scale-up risk.
Ronawk’s Bio-OS will do for biological manufacturing what Nvidia™ GPUs did for AI computing.
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